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.     

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.     

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.     

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.     

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 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.     

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.     

Estradiol-induced progesterone receptor synthesis in normal and diabetic ovariectomized rat uterus

Estrogen stimulation of progesterone-receptor (Prog R.) synthesis is an important parameter of the sex hormones activity at the uterine level. Experimental diabetes in the rat has been shown to perturb protein synthesis in some tissues and to reduce, under certain circumstances, estrogen and androgen activity on their respective target tissues. The present work tended to evaluate the effect of streptozotocin diabetes on estradiol (E2) stimulation of Prog. R and on Prog R. kinetics in the rat uterus. Two groups of diabetic rats were primed for three consecutive days with 5 microg. E2 s.c. (EP). One group received an acute i.p. injection of progesterone (P), 1 h before sacrifice (Inj), the other group did not (n Inj). Two other groups, not primed with E2 (nEP) were similarly injected or not with P. Four groups of non diabetic animals served as controls. Estrogen priming induced a 20-25% increase in DNA content, both in controls and in diabetics. Protein content was also increased to almost the same extent in diabetics and controls; protein concentration remained however slightly lower in cytosol of EP diabetics as compared to controls. Prog R. increased about 7-fold in cytosol and 4-5-fold in nuclei of EP control and diabetic groups. Cytosol to nuclei ratios of Prog R. decreased similarly in Inj. EP diabetics and controls, compared to the corresponding n Inj. groups. It is concluded that estrogen priming stimulated Prog R., total protein and DNA synthesis to the same extent in diabetic as in control rats Prog R. kinetics was unaltered in diabetics. This finding might be relevant to situations like early pregnancy, when Prog R. levels change rapidly and specifically in relation with the time and the site of implantation.     

Induction of catechol-O-methyltransferase in the luminal epithelium of rat uterus by progesterone

We performed light microscopic immunocytochemical observations of the localization of catechol-O-methyltransferase (COMT) in rat uterus, using a rabbit anti-rat serum specific for the soluble form of rat liver COMT, biotinylated goat anti-rabbit immunoglobulin, and peroxidase conjugated with streptavidin. In the non-pregnant rat, COMT was minimal but detectable in the uterine luminal and glandular epithelium, with greater amounts present in uteri from rats in estrus than those in diestrus. In early pregnancy a robust accumulation of COMT was observed in the luminal epithelium. To more precisely define both the timing and the factors contributing to the appearance of COMT, uteri were examined on Days 1-5 in pregnant and pseudopregnant rats. Accumulation of COMT in the luminal epithelium was observed by Day 3 in uteri from pregnant and pseudopregnant rats and by Day 4 in lactating post-partum rats. No immunostaining of COMT was observed in uteri from non-lactating post-partum rats. Ovariectomy on Day 0 or 1 but not on Day 2 of pregnancy prevented the appearance of COMT on Day 4. Progesterone treatment immediately after ovariectomy on Day 0 or 1 of pregnancy restored the COMT.     

Prostaglandin production by isolated cells of rat uterus incubated with or without progesterone

The production of prostaglandins F2 alpha and 6-keto F1 alpha in vitro by the luminal epithelial and the residual (mainly stomal) cells isolated from the uteri of proestrous rats have been measured. The basic procedure involved culturing the cells overnight, washing, and then incubating for 2 h when the amounts of prostaglandins released into the medium were determined by radioimmunoassay. No significant change occurred when the epithelial cells were incubated in the combined presence of arachidonic acid and progesterone for the short-term culture compared to being incubated with arachidonic acid alone. However, when progesterone was incorporated into the medium for the overnight culture, a significant increase occurred in 6-keto PGF1 alpha production by the epithelial cells, without any change in PGF2 alpha, compared to when the cells were incubated overnight in the absence of progesterone. It is suggested that overnight treatment of the epithelial cells with progesterone increases the amount or activity of prostacyclin synthetase, responsible for converting PGH2 endoperoxide to PGI2. Very low levels of prostaglandin production were found for the residual cells.