Progesterone-binding components of chick oviduct. IX. The kinetics of nuclear binding.

A cell-free system was used to study the kinetics of progesterone-receptor interaction with purified nuclei prepared from estrogen-primed chick oviducts. The binding process was a saturable phenomenon in both target and nontarget tissues. More nuclear acceptor sites were available to target tissue (similar to 9000 sites per oviduct nucleus) than in nontarget tissues (similar to 1000 to 3000 sites per nucleus), but the binding constant was essentially the same (Kd similar to 10-8 M). A second much smaller class of higher affinity sites (Kd similar to 10-11M) may exist. Its presence was detected by Scatchard plot nonlinearity at very low concentrations of added receptor-hormone complex (similar to 10-10 to 10-12M). The current study focused on the prevalent class of acceptor sites which was more readily detectable. Receptor binding to these sites was highly sensitive to salt. More sites were exposed at 25 degrees than at 0 degrees. Binding to these sites was inhibited in a nonselective fashion by the addition of protein. Although receptors may be activated by temperature or conditions of high ionic strength, these conditions could not capacitate more than 30 to 40% of the progesterone-receptor proteins for binding. Rate studies suggested that temperature plays a minimal role in nuclear uptake of activated receptors. Such a finding is consistent with a diffusion-limited uptake process.     

Progesterone-binding components of chick oviduct. VIII. Receptor activation and hormone-dependent binding to purified nuclei.

A cell-free system prepared from the estrogen-primed chick oviduct was developed and used to study the uptake of cytoplasmic progesterone-receptor complex by isolated nuclei. The receptor and purified nuclei were shown to be stable at 25 degrees, but not at 37 degrees. Thus, nuclear incubations were routinely performed at 25 degrees. Such incubations revealed greater nuclear uptake of the cytoplasmic hormone-receptor complex as compared to control incubations performed at 0 degrees. The uptake process showed a quantitative preference for oviduct nuclei. No net uptake occurred during 0 degrees incubations when the nuclei were preincubated in the absence of cytoplasmic components at 25 degrees. In contrast, the temperature requirement was partially removed by preincubation of the hormone-receptor complex at 25 degrees prior to incubation with nuclei at 0 degrees. Nuclear uptake was not accompanied by measurable alterations in the sedimentation properties of the progesterone receptor. The activation and nuclear uptake of receptor was clearly dependent upon prior binding of steroid hormone to the receptor indicating that the active nuclear form of the receptor could not be generated in the absence of the hormone. Receptor precipitation with ammonium sulfate also partially removed the temperature requirement for nuclear binding. In contrast to temperature activation, ammonium sulfate precipitation activated the receptor in the absence of hormone. It thus seemed likely that temperature and salt activation of receptor occurred via different mechanisms. Although we were able to destroy up to 60% of the nuclear DNA content by treatment with DNase prior to nuclear incubation, some 80 to 85% of the receptor-binding capacity was still present in the treated nuclei. Thus, chick progesterone receptors apparently bind to a relatively DNase-resistant portion of the oviduct genome. The properties of this system indicate its value for further investigation into the initial events of progesterone action in the chick oviduct.     

Progesterone-binding sites of the chick oviduct receptor. Presence of a weaker ligand site which is destroyed by phosphatase treatment

Titration of chick progesterone receptor over a wide range of [3H]progesterone concentration (0.15 to 90 nM) shows two distinct types of binding sites in cytosol and in partially purified receptor samples prepared from oviducts of estrogenized chicks. The difference in affinity between the two sites (Kd = 1 nM; Kd = 25 nM) is sufficient to allow analysis by Scatchard plot methods. Ligand competition studies show that both sites have the same relative specificity for progesterone compared to other steroids. Both sites seem to be on the same receptor molecule as shown by their copurification and chromatographic properties. No cooperativity between the two sites has been detected in analysis using either rate kinetics or equilibrium methods. Thus, the function of the low affinity sites is not apparent at this time; it does not appear to function as a "helper" site which influences binding to the high affinity site previously described. The binding constant of the low affinity site is sufficiently strong to allow potential occupancy of these sites in vivo, at least at certain stages of the female reproductive cycle. The hormone-binding activity of the low affinity site can be destroyed after in vitro treatment with alkaline phosphatase, but the high affinity site remains functional under these conditions. Inhibitors of the enzyme block the inactivation. Furthermore, preliminary data in vivo suggest that estrogen administration to the animal can influence the relative titer of the low affinity sites.     

Progesterone binding components of chick oviduct. X. Purification by affinity chromatography.

