3D models of lamprey progesterone receptor complexed with progesterone, 7α-hydroxy-progesterone and 15α-hydroxy-progesterone

Sea lamprey, a basal vertebrate, contains a progesterone receptor [PR]. An unusual property of lamprey is that gonadotropin-releasing hormone induces synthesis of 15α-hydroxy-progesterone [15α-OH-P] instead of progesterone. There also is indirect evidence for 7α-OH-P in lamprey serum. To determine if there is a structural basis for the binding of 7α-OH-P and 15α-OH-P to lamprey PR, we constructed 3D models of the lamprey PR complexed with progesterone, 7α-OH-P and 15α-OH-P. These 3D models reveal that Met-277 in lamprey PR has a specific interaction with the 15α-hydroxyl on 15α-OH-P and with Met-192, which also contacts the 15α-hydroxyl group. We also find that 7α-OH-P has favorable contacts with side-chains in lamprey PR. BLAST searches reveal that Met-277 on lamprey PR is unique among vertebrate PRs. This unique site on lamprey PR could be a target for compounds to control reproduction in sea lamprey, an environmental pest in Lake Michigan.     

Is progesterone a pre-hormone in the CNS?

In this paper, experimental evidences have been presented indicating that progesterone per se appears to be a powerful modulatory steroid of presynaptic striatal dopaminergic terminals of the central nervous system of the rat. This effect of the progesterone signal is concentration as well as infusion mode dependent. Low pulsatile doses of the steroid positively modulate the mechanism by which dopamine terminals respond to amphetamine stimulation and increase tissue dopamine concentration. Whereas, continuous and/or high doses of this steroid negatively modulate the response of the dopamine terminals to amphetamine stimulation and decreases tissue dopamine concentration. This effects occurs through a membrane mediated mechanism either upon the dopamine neuron directly and/or upon an interneuron. Pregnanolone a 5- beta-3 beta-metabolite of progesterone known to activate the hypothalamic LHRH neural apparatus at the level of the hypothalamus of ovariectomized estrogen primed rats in both in vitro as well as in vivo preparations was completely ineffective at the level of the corpus striatum of similar animal preparations. Therefore, it is reasonable to assume that site specific mechanisms exist within the central nervous system which may control differentially the final action of progesterone. In the hypothalamus, pregnanolone appears to be the final signal for its action on the LHRH neural apparatus, whereas in the corpus striatum, the steroid per se, and dependent on the modality and/or the strength of the signal can either directly or indirectly up-regulate (stimulatory component) or down-regulate (inhibitory component) the activity of striatal dopaminergic terminals.     

16α-hydroxylation of progesterone by a strain ofactinomyces globosus

В систематическое исследование трансформации свойств Actinomycetes было было установлено, что 17 из 76 видов тестировани е преобразованы прогестерон по 16 га-гидрокси -п рогестерона. Оптимальные условия для этой трансформац ии были изучены этой трансформации б ыли изучены следующие результат ы:

1. (1)
   Оптимальное рН для данного типа трансформации была 6–7. На нижней hydroxylation ценностей была меш ает.     
   
   

Aromatic β-amino-ketone derivatives as novel selective non-steroidal progesterone receptor antagonists

A novel class of non-steroidal progesterone receptor antagonists with aromatic β-amino-ketone scaffold have been synthesized and characterized with high binding affinity and great selectivity for the cognate receptors. Among them, compound 22 was shown to be the most potent progesterone receptor antagonist in cotransfection assay and a murine model of ligand-induced decidualization.     

11α-Hydroxylation of progesterone by gel-entrapped living Rhizopus stolonifer mycelia

Summary Spores of Rhizopus stolonifer were immobilized aseptically by entrapment with photo-crosslinkable resin prepolymers, urethane prepolymers or several kinds of polysaccharides. The entrapped spores were allowed to germinate and develop in situ. The immobilized living mycelia so obtained were induced for the steroid 11-hydroxylation system and examined for their activity to hydroxylate progesterone at 11-position in a buffer system containing 2.5% of organic cosolvent. Of various water-miscible organic cosolvents, methanol was found to be most effective in terms of the activity of the entrapped mycelia and the solubility of the product, 11-hydroxyprogesterone. Though all the living mycelia entrapped in different gels exhibited the hydroxylation activity, mycelia entrapped in photo-crosslinked gels showed the maximum activity which was rather higher than that of the free mycelia. The net-work size of the photo-crosslinked resins, namely the chain length of the photo-crosslinkable resin prepolymers, affected markedly the mycelial growth in gels, and subsequently, the hydroxylation activity of the entrapped mycelia. Entrapment significantly enhanced the operational activity and stability of the 11-hydroxylation system in the mycelia, and permitted the intermittent reactivation of the system by incubating the entrapped mycelia in potato-dextrose broth.     

