Progesterone in the uterus. VII. Uptake of cortisol and incorporation of progesterone under simultaneous application of cortisol into rat uterus

After injection of radioactively labelled Cortisol, the distribution of the radioactivity in the subcellular fractions of the rat uterus (nuclei, mitochondria, microsomes and 105 000 × g supernatant) was studied. In all fractions, radioactivity was observed and maxima were found 10, 20 and 50 min. after injection of the labelled hormone. Radioactivity was measured in all subcellular fractions even 180 min. after application of labelled cortisol. Additionally, radiolabelled progesterone and unlabelled cortisol in the ratio 1:1 or 1:2 (moles:moles) were injected into the animals. Studying the uptake of labelled progesterone in the subcellular fractions of the uterine tissue, revealed that no competition of unlabelled cortisol could be observed 10, 20 and 50 min. after application of the hormone mixture, compared with the control experiments. The results of this study give evidence that the progesterone uptake into rat uterus is specific and cannot be influenced by unlabelled cortisol.     

Progesterone in the uterus. X. Dependence of the in vitro progesterone metabolism on progesterone binding in rat uterus

The effect of estrogen pretreatment was stud-ed on the in vitro metabolism and binding of progesterone in uteri of ovariectomized rats in order to prove the dependence of the metabolism of progesterone on its binding. For this purpose, the extent of progesterone binding was varied in uterine tissue by different estrogen treatment of the rats and compared with the metabolism under the same conditions. The protein content determined in 100 mg tissue was used as parameter indicating the success of the pretreatment. Estrogen exposure of the rats for 30 or 45 hrs. caused a rise of protein amount in uterine tissue which was accompanied by an increase of binding sites of progesterone binding components. The binding sites were determined by charcoal adsorption technique and SCATCHARD-analysis. Under nearly the same success of estrogen pretreatment, the increase of the portein amount and with it the rise of binding sites reduced the amount of progesterone metabolites in uterine tissue. The metabolites were determined by quantitative TLC-analysis of the recovered compounds from uterine segments after incubation with radioactive progesterone. Additionally, an enlarged metabolic rate could be observed after saturation of binding components. It is concluded from the results of these experiments that progesterone binding components are factors limiting the enzymatic conversion of progesterone in rat uterus.     

Progesterone in the uterus. V. Correlation of the in vitro progesterone metabolism with the progesterone binding in rat uterus.

Incubations of rat uterine segments with varying 3H-progesterone concentrations were performed to study the hormone uptake by the tissue. The radioactivity of the uterus and the nutrient medium were plotted in form of a SCATCHARD plot. Additionally, the binding capacity of the uterine cytosol was measured. In both systems, the hormone was found to be associated with two components which differ from each other in their association constants. The progesterone metabolism occuring at a hormone concentration of 10(-6)M and more in the incubation medium is discussed with respect to the affinity and the capacity of the hormone binding components.     

Progesterone in the uterus. IV. Dependence of the in vitro progesterone metabolism in the rat uterus on the progesterone concentration.

After incubation of uterine segments of normal rats with various 3H-progesterone concentrations in nutrient medium, different patterns of radioactive steroids were obtained in uterine tissue. Using hormone concentrations of less than 5 X 10(-7)M progesterone metabolites could not be detected in the tissue. A series of metabolites appeared with progesterone concentrations of 10(-6)M and higher. Six radiometabolites were identified and two were characterized.     

Effect of estradiol and progesterone on the rate of incorporation of radiolabeled arachidonate into phospholipids and triglycerides of the rat uterus