Cytoplasmic progesterone receptors of chick oviduct have been purified in 8% yield by steroid affinity and ion exchange chromatography. The affinity resin, deoxycorticosterone-bovine serum albumin-Sepharose, binds progesterone receptors with high affinity (KD equals 8 times 10-minus 10 M) and its use resulted in a greater than 2000-fold purification over the starting material in a single step. DEAE-Sephadex A-50 chromatography was then used to achieve final purification. NA dodecyl-SO4 gel electrophoresis and DEAE-cellulose chromatography showed that the purified receptors contained both of the previously described 4 S progesterone binding components in near equal amounts. Na dodocyl-SO4 gel electrophoresis also showed that these components consisted of single polypeptide chains with molecular weights of 110, 000 (A component) and 117, 000 (B component). There was no evidence for subunits of lower molecular weight. The purified materials have identical hormone-binding kinetics and steroid specificity to crude cytosol receptors. The isolated receptors retain the three biologically important properties exhibited by progesterone binding components present in cruder preparations: they bind specifically to (a) nuclei (KD equals 1.1 times 10-minus 9 M, 10, 000 sites per nucleus); (b) chromatin (KD equals 3 times 10-minus 9 M, 2000 sites per pg of DNA-);and (C) DNA.     

Nuclear origin of progesterone receptor of the chick oviduct cytosol

Summary Progesterone receptor (PR) was studied immunoelectron microscopically from fixed vibratome sections of the chick oviduct and biochemically from the fractionated oviduct homogenate. Immunoelectron microscopically both unoccupied and occupied PR were localized inside the nuclei. Only a few cells showed PR immunoreactivity in the endoplasmic reticulum which probably represents newly synthetized PR. Biochemically unoccupied PR was in the cytosol fraction. The cytosol and nuclear PR as well as the non-transformed 8S-form and the transformed 4S-forms of cytosol PR were recognized by the anti-PR antibody (IgG-RB). The lack of PR immunostaining in the cytoplasm is therefore not due to lack of recognition by IgG-RB. We propose that in the chick oviduct progesterone receptor is a nuclear protein but synthetized in the endoplasmic reticulum.     

Nuclear origin of progesterone receptor of the chick oviduct cytosol. An immunoelectron microscopic study

Progesterone receptor (PR) was studied immunoelectron microscopically from fixed vibratome sections of the chick oviduct and biochemically from the fractionated oviduct homogenate. Immunoelectron microscopically both unoccupied and occupied PR were localized inside the nuclei. Only a few cells showed PR immunoreactivity in the endoplasmic reticulum which probably represents newly synthetized PR. Biochemically unoccupied PR was in the cytosol fraction. The cytosol and nuclear PR as well as the non-transformed 8S-form and the transformed 4S-forms of cytosol PR were recognized by the anti-PR antibody (IgG-RB). The lack of PR immunostaining in the cytoplasm is therefore not due to lack of recognition by IgG-RB. We propose that in the chick oviduct progesterone receptor is a nuclear protein but synthetized in the endoplasmic reticulum.     

Nuclear binding of progesterone in chick oviduct. Multiple binding sites in vivo and transcriptional response.

1. Varied doses of labelled or unlabelled progesterone were injected into immature chicks which had previously been stimulated with oestrogen. The concentrations of nuclear bound [3H]progesterone were correlated with the effects of the hormone on endogenous RNA polymerase I and II activities in isolated oviduct nuclei. 2. The extent of nuclear localization of [3H]progesterone in oviduct (a progesterone target tissue) was shown to be much greater than in lung (non-target tissue). The conccentration of bivalent cations in solvents used in the nuclei isolations has a marked effect on the amount of bound hormone in the nuclei. 3. Evidence for the existence of several classes of binding sites for progesterone in the oviduct nuclei is given. These classes represent about 1000) 10000 and 100000 molecules of the hormone per cell nucleus and are saturated by injecting approx. 10, 100 and 1000 mug of progesterone respectively. 4. When saturation of the first (highest affinity) class of nuclear sites occurs, a marked inhibition in RNA polymerase II (but not RNA polymerase I) activity was observed. When the second class of sites was saturated, alterations in both RNA polymerase I and II activities were observed. Binding to the third class of nuclear binding sites was not accompained by further changes in polymerase activity. It is suggested that the first two classes of nuclear binding sites may represent functional sites for progesterone action in the chick oviduct.     

Fractionation of chick oviduct chromatin. Nuclease-resistant deoxyribonucleic acid.