Synchronization of estrus in post-partum mares with progesterone and estradiol 17β

Thirty-six mares which foaled over a 10-day period were given 1 to 10 daily intramuscular injections of a combination of 150 mg. progesterone and 10 mg. estradiol 17β. The first injection was given within 18 hours after parturition. Because individual mares foaled on different dates during the 10 day period, commencement of treatment varied, but treatment for all mares ceased on the same day. Teasing and breeding began seven days after the final treatment. The mares were teased daily for 10 days and artifically inseminated every second day until ovulation occurred. The mean interval from the end of treatment to beginning of estrus was 9.4 days (range 7 to 14) and 33 of 26 mares (94.7%) ovulated 10 to 16 days after the final treatment. Both estrus and ovulation were effectively synchronized, resulting in a first estrus pregnancy rate of 80.6% (29 of 36).     

Aromatic esters of progesterone as 5α-reductase and prostate growth inhibitors

The aim of this study was to determine the biological activity of 4 steroidal derivatives (9a, 9b and 10a, 10b) prepared from the commercially available 17α acetoxyprogesterone, where 9a, 9b, have the Δ⁴-3-oxo structure and 10a and 10b an epoxy group at C-4 and C-5.

These steroids were tested as inhibitors of 5α-reductase enzyme, which is present in androgen-dependent tissues and converts testosterone to its more active reduced metabolite dihydrotestosterone.

The pharmacological effect of these steroids was demonstrated by the significant decrease of the weight of the prostate gland of gonadectomized hamsters treated with testosterone plus finasteride or with steroids 10a and 10b. For the studies in vitro the IC₅₀ values were determined by measuring the steroid concentration that inhibits 50% of the activity of-5α-reductase. In this study we also determined the capacity of these steroids to bind to the androgen receptor present in the rat prostate cytosol.

The results from this work indicated that compounds 9a, 9b, 10a, and 10b inhibited the 5α reductase activity with IC₅₀ values of 360, 370, 13 and 4.9 nM respectively. However these steroids did not bind to the androgen receptors since none competed with labeled mibolerone. Steroid 10b, an epoxy steroidal derivative containing bromine atom in the ester moiety, was the most active inhibitor of 5α-reductase enzyme, present in human prostate homogenates with an IC₅₀ value of 4.9 nM and also showed in vivo pharmacological activity since it decreased the weight of the prostate from hamsters treated with testosterone in a similar way as finasteride.     

A progesterone receptor affinity chromatography reagent: 17α-Hexynyl nortestosterone sepharose

Several affinity chromatography reagents have been proposed for purification of progesterone receptor (PgR), and significant results have been achieved with some of these. None, however, have approached the results achieved in affinity chromatography of estrogen receptor. We have therefore synthesized a number of new 19-nortestosterone derivatives capable of chemically stable linkage with Sepharose beads, and have identified one with very high PgR affinity for further study. We first synthesized the epoxides of 17α-allyl nortestosterone, by analogy with the estradiol derivatization of Greene and Jensen. The relative affinity of these epoxides for PgR from T47D human breast cancer cells, however, was only around 5% that of R5020, and affinity beads prepared from them bound very little PgR. We then reacted appropriately protected 17α-ethynyl-nortestosterone with a series of diiodo alkanes, and found that 17α-(6'-iodohex-1'-ynyl)nortestosterone had an affinity of 22% relative to R5020, equal to the affinity of progesterone itself. Reaction with Thiopropyl-Sepharose 6B yielded hexynyl-nortestosterone-Sepharose beads with a ligand density of about 7 micromoles/ml beads. One-hundred μl of these beads adsorbed 71% of the PgR present in 1 ml ofcytosol from T47D cells. This adsorption was inhibited by 10 μM progesterone but not Cortisol, indicating the specificity of the binding. Comparisions with NADAC and Sterogel, other affinity beads used for PgR purification, show that the former takes up much less receptor, while the latter takes up and releases similar amounts of receptor but more extraneous protein, and is less stable. We therefore believe that hexynyl-nortestosterone-Sepharose, having a high density of a high affinity ligand, and having chemically and biochemically stable covalent bonds, should be a good reagent for affinity purification of PgR.     

