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.     

Estrogen and progesterone receptors in mammary tumors induced in rats by simultaneous administration of 17ß-estradiol and progesterone

Mammary tumors were promoted in male rats of the Wistar WAG strain by continuous and simultaneous administration of 17ß-estradiol and progesterone. Tumor induction and growth were dependent on estradiol and on progesterone. Their histological features were comparable with those of human breast cancers. Hormone receptors were present in tumor cells. Estradiol receptor was found in 95% of them, at a higher level in nuclei than in cytosol. Progesterone receptor was present in 75% of tumors. In all cases, the level of androgen receptor was low.     

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.     
   
   

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.     

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.     

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.     

Serum progesterone concentrations,follicular development and time of ovulation using a new progesterone releasing device (DICO®) in sheep

Four experiments were conducted to evaluate the effectiveness of a new controlled drug releasing device containing 0.3 g progesterone (DICO^®) on ovarian control in sheep. In experiment 1, serum progesterone concentrations induced by a 14 days treatment of DICO^® (n = 9) and CIDR-G^® (n = 9) were compared in ovariectomized ewes. Both devices induced similar responses and no differences were recorded. In experiment 2, the onset of oestrus and the time of ovulation obtained after 14 days treatment with DICO^® (n = 8) and CIDR-G^® (n = 7) were compared in cyclic ewes. Both devices induced oestrus and ovulation in all of the ewes. The onset of oestrus (34.5 ± 2.8 and 30.0 ± 7.7 h), the time of ovulation (60.0 ± 9.1 and 54.9 ± 6.4 h), the ovulation rate (1.3 ± 0.5 and 1.4 ± 0.5), the follicular diameter at ovulation (7.0 ± 0.8 and 7.3 ± 1.1 mm), and the lifespan of the ovulatory follicles (8.6 ± 2.2 and 10.0 ± 2.9 days) were similar for the DICO^® and CIDR-G^® devices, respectively. In Experiment 3, the re-utilization of DICO^® devices inserted for 6 days (i.e. short-term protocol) was evaluated in ovariectomized ewes. The females received a re-used (previously used for 6 days; n = 11) or a new DICO^® (n = 11) for a period of 6 days. The re-used DICO^® devices induced a lower serum progesterone concentration than the new devices (P < 0.05). However, the re-used DICO^® device maintained serum progesterone concentrations above 7.1 nmol/L (i.e. >2 ng/ml) throughout treatment. In Experiment 4, the administration of eCG treatment at DICO^® withdrawal was evaluated in cyclic ewes. The short-term protocol using DICO^® devices for 6 days was applied with (n = 8) or without (n = 7) 300 IU eCG at the time of device withdrawal. The administration of eCG advanced ovarian follicular development, synchronizing the onset of oestrus at 36 h and the time of ovulation at 60 h from device withdrawal. In conclusion, data from these experiments show the use of DICO^® or CIDR-G^® devices containing 0.3 g of progesterone to have a similar efficiency in controlling serum progesterone concentrations, follicular development and the time of ovulation in sheep. The re-use of the devices, associated with the short-term protocol for 6 days is possible, although further studies on induced fertility rates are warranted.     

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.     

Initiation of human parturition—VII partial characterization of progesterone metabolizing enzymes of human amnion and chorion laeve

