Judith Saffran

Metabolism of progesterone in rat uterus

Abstract 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 −9 M) progesterone concentration the rate of ring A reduction was decreased in estrogen stimulated uteri. At a high progesterone concentration (3 × 10 −6 M) 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.

The effect of progesterone in vivo on the subcellular distribution and properties of progesterone receptors in guinea pig uterus

Abstract The distribution of progesterone receptors between cytosol and nuclear fractions of guinea-pig uterus was determined after the injection of 1 mg/kg and 10 mg/kg progesterone. After the lower dose, the concentration of cytoplasmic receptors decreased to approx. 2 3 of the preinjection level within 30–60 min and then gradually increased to almost the control value by 24 h. The nuclear receptors increased by an equivalent amount within 30–60 min and were retained at a slightly lower concentration for 4 h. Nuclear receptors decreased to the control level within 8 h and were not changed further. After 10 mg/kg, the cytoplasmic receptors decreased to approx. 1 2 the control value within 30 min and thereafter continued to decrease gradually. The nuclear receptors increased correspondingly to a maximum after 30 min and remained at somewhat less than the maximum concentration for 4 h. By 8 h the concentration of nuclear receptors had decreased to the control level. The properties of the cytoplasmic receptor (sedimentation coefficient of 7S (unactivated) and 5.5S (activated); K D 6–9 nM) were not changed after the administration of progesterone. However, the properties of the nuclear receptor changed in some respects. When exchange with [ 3 H]-R5020 was carried out on nuclei suspended in 5 mM phosphate buffer, at least 80% of the specifically bound 3 H was found in the supernatant. The tritium was not absorbed by dextran-coated charcoal and on sucrose density gradient centrifugation, a binding peak with a sedimentation coefficient of 3.5S was seen. Binding was temperature sensitive and was suppressed by unlabeled steroids in the following order: progesterone > 5α-pregnanedione testosterone cortisol. The nuclear receptor, extracted by 0.3–0.5 M KCl, also had a sedimentation coefficient of 3.5S on sucrose gradients in 5 mM phosphate. Competition by unlabeled steroids was similar to that seen with the receptor soluble in 5 mM phosphate.

Nuclear binding of guinea pig uterine progesterone receptor in cell-free preparations.

Abstract The transfer of guinea pig uterine progesterone receptors from cytosol to nuclei, in cell-free incubations, was studied using an exchange method to measure receptors. In order to validate the exchange measurement of the nuclear receptors, the direct nuclear uptake of [ 3 H]-progesterone-receptor (RcP ∗ )† and [ 3 H]-R5020-receptor (RcR5020 ∗ ) was compared with the uptake of unlabeled RcP or RcR5020, followed by exchange of RnP or RnR5020 with the corresponding [ 3 H]-steroid. Under a variety of experimental conditions the amounts of receptor measured by both methods were very similar. We found that temperature-activated cytosol receptor was bound by nuclei in the absence of added progesterone. The addition of 10 −9 and 10 −8 M progesterone increased receptor translocation, but higher concentrations of progesterone decreased receptor binding by nuclei. Other steroids that were bound by the 7S progesterone receptor of uterine cytosol and translocated to nuclei (5α-pregnanedione, R5020) also increased receptor binding by nuclei at concentrations of 10 −9 and 10 −8 M, but decreased binding at higher concentrations. Cortisol, corticosterone, testosterone and 17β-estradiol had no effect at concentrations below 10 −8 M, but at higher concentrations increased progesterone receptor binding by nuclei. Compounds with no apparent binding affinity for receptors in uterine cytosol (5β-pregnane 3α,20α-diol, 20α-hydroxy-4-pregnen-3-one) had no effect on receptor translocation. When nuclei were incubated with cytosol in the absence of progesterone, the nuclear receptor subsequently bound [ 3 H]-progesterone and [ 3 H]-R5020 rapidly at 0°C. Equilibrium was reached within l h. When nuclei were incubated with progestin-receptor complexes of cytosol, the nuclear receptor exchanged slowly with [ 3 H]-progestins at 0°C. Four hours were required for exchange with [ 3H -progesterone. Exchange with [ 3 H]-R5020 was only partial (40%) after 24 h.

Binding of progesterone by rat uterus in vitro

Abstract Rat uteri and abdominal muscle were incubated with 3 H-progesterone. Non-specific tritium uptake was seen in both tissues, but evidence for specific binding was found only in the uterus. Pretreatment with β-estradiol increased uterine binding. In the presence of progesterone, deoxycorticosterone, 19-norprogesterone, testosterone and several synthetic progestational agents tritium uptake by the uterus was reduced significantly. β-Estradiol, corticosterone or the 5α-pregnane uterine metabolites of progesterone did not reduce tritium uptake.

Effects of Progestins on the Progesterone Receptor in Guinea Pig Uterus

We examined the effects of progesterone and some synthetic progestins and other steroids on the physical properties of the progesterone receptor of guinea pig uterine cytosol and on the binding of the receptor by nuclei. Progestational potency seemed to correlate with the ability to keep the receptor in the 7S form and to prevent dissociation into smaller subunits. The rate of activation prior to nuclear binding was slower with steroids of increasing progestational activity. Therefore activation in vitro may be unrelated to biological activity. Concentration of the cytosol led to a decrease in the equilibrium association constant. The extent of the decrease was less with progesterone than with its metabolite, 5 alpha-pregnanedione. When cytosol and nuclei were incubated in the absence of ligand measureable progesterone receptor was bound by the nuclei. The uncomplexed nuclear receptor bound [3H]-progesterone of [3H]-R5020 rapidly at 0 degrees, but progesterone-receptor complexes exchanged [3H]-progestin slowly at 0 degrees. Progesterone increased the amount of nuclear receptor at concentrations of 10(-9) and 10(-8)M, but decreased binding at higher concentrations. 5 alpha-Pregnanedione had the same effect as progesterone, but other metabolites of progesterone that had little affinity for the 7S progesterone receptor in cytosol had no effect on nuclear binding at any concentration. Glucocorticoids, testosterone and estradiol-17 beta increased the nuclear binding of the progesterone receptor when present at concentration of 10(-8)M and greater.

Mammalian Progesterone Receptors: Biosynthesis, Structure and Nuclear Binding

Because estrogen and progesterone receptors may be involved in the endocrine sensitivity of certain breast and endometrial carcinomas (1,2), considerable interest has been generated in the study of steroid receptors in hormonally sensitive tumors. In this chapter, we will review some recent progress on three aspects of receptor biology as applied to mammalian progesterone receptors: regulation of receptor biosynthesis, structure of the receptor and nuclear binding of the steroid-receptor complex.

Hormonal Modulation of Progesterone Receptors

According to current theories of hormone action, all the steroid hormones, including vitamin D, exert their effects in a similar way (Buller and O’Malley, 1976; Chan and O’Malley, 1976; DeLuca, 1979; Gorski and Gannon, 1976; Yamamoto and Alberts, 1976). There is general, but not universal, agreement that the hormone diffuses passively through the cell membrane (Baulieu, 1978). After entering the target cell, the hormone binds to a cytoplasmic receptor protein that is present only in target cells and that is fairly specific for the hormone. The binding is of high affinity, with equilibrium dissociation constants in the neighborhood of 10-9 M. Because there is a limited number of receptor molecules per cell, the binding of the hormone is saturable. The complex of hormone and receptor then undergoes a process of “activation” or “transformation” that enables it to enter the nucleus and bind to chromatin. This binding initiates the biochemical events that are characteristic of the hormone. There is increased transcription, resulting eventually in an increase in protein synthesis.