Sex-specific modification of progesterone receptor expression by 17β-oestradiol in human cardiac tissues

Background

Although circulating levels of sexual hormones in elderly men and women are low and quite similar, the adaptation of the elderly heart to stress differs between the sexes. We have hypothesized that the effects of sexual hormones in the heart may differ in men and women. Here, we assessed whether 17β-oestradiol regulates gene expression in the human heart in a sex-dependent manner. We selected the progesterone receptor as a well studied 17β-oestradiol target that may be pathologically linked to cardiac remodelling.

Methods

In order to assess the ex vivo effects of 17β-oestradiol in intact human cardiac tissues, we developed a 24-h model for the culture of human atrial myocardium. We verified tissue viability after 24 h in culture with two standard assays to determine the degree of apoptosis and metabolic activity of cardiac tissues. Progesterone receptor mRNA and protein level were measured after 24-h treatment of tissues with 17β-oestradiol. Statistical analysis was performed by the Mann-Whitney U test and two-way ANOVA.

Results

We established a tissue culture model that allows for the study of viable human cardiac tissue over a 24-h period. After 24 h, cultured cardiac tissues revealed low apoptosis, retained their metabolic activity and, therefore, remained viable. Treatment with 17β-oestradiol led to an induction of the progesterone receptor mRNA level in female (P = 0.001) but not in male tissues. Similarly, there was an increase in the level of progesterone receptor protein in female tissues (P = 0.03), while a decreasing trend was observed in male tissues (P = 0.079) exposed to 17β-oestradiol.

Conclusions

Our novel finding may offer a molecular explanation for the sex-specific differences observed in cardiac remodelling. The culture model we established for human cardiac tissue will facilitate the study of cellular processes in health and disease and will be of use for pharmacological testing.     

The influence of β-cyclodextrin on the kinetics of progesterone transformation by Rhizopus nigricans

The process of progesterone 11α-hydroxylation by the pelleted growth form of the filamentous fungus Rhizopus nigricans has been described with a mathematical model, based on Michaelis-Menten enzyme kinetics and the rate of substrate dissolution. It was confirmed that the low water solubility of steroids is the limiting step of this process at high steroid concentrations. In order to overcome this problem, β-cyclodextrin, which is known to form inclusion complexes with these organic compounds, was added to the production medium. The phase solubility of the steroid-β-cyclodextrin system was investigated and the effect of β-cyclodextrin addition on progesterone biotransformation evaluated. Enhancement of steroid solubility was demonstrated and nearly two-fold increase in reaction rate was found in the presence of β-cyclodextrin.     

Antagonist action of progesterone at σ-receptors in the modulation of voltage-gated sodium channels

σ-Receptors are integral membrane proteins that have been implicated in a number of biological functions, many of which involve the modulation of ion channels. A wide range of synthetic ligands activate σ-receptors, but endogenous σ-receptor ligands have proven elusive. One endogenous ligand, dimethyltryptamine (DMT), has been shown to act as a σ-receptor agonist. Progesterone and other steroids bind σ-receptors, but the functional consequences of these interactions are unclear. Here we investigated progesterone binding to σ(1)- and σ(2)-receptors and evaluated its effect on σ-receptor-mediated modulation of voltage-gated Na(+) channels. Progesterone binds both σ-receptor subtypes in liver membranes with comparable affinities and blocks photolabeling of both subtypes in human embryonic kidney 293 cells that stably express the human cardiac Na(+) channel Na(v)1.5. Patch-clamp recording in this cell line tested Na(+) current modulation by the σ-receptor ligands ditolylguanidine, PB28, (+)SKF10047, and DMT. Progesterone inhibited the action of these ligands to varying degrees, and some of these actions were reduced by σ(1)-receptor knockdown with small interfering RNA. Progesterone inhibition of channel modulation by drugs was consistent with stronger antagonism of σ(2)-receptors. By contrast, progesterone inhibition of channel modulation by DMT was consistent with stronger antagonism of σ(1)-receptors. Progesterone binding to σ-receptors blocks σ-receptor-mediated modulation of a voltage-gated ion channel, and this novel membrane action of progesterone may be relevant to changes in brain and cardiovascular function during endocrine transitions.     

Regulation of maternal ACTH in ovine pregnancy: does progesterone play a role?