To obtain information on the effect of estradiol (E) and progesterone (P) on the overall rate of the acylation and deacylation reactions with [3H]arachidonate ([3H]AA) in the rat uterus, we investigated the effect of chronic treatment of ovariectomized rats with 2-200 micrograms/day E-dipropionate (EPP) and with 2 mg P alone or with the combination of these steroids on the extent and the time course of the in vitro incorporation of [3H]AA into various phospholipids (PLs) and the triacylglycerol fraction (TG). The results demonstrate that physiologic doses of E leads to a rapid equilibrium of the deacylation-acylation cycle only in the case of phosphatidylinositol (PI) whereas at high (200 micrograms/day) dose level it exerts the same effect on phosphatidylcholine (PC), phosphatidylethanolamine (PE) as well as on TG. P alone has no remarkable effects in the ovariectomized rat, but it decreases markedly the incorporation of [3H]AA into PI and TG in intact animals. Furthermore, P decreases the incorporation of [3H]AA into PI in the ovariectomized rat treated with 2 micrograms/day EPP as well as attenuating the enhanced labeling of PC, PE and PI caused by higher doses. Time-course studies provide evidence that all of these effects of progesterone can be accounted for by its ability to decrease the rate of deacylation and, therefore, to prolong the time needed to attain equilibrium in the rates of acylation and deacylation reactions of the various lipids with arachidonate. These data offer a novel outlook on the regulatory role of progesterone and estrogens in uterine function.     

Progesterone binding in the immature mouse and rat uterus

The use of a highly active progestin, 17, 21-dimethyl-19-nor-4, 9-pregnadiene-3, 20-dione (R 5020), as a tag has established that progesterone binds to a specific “7–8S” uterus cytosol component in both the immature mouse and rat.     

Metabolism of progesterone in rat uterus

The in vitro metabolism of progesterone was studied in uteri of untreated and estrogen stimulated immature rats. In intact uteri the rate of metabolism varied with the hormonal status of the animal in a concentration dependent manner. At a low (3 × 10^?9M) progesterone concentration the rate of ring A reduction was decreased in estrogen stimulated uteri. At a high progesterone concentration (3 × 10^?6M) the rate of ring A reduction was increased after estrogen treatment. The rate of reduction of the C20 ketone was increased after estrogen treatment at all concentrations of incubated progesterone. In dilute homogenates of uterus, estrogen stimulation always increased the rate of progesterone metabolism.Estrogen stimulation results in increased concentration of progesterone receptor in the uterus. It is proposed that increased activity of ring A reductases also occurs. The relative influence of these two factors on the metabolism of progesterone is dependent on the progesterone concentration in the incubation medium.     

Binding of progesterone by rat uterus in vitro

Circular dichroism (CD) spectra have been determined for chromatin fractions obtained by ECTHAM-cellulose chromatography. The molecular ellipticity at the positive long wavelength maximum is about 3000 deg cm²/dmol for early-eluted chromatin fractions, thought to be relatively repressed $\text{in vivo}$, and 5000–6000 deg cm²/dmol for late-eluted chromatin fractions, those thought to be preferentially transcribable $\text{in vivo}$. CD bands in the peptide bond spectral region also differ for the two chromatin fractions, early-eluted chromatin having a more helical conformation for proteins. In addition to previously known differences in protein content, the biological activity of a native chromatin fraction can now be correlated with the conformation of its DNA.     

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.     

Progesterone binding in rabbit oviduct and uterus.

Progesterone binding of high affinity with a dissociation constant of 10(-9) M was identified in cytosol of rabbit oviduct and uterus. Macromolecules with sedimentation coefficients of 7-8 S and 4-5 S were present. Progesterone receptor concentration was two to fivefold lower in the oviduct when compared with the uterus. The receptor concentration declined steadily from 3 hr until 144 hr after mating in the uterus; however, the decline in oviductal receptor was not significant until the sixth day of pregnancy. Progesterone receptor concentration in rabbit oviduct and uterus in estrus and early pregnancy was greater than estradiol receptor levels.     

Progesterone in the uterus. VI. On the question of the in vitro progesterone metabolism in human endometrium and myometrium

Human endometrial and myometrial tissue pieces were incubated with radioactively labeled progesterone in nutrient medium for 20 min., 1 hr and 2 hrs. The only compound extracted from the tissue pieces and the nutrient fluids was identified to be progesterone by TLC, chemical reactions and crystallization experiments. Radiometabolites could not be detected in the tissue pieces and in the nutrient fluids under the experimental conditions applied ( 10^?7 M 1,2-³H-progesterone in the incubation medium). This result is comparable with recent findings on the in vitro progesterone metabolism by rat uterine tissue.     