The kinetics of the metabolism of progesterone by the reducing enzymes present in the human fetal membranes was evaluated utilizing tissue samples obtained from early and late pregnancies. The specific activities of 5α-reductase, 3β- and 20α-hydroxysteroid oxidoreductase were greater in amnion and chorion laeve tissue obtained from a 16 weeks pregnancy than in those obtained from a 36 1/2 week and from a term pregnancy. Amnion and chorion laeve tissue 20α-hydroxysteroid oxidoreductase enzymes utilized either NADH or NADPH as the source of reducing equivalents (K_m = 0.2–0.3 mM) while amnion tissue 5α-reductase activity required NADPH as the obligatory cofactor (K_m = 0.1mM). The apparent pH optima for the 20α-hydroxysteroid oxidoreductase present in amnion and chorion laeve tissues were similar, while the apparent pH optima for the 5α-reductase enzyme was slightly higher in chorion laeve than in amnion tissue. In 15 min incubations, the apparent maximal activity of amnion 5α-reductase was found at a temperature of 49°C. whereas chorion laeve tissue 3β- and 20α-hydroxysteroid oxidoreductase had maximal activity at 37°C. From progesterone saturation kinetics an apparent K_m of 0.20–0.25 mM for amnion and chorion laeve 20α-hydroxysteroid oxidoreductase, and an apparent K_m of 0.4–1.2 μM for amnion and chorion laeve tissue 5α-reductase activity was computed. Amnion 5α-reductase activity appeared to be particulate in nature.     

Effect of progesterone pretreatment on the uptake of estradiol-17β by the uterine epithelium of the rat

Summary Progesterone pretreatment of ovariectomised rats resulted in a moderate (44%) lowering of the level of nuclear estradiol receptors found in the uterine epithelium 2 h after a single injection of this estrogen.     

Do small and large luteal cells of the sheep interact in the production of progesterone?

Corpora lutea from cyclic ewes were dissociated by collagenase and trypsin/EGTA treatments, and enriched fractions of small and large luteal cells were prepared on gradients of Ficoll. These fractions were incubated separately or remixed before incubation. Colchicine, cytochalasin B and the calcium channel-blocker verapamil significantly reduced progesterone production by both small and large luteal cell fractions, while isoprenaline stimulated an increase in progesterone production by large luteal cell fractions only. When fractions of small and large luteal cells were remixed, no more and no less progesterone was produced than would have been predicted from equivalent fractions incubated separately. There was therefore no evidence of synergism between small and large luteal cells in the production of progesterone. Prostaglandin F-2 alpha, which can inhibit LH-stimulated progesterone production by ovine luteal tissue in vitro, had no effect on LH-stimulated progesterone production by small luteal cell fractions, but significantly inhibited that by enriched fractions of large luteal cells. Since large luteal cell fractions were contaminated with small luteal cells, which are probably responsible for the progesterone-secretory response of these fractions to LH, it was concluded that the inhibition of LH-stimulated progesterone production by small luteal cells is dependent on the presence of large luteal cells. Oxytocin added to large and small luteal cell fractions did not affect progesterone production by either fraction. It was therefore concluded that the inhibitory action of PGF-2 alpha on LH-stimulated progesterone production may require the interaction of large and small luteal cells, but that oxytocin is not likely to be an intermediary in this interaction.     

Reciprocal interactions of progesterone and 17α-OH-Progesterone as exogenous substrates of rat adrenal 21-Hydroxylase

Due to the small concentration and activity of 17α-hydroxylase present in the rat adrenal, the main corticoids secreated in the rat are DOC, B_k, A_k, 18-OH-DOC and aldosterone, formed directly from progesterone (I). Because of the limited amounts of 17α-OH-progesterone (II) available, the biosynthesis of S_R, F_k and E_k is restricted. Since 21-OH steroid hydroxylase (21-OH-ase) uses both I and II in corticoid biosynthesis in other species, it was considered of interest to study the comparative interactions which could exist between these two precursors and the rat adrenal 21-OH-ase, determining enzymatic constants for I and II (usual and unusual substrates, respectively). Homogenized adrenals from normal rats were incubated with various combinations of concentrations of I-7-³H and/or II-¹⁴C, acting as substrates and/or inhibitors of 21-OH-ase. The results showed that 21-OH-ase uses II almost as efficiently as I. The K_m values were about the same for both I and II (13.9 and 14.2 x 10⁻⁶ M/L), respectively, however, the V_max values were 54.6 and 26.0 x 10⁻⁷ M/L/min for I and II, respectively. The amounts of I required to saturate the 21-OH-ase was double that of II. Further kinetic studies showed that both I and II inhibit the 21-hydroxylation of the other in a reciprocal fashion. While II inhibits the 21-hydroxylation of I by competitive inhibition, I inhibits the 21-hydroxylation of II through a mixed type of inhibition. The results suggest that, rather than the existence of two different specific enzymes (one for I and another for II) as it has been postulated by others, it seems that we are dealing with a 21-hydroxylation system with two active sites. One site used only I and the other site uses I and/or II indistinctively.     