Pregnancy is characterized by increased plasma adrenocorticotropic hormone (ACTH) and cortisol. Studies suggest that progesterone acts as an antagonist at mineralocorticoid receptors. Therefore, we tested the hypothesis that chronic progesterone, produced by treatment of nonpregnant ewes or during pregnancy, will result in increased plasma ACTH relative to the plasma cortisol concentrations. We studied three groups of ewes: ovariectomized nonpregnant, nonpregnant treated with progesterone, and pregnant ewes. In two series of studies, ewes were adrenalectomized and replaced with 0.35 mg x kg(-1) x day(-1) or 0.5 mg x kg(-1) x day(-1) cortisol. In both studies, aldosterone was infused at 3 microg x kg(-1) x day(-1). In the first study, additional infusions of cortisol over 24 h were used to increase daily replacement doses to 0.5, 1, or 1.5 mg x kg(-1) x day(-1), and intact pregnant and nonpregnant ewes were studied with infusions of cortisol at 0, 0.5, and 1 mg x kg(-1) x day(-1). In adrenalectomized ewes chronically replaced to 0.35 mg x kg(-1) x day(-1) cortisol, plasma ACTH concentrations were decreased significantly in the nonpregnant progesterone-treated ewes compared with the ovariectomized nonpregnant ewes. With 0.5 mg x kg(-1) x day(-1) cortisol, plasma ACTH levels were greater in pregnant ewes than in nonpregnant ewes with or without progesterone. Overall plasma ACTH levels at 0.35 mg x kg(-1) x day(-1) were significantly related to the plasma protein concentration, suggesting that the ACTH levels in the hypocorticoid ewes are most closely related to plasma volume. Across all steroid doses, ACTH was positively related to plasma proteins and progesterone, and negatively related to cortisol. We conclude that increased progesterone does not alter the feedback relation of cortisol to ACTH, but may modulate ACTH indirectly through plasma volume.     

Interactions of progesterone with D-amino acid oxidase—different effects on apo- and holo-enzyme

A simple method using charcoal treatment was developed for the preparation of apo-D-amino acid oxidase from rat kidney homogenates. This apo-D-amino acid oxidase was used to study the effect of progesterone on the apo- and holo-enzyme. Progesterone inhibited the activity of D-amino acid oxidase, when the apo-enzyme, preincubated with saturating amounts of FAD was used; this effect varied with FAD concentration. Progesterone did not inhibit the activity when added to a mixture of non-preincubated apo-enzyme and FAD; this suggests that progesterone has different effects on apo- and holo D-amino acid oxidase. Preliminary report presented at the V International Congress of Hormonal Steroids, 29 Oct–4 Nov. 1978, New Delhi.J. Steroid Biochem. 9, 832, (abstract 94) 1978.     

Biotransformation of progesterone to 11-α-hydroxyprogesterone by different morphological forms ofRhizopus arrhizus

Summary Medium supplementation with carboxymethyl cellulose resulted in production ofRhizopus arrhizus mycelium with an increased specific capacity to biotransform progesterone to 11--hydroxyprogesterone. This increase may be attributed to the observed differences in morphology. Morphologies of the control and CMC-grown mycelia were clumped, pelleted and dispersed respectively. Carbopol-grown mycelium, which manifested a clumped, dispersed morphology, intermediate between that of the control and CMC-grown mycelia, had a lower progesterone transforming capacity.     

Vein Patterning 1-encoded progesterone 5β-reductase: Activity-guided improvement of catalytic efficiency

Progesterone 5β-reductases (P5βR; EC 1.3.99.6) encoded by Vein Patterning 1 (VEP1) genes are capable of reducing the CC double-bond of a variety of enones enantioselectively. Sequence and activity data of orthologous P5βRs were used to define a set of residues possibly responsible for the large differences in enzyme activity seen between rAtSt5βR and rDlP5βR, recombinant forms of P5βRs from Arabidopsis thaliana and Digitalis lanata, respectively. Tyrosine-156, asparagine-205 and serine-248 were identified as hot spots in the rDlP5βR responsible for its low catalytic efficiency. These positions were individually substituted for amino acids found in the strong rAtSt5βR in the corresponding sites. Kinetic constants were determined for rDlP5βR and its mutants as well as for rAtSt5βR using progesterone and 2-cyclohexen-1-one as substrates. Enzyme mutants in which asparagine-205 was substituted for methionine or alanine showed considerably lower km and higher K_cat/k_m values than the wild-type DlP5βR, approaching the catalytic efficiency of strong P5βRs. The introduced mutations not only lead to an improved capability to reduce progesterone but also to altered substrate preference. Our findings provided structural insights into the differences seen among the natural P5βRs with regard to their substrate preferences and catalytic efficiencies.     