Tissue- and hormone- dependent progesterone receptor distribution in the rat uterus

Background  

Estradiol (E2) and progesterone (P) are well known regulators of progesterone receptor (PR) expression in the rat uterus. However, it is not known which receptor subtypes are involved. Little knowledge exist about possible differences in PR regulation through ERalpha or ERbeta, and whether the PR subtypes are differently regulated depending on ER type bound. Thus, in the present study PR immunostaining has been examined in uteri of ovariectomized (ovx) rats after different treatments of estrogen and P, in comparison with that in immature, cycling, and pregnant animals.     

Interaction of antiprogestins with progesterone receptors in rat uterus

Cytosolic and nuclear progesterone receptors (PRc and PRn) under antiprogestin treatment were measured in rat deciduoma and compared with values for contralateral (nondeciduomatous) rat uterine tissue. Uterine PRc and PRn of the progesterone treated group were 101 +/- 8.7 and 4770 +/- 590 fmol/mg DNA respectively. After treatment with antiprogestins STS-557, 5 alpha-DNE, (5 alpha-dihydronorethisterone), 5 alpha-DNG (5 alpha-dihydronorgestrel), RU-22092 and RU-16556, PRc in the nondeciduomatous control horn ranged from 127 to 377 fmol/mg DNA and PRn from 2785 to 17925 fmol/mg DNA. In the decidual tissue, PRc decreased significantly (4.6 +/- 0.8 fmol/mg DNA) on 5 alpha-DNG treatment as compared with the progesterone alone treatment group (147 +/- 3.8). PRn in decidual tissue also decreased maximally on 5 alpha-DNG treatment. These results suggest that the interaction of antiprogestins may not be identical in control uterine tissue and in deciduoma.     

Metabolism of progesterone in subcellular fractions of rat uterus

The metabolism of progesterone and 5α-pregnane-3,20-dione was studied in subcellular fractions of uterus from untreated and estradiol-17β treated immature rats. The reduction of progesterone to 5α-pregnane-3, 20-dione took place in all the particulate fractions of the uterus. The nuclear 5α-reductase accounted for the greatest fraction of enzymatic activity and was stimulated by estradiol treatment in vivo. The 5α-reductase activity in the mitochondrial and microsomal fractions was not increased after estradiol treatment. The reduction of 5α-pregnane-3,20-dione to 3α-hydroxy-5α-pregnan-20-one occurred mainly in the soluble fraction and was only slightly stimulated by estradiol. It proceeded much more rapidly than the reduction of progesterone to pregnanedione. Progesterone was also reduced to 20α-hydroxy-4-pregnen-3-one by a soluble enzyme whose activity was increased after estradiol-17β treatment.     

Uptake of corticosterone into isolated rat liver cells: Possible involvement of Na/K-ATPase

Isolated rat hepatocytes posses a saturable glucocorticoid uptake system with high affinity (K_d value = 2.8 ± 0.7 × 10⁻⁸ M; 318,000 ± 80,000 binding sites per cell; 317 fmol/mg protein). The initial rates of uptake decrease by about 30–40% if the cells are incubated simultaneously with [³H]corticosterone and either SH-reagents (N-ethylmaleimide and p-chloromercuriphenylsulphonate, 1 mM), metabolic inhibitors (2,4-dinitrophenol, 1 mM; and antimycin, 0.1 mM) or the Na⁺/K⁺-ATPase-inhibitors, ouabain and quercetine. These Na⁺/K⁺-ATPase-blockers exert half-maximal inhibition at 3 × 10⁻⁷ and 3 × 10⁻⁶ M, respectively. A slight increase in K⁺ concentration and a corresponding decrease in Na⁺ in the medium leads to a significant reduction in the initial uptake rate. The uptake system from the rat hepatocytes shows a clear steroid specificity, being different from the intracellular receptor. Corticosterone and progesterone are the strongest competitors, cortisol, 5- and 5β-dihydrocorticosterone, 11-deoxycorticosterone, cortisone and testosterone have an intermediate effect and only weak competition is exerted by dexamethasone and by the mineralocorticoid, aldosterone. Estradiol and estrone sulphate as well as the synthetic glucocorticoid triamcinolone acetonide are unable to inhibit initial corticosterone uptake.