Chromatin isolated from several chick tissues was treated with micrococcal nuclease. A limited degree of tissue specificity of chromatin DNA resistance to nuclease digestion was observed. No difference in the extent of nuclease resistance of chromatin DNA was detected during oestrogen-induced oviduct differentiation. This suggested that the amount of non-histone chromosomal protein does not play an important role in the sensitivity of chromatin DNA to nuclease digestion. Studies of nuclease resistance of chromatin DNA after dissociation and reconstitution of chromatin proteins and ethanol extraction of chromatin indicate that the histones protect the DNA from nuclease attack. Slow thermal denaturation of nuclease-resistant DNA suggests that the protected DNA sequences may be (A+T)-rich, and the (G+C)-rich satellites present in total chick DNA are sensitive to nuclease.     

The chick oviduct in tissue culture. I. Initial characterization of growing primary oviduct tissue cultures

A simple three-enzyme treatment of collagenase, dispase and hyaluronidase on finely minced chick oviduct yields clumps of 50-150 cells. These cells attach to collagen-treated dishes and survive in culture for at least 2 weeks without subculturing. Oviduct cell cultures can also be induced to grow. Estradiol or epidermal growth factor (EGF) induce a 40% increase in cells in 4 days when cultures are grown in serum levels that do not support growth. Serum from estrogen-stimulated chicks promotes rapid cellular proliferation (doubling times of 1-2 days). Sera from estrogen withdrawn chicks, laying hen or horse do not support as rapid proliferation. The oviduct growth-promoting factors in serum from estrogen-stimulated chicks are not steroids or fibroblast growth factors (FGF). Removal of steroids from these sera by charcoal treatment or delipidization does not decrease the rate of growth. The addition of 1-100 nM estradiol does not increase a serum's ability to promote growth. Purified FGF or platelet-derived growth factor (PDGF) do not induce oviduct proliferation. These results were reproduced in oviduct cell cultures started from estrogen-stimulated and withdrawn chicks as well as laying hens. Thus the factors in serum from estrogen-stimulated chicks that promote rapid oviduct growth are induced by estrogen treatments in vivo, but do not seem to be only steroids.     

Inhibition of chick embryo hepatic uroporphyrinogen decarboxylase by components of xenobiotic-treated chick embryo hepatocytes in culture. II.

A variety of xenobiotics, viz., 3,3',4,4'-tetrachlorobiphenyl (TCBP), sodium phenobarbital (PB), 3,5-diethoxycarbonyl-2, 4,6-trimethylpyridine (OX-DDC), and nifedipine, cause a decrease in uroporphyrinogen decarboxylase (UROG-D) activity, accompanied by uroporphyrin accumulation, in chick embryo hepatocytes in culture. In this study the activity of 17-day-old chick embryo hepatic UROG-D was determined by measuring the conversion of pentacarboxylporphyrinogen I to coproporphyrinogen I, and it was shown that a UROG-D inhibitor, previously reported to accumulate in TCBP-treated and PB-treated chick embryo hepatocytes in culture, also accumulates in OX-DDC-treated and nifedipine-treated chick embryo hepatocytes in culture. It was concluded that the accumulation of a UROG-D inhibitor provides an explanation for the UROG-D inhibition observed in this culture system with xenobiotics that cause uroporphyrin accumulation. Studies of the UROG-D inhibitory fraction isolated from the 10,000 x g, 40,000 x g, and 100,000 x g supernatant fractions of cultured chick embryo hepatocyte homogenate led to the conclusion that the UROG-D inhibitor is derived from a soluble component of the homogenate.     

Testosterone binding in the chick oviduct

A novel androgen receptor was observed in estrogen-stimulated chick oviducts but not in unstimulated oviducts. This binding component showed a preference for androgens and could therefore be distinguished from oviduct receptors for estadiol and progesterone. Testosterone was tightly bound having a dissociation constant (Kd) of 2.7 × 10^?10 M. Sucrose gradient centrifugation, under low ionic strength conditions, showed testosterone to be bound as an 85 complex. These binding properties, plus the estrogen dependency of this component, suggest its role as a biological receptor for androgens.     

Oestrogen receptors in chick oviduct. Characterization and subcellular distribution.

Macromolecular components with properties of oestrogen receptors have been identified in the 0.5 M KCl nuclear soluble, the nuclear insoluble and the cytosol fractions of laying hen and immature (2--4 weeks, untreated by hormone) chicken oviduct. 7n the 0.5 M KCl extract of laying