Analysis of tissue progesterone residues by gas chromatography — Mass spectral and radioimmunological detection methods

Methods for the extraction, isolation and analysis of tissue concentrations of progesterone suitable for studying residue levels from livestock treated with this steroid for the control and synchronization of estrus are presented. The system employs biphasic partitioning for the extraction and silica gel chromatography for the isolation and demonstrates 80 to 90% recovery of ¹⁴C-labeled progesterone added as an internal standard. Residue analysis of fat, kidney, liver and muscle tissue samples from ovariectomized non-treated and progesterone treated ewes are compared employing a competitive inhibition radioimmunoassay system which appears to be less specific for progesterone than the gas chromatography-mass spectrometry method employing selective ion monitoring detection.     

Localization of the human progesterone receptor gene to chromosome 11q22–q23

Summary The human progesterone receptor gene was mapped by in situ hybridization using two cDNA probes corresponding to the 5′ and 3′ part of the coding sequence. This gene was localized to 11q22-q23.     

Treatment of growth hormone excess in dogs with the progesterone receptor antagonist aglépristone

Acromegaly or hypersomatotropism in dogs is almost always due to progestin-induced hypersecretion of GH originating from the mammary gland. The aim of this study was to investigate whether aglépristone, a progesterone receptor antagonist, can be used to treat this form of canine acromegaly. In five Beagle bitches hypersomatotropism was induced by administration of MPA for over 1 year. Subsequently, aglépristone was administered. Blood samples were collected before MPA administration, immediately before, during, and 3.5 and 5.5 weeks after the last administration of aglépristone for determination of the plasma concentrations of GH and IGF-I. In addition, blood samples for the determination of the 6-h plasma profile of GH were collected before MPA administration, before aglépristone administration, and 1 week after the last aglépristone treatment. MPA administration resulted in a significant increase of the mean plasma IGF-I concentration, whereas analysis of the pulsatile plasma profile demonstrated a trend (P=0.06) for a higher mean basal plasma GH concentration and a higher mean AUC(0) for GH. Treatment with aglépristone resulted in a significant decrease of the mean plasma GH and IGF-I concentrations. Analysis of the pulsatile plasma profile showed a trend (P=0.06) for a lower mean basal plasma GH concentration and a lower mean AUC(0) for GH 1 week after the last aglépristone treatment compared with these values before aglépristone administration. Three and a half and 5.5 weeks after the last aglépristone administration the mean plasma IGF-I concentration increased again. In conclusion, aglépristone can be used successfully to treat dogs with progestin-induced hypersomatotropism.     

Luteal progesterone and prostaglandin F2α production invitro during fluprostenol-induced luteolysis in the mare

Prostaglandin F₂α (PGF₂α) release $\text{in}$$\text{vitro}$ by luteal tissue from mares was quantified to determine if exogenous prostaglandin analog increased endogenous luteal PGF₂α production during induced luteolysis. On day 8 after ovulation, luteal tissue was collected by flank laparotomy and endometrium was collected by uterine biopsy. Mares were assigned to one of four treatments: (1) no intramuscular injection at 0-hr (n = 5), (2) 250 μg Fluprostenol (ICI 81008 PGF₂α analog) at 4-hr (n = 4), (3) 250 μg Fluprostenol at 12-hr (n = 5), or (4) 250 μg Fluprostenol at 28-hr (n = 5) prior to tissue collection at laparotomy. Blood was collected from a jugular vein at laparotomy. Luteal and endometrial tissues (100-mg minces) were incubated in duplicate in 5 ml of Krebs-Ringer bicarbonate buffer (pH 7.4) in an ice bath in an air atmosphere or at 37°C in an atmosphere of 95% O₂:5% CO₂. The incubation treatments consisted of: no treatment, indomethacin 1.3 × 10^?4M, 1 μg/ml of arachidonic acid, 10 μg/ml of Fluprostenol, and 100 μM dbc-AMP (Fluprostenol was not added to endometrial tissue incubations). The injection of Fluprostenol induced luteolysis in these mares as indicated by decreased plasma progesterone and luteal tissue progesterone production (P<0.01). Luteal PGF₂α production was only detectable in tissue from mares that had been injected with Fluprostenol; production reached a maximum by 12 hr post-injection and had returned to pre-treatment levels by 28 hr (P<0.01). Endometrial tissue produced PGF₂α, but this activity was not significantly affected by injection of mares with Fluprostenol. Increased production of PGF₂α by luteal tissue of mares during PGF₂α analog induced luteolysis was similar to that observed in the pig and ewe.     

Regeneration of the progesterone 11α-hydroxylase of Rhizopus nigricans by the use of periodate

Summary The cell-free progesterone 11-hydroxylase enzyme of Rhizopus nigricans can be directly regenerated by periodate oxidation. This permits action of the enzyme over a period of hours with an activity similar to that in the presence of an NADPH generating system.