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.     

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.     

Progesterone in the uterus. VIII. Uptake of corticosterone and incorporation of progesterone under concurrent injection of corticosterone into rat uterus

A study of the subcellular distribution of radioactivity in rat uterus after injection of labelled corticosterone showed that the radioactivity was observed in all fractions from 5 min. to 120 min. A maximum uptake was observed 10 min. after application of the labelled steroid. Competitive uptake of radioactive progesterone and unlabelled corticosterone was assayed 10 min. after injection of the hormone mixture. The ratio between radioactive progesterone and unlabelled corticosterone was 1 : 1 and 1 : 2 (moles:moles), respectively. Compared with control experiments with rats which had received radioactive progesterone alone, the results gave evidence that progesterone found in all subcellular fractions and in the total homogenate was not depressed by unlabelled corticosterone. However, unlabelled progesterone reduced the tritiated progesterone in uterine tissue. This observation demonstrates that the uptake of progesterone by rat uterus is specific.     

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. 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.     

Progesterone metabolism in human saliva in vitro.

Human salivary glands are known to be able to metabolize progesterone as well as other steroid hormones. The rate of progesterone metabolism in the salivary glands is so low that it is not thought to affect salivary progesterone concentrations. On the other hand it is usually recommended that saliva should be frozen quickly after the collection to prevent any kind of metabolism in saliva. When saliva is collected at home e.g. delayed freezing or partial thawing during to transport to laboratory may create circumstances where progesterone metabolism may occur. However, it is not known to which extent progesterone metabolism continues in saliva and whether this continued metabolism of progesterone affects salivary hormone levels. Paraffin-stimulated salivary samples were collected from female (N = 6) and male (N = 6) dental students and perimenopausal women (N = 8). The salivary samples were incubated with 14C-progesterone for 2 h at 37 degrees C in a shaking water bath. Metabolites were analyzed using thin-layer chromatography and autoradiography and quantified by liquid scintillation counting. Human saliva was found to be able to metabolize progesterone, but its metabolic activity was very low, 9.3 and 6.8 pmol/ml/h in young adults and perimenopausal women, respectively. Metabolic activity was higher in whole saliva than in the corresponding activities of the supernatant or sonicated fraction of the same saliva. The supernatant fraction, which was thought to be mainly representative of glandular saliva, was metabolically least active. The polar metabolites of progesterone predominated in all incubations. The metabolic activity of saliva is probably mainly due to its cellular content and the contribution of this activity to salivary progesterone concentrations is not significant.     

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.     

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.     

Arachidonic acid metabolism in isolated rat aorta. Dependence of prostacyclin biosynthesis on extracellular potassium concentration

Slices of rat aorta were incubated in Krebs-Ringer bicarbonate buffer for measurements of immunoreactive 6-ketoprostaglandin F1 alpha, thromboxane (TX) B2, prostaglandin (PG)E2, and PGF2 alpha, and in Tris buffer (pH 9.3) for determination of prostacyclin (PGI2)-like activity. No significant generation of TXB2, PGE2, or PGF2 alpha by rat aortic tissue could be detected. The time-dependent release of 6-keto-PGF1 alpha Krebs-Ringer bicarbonate buffer closely correlated with PGI2 generation in alkaline Tris buffer. During a 30-min incubation period, 6-keto-PGF1 alpha, release was 79.8 +/- 3.3 pmol/mg at a buffer potassium concentration of 3.9 mmol/liter and significantly increased by 23% to 98.3 +/- 8.5 pmol/mg (P less than 0.025) in the absence of potassium in the incubation medium. A smaller decrease in buffer potassium concentration to 2.1 mmol/liter and an increase to 8.8 mmol/liter did not significantly alter aortic 6-keto-PGF1 alpha release. Changes in the incubation buffer sodium concentration from 144 mmol/liter to either 138 or 150 mmol/liter at a constant potassium concentration of 3.9 mmol/liter did not alter the recovery of 6-keto-PGF1 alpha. Our results support the concept that PGI2 is the predominant product of arachidonic acid metabolism in rat aorta. They further show that PGI2 can be recovered quantitatively as 6-keto-PGF1 alpha under the present in vitro conditions. In addition, this in vitro study points to the potassium ion as a modulator of vascular PGI2 synthesis with a stimulation at low potassium concentrations.     

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.     

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.