Effect of incubation with NADPH generator on progesterone bound to nuclei of the rat uterus.

The metabolism of progesterone bound to uterine nuclear progesterone receptors was accelerated in vitro by incubation at 25°C for 30min with NADPH generator. The characteristics of progesterone metabolites and receptors were examined by 3H-R5020 exchange assay. Progesterone bound nuclei were prepared from uteri of estradiol-primed progesteronetreated ovariectomized rats. The specific nuclear R5020 binding sites examined by exchange assay were identified as specific progesterone receptors by the binding specificity of R5020 to progesterone receptors, their high affinity for progesterone, and the increase in the number of specific binding sites in response to a progesterone injection. Major metabolites of progesterone specifically bound to nuclear receptors by incubation with the cofactor were 5α-dihydroprogesterone (5α-DHP) and 3α-hydroxy-5α-pregnan20-one. The relative binding affinity of the progesterone receptor for 5α-DHP at 0°C was 27.5% of progesterone, but 3α-hydroxy-5α-pregnan-20-one had no binding affinity for the receptor. Incubation of the nuclei in the presence of NADPH generator decreased the number of R5020 binding sites from 143 fmol to 25 fmol per 100μg DNA, and increased the dissociation constant of the receptor with progesterone from 2.56nM to 7.78nM. Results suggest that after gene activation, the specific binding sites of progesterone to nuclear receptors may be denatured in the presence of NADPH, causing both progesterone and receptors to dissociate from the receptors and chromatins, respectively, and that the dissociated progesterone may be converted to 5α-reduced pregnanes which have lower affinity for the receptor. A steroid hormone taken up by the target cells is bound to the specific cytoplasmic receptor. The cytoplasmic receptor-hormone complex is transported to the nucleus, bound to specific active sites on the chromatin, and activates genetic transcription (O'Malley and Means, 1974). These processes are generally accepted as the mechanism of steroid hormone action. However, the mechanism for the removal of the hormone from the nucleus is still unknown. In the case of testosterone, the steroid is transformed to the active androgen, 5α-dihydrotestosterone, which can be selectively retained by rat prostate cell nuclei (Liao and Fang, 1969; Wilson and Gloyna, 1970). But the progesterone-receptor complex in the chick oviduct enters the nucleus without being metabolized (Schrader et al., 1972). It has been revealed that the rat uterus is able to convert progesterone to 5α-dihydroprogesterone (5α-DHP), and that the nuclear fraction accounts for a large portion of the 5α-reductase activity (Armstrong, 1971; Saffran et al., 1974). Although the significance of the reduction of progesterone to 5α-DHP in the nucleus is unknown, it may be associated with the behavior of the progesterone after gene activation. Saffran et al. (1978) mentioned the Received July 2, 1981 32 MORIOKA et al. Endocrinol. Japon. February 1982 possibility that the reduction of progesterone to 5α-DHP in the nucleus may decrease the steroid-receptor binding affinity and release the progesterone receptor. Clark and Peck (1979) hypothesized that the metabolism of progesterone to 5α-DHP may play an important regulatory role in uterine hormone action by reducing the effectiveness of progesterone. This investigation examines the characteristics of the reduction products of progesterone bound to nuclear receptors in the rat uterus which were incubated with an NADPH generating system to accelerate the reduction of progesterone to 5α-DHP, and discusses the possible mechanisms for the removal of progesterone from the nucleus after gene activation. Materials and Methods Chemicals 3H-R5020 (17α , 21-dimethyl-19-nor-pregna-4, 9-diene-3, 20-dione; specific activity: 87.0Ci/mmol) and 1, 2-3H-progesterone (specific activity: 55.7Ci/mmol) were purchased from New England Nuclear Corp. (Boston, MA). The purity of the labeled steroids was checked by thin layer chromatography on silica gel plates, using benzene-ethyl acetate (7 : 3), and a hexaneethyl acetate system (7 : 3). Unlabeled steroids were purchased from Sigma chemical Co., (St. Louis, MO). Steroids were dissolved in an absolute ethanol solution. The buffer used in this study was a 10mM Tris-HCl (pH 7.4) containing 1.5mM EDTA, 1mM β-mercaptoethanol and 10% (v/v) glycerol (TEMG buffer). The NADPH generating system consisted of 0.98mM NADP, 9.52mM glucose-6-phsophate and 2.9U glucose6-phosphate dehydrogenase per ml. Preparation of crude uterine nuclear suspension Female rats of the Sprague-Dawley strain bred in our laboratory were used. They were maintained on a 14h light schedule (0500 to 1900h) at 24±2°C and fed with commercial rat chow and water ad libitum. All rats were ovariectomized at 50-to 60-days of age, and 2weeks later injected sc daily with 5μg 17β-estradiol for 3 consecutive days. The rats were injected with 2mg progesterone 24h after the last estradiol injection and killed 1h later by decapitation. The uteri were immediately removed, dissected longitudinally, rinsed in ice-cold buffer, blotted on a towel and weighed on a torsion balance. The uterine strips were finely minced and homogenized in 5 volume (w/v) of buffer in a glass homogenizer with a Teflon-pestle. The homogenate was filtered through 4 layers of gauze, and the filtrates were centrifuged at 800×g for 10min. The crude nuclear pellet was washed twice with 3ml of TEMG buffer each time, and each time centrifuged for 10min (800g). The crude nuclear suspension was prepared by suspending the washed pellet in buffer at a concentration equivalent to 200mg uterine wet weight per ml. All processes were carried out in a cold room maintained at 0-4°C. The crude nuclear suspension was stored at -80°C until use. Nuclear binding sites of the preparations stored in this way were stable for at least 3weeks; 94±0.9% (M±SEM) of the initial specific binding sites have remained after the storage