Effects of oestradiol-17β and progesterone on total and nuclear-protein synthesis in epithelial and stromal tissues of the mouse uterus, and of progesterone on the ability of these tissues to bind oestradiol-17β

1. A method is described for separating uterine epithelium that is 80% pure and connective-tissue stroma that is 60% pure. This was used to study the effects of steroid hormones on total and nuclear-protein synthesis in these tissues. 2. Oestradiol-17beta given alone produces mitoses in the epithelium but not in the stroma. It stimulated incorporation in vitro of [(14)C]lysine into total protein, histones and acidic nuclear proteins to a greater extent in epithelium than stroma. Incorporation into acidic nuclear proteins was most markedly stimulated, reaching four to six times the normal value 4h after treatment, and then declining rapidly. This peak was only seen in epithelial preparations. 3. After pretreatment with progesterone, oestradiol-17beta has the reverse effect, producing mitoses only in stroma. Progesterone alone had no effect on the amounts or rates of incorporation of [(14)C]lysine into stromal nuclear proteins, but changes after oestradiol-17beta treatment were similar to those seen in epithelium with oestradiol-17beta alone. In the epithelium, progesterone alone depressed incorporation into histones and acidic nuclear proteins, but did not abolish the subsequent response to oestradiol-17beta. With this treatment there was a rapid, large and transient increase in incorporation into epithelial total protein not seen with oestradiol-17beta alone. 4. Progesterone had no qualitative effect on the distribution of specific oestrogen-binding proteins, as judged by sucrose-density-gradient centrifugation. However, progesterone treatment increased the uptake in vivo of [6,7-(3)H]oestradiol-17beta by stroma, and it is possible that this is important although the differences were not apparent after labelling in vitro.     

Effects of gonadotropin-exposed medium with high concentrations of progesterone and estradiol-17β on in vitro maturation of canine oocytes

During the process of maturation in the oviduct, canine oocytes in the germinal vesicle stage are exposed to decreasing levels of estradiol-17β and increasing levels of progesterone. However, hormone concentrations in the microenvironments in which they act are higher than serum concentrations. Therefore, the aim of the present study was to compare the meiotic competence of canine oocytes harvested from anestrous bitches in culture medium containing high concentrations (20 μg ml⁻¹) of estradiol-17β and/or progesterone in association to gonadotropins (luteinizing hormone and follicle-stimulating hormone) using three different maturation periods (48, 72, and 96 h). Oocytes were cultured in tissue culture medium (TCM-199) and arranged in four experimental groups: group control, group E2 (estradiol-17β), group P4 (progesterone), and group E2 + P4. Regardless of the maturation period, groups P4 and E2 + P4 presented statistically higher rate of germinal vesicle breakdown oocytes compared to the group control and group E2. There were no significant differences among groups on germinal vesicle, metaphase I, metaphase II, and degenerated or unidentifiable oocytes rates. The mean percentage of metaphase II oocytes was higher at 96 h when compared to 72 h. Results of the present research indicate no influence of estradiol-17β supplementation, unless in association with progesterone. There is an evidence of the positive effect of progesterone on germinal vesicle breakdown. Results also showed that extended periods of in vitro maturation affect positively maturation rates to metaphase II of low competent oocytes harvested from anestrous bitches, independent of the maturation media. In conclusion, high concentrations of steroids, especially progesterone, have positive effect on in vitro oocyte maturation when the oocytes are derived from the anestrous status.     

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.     

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.     

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.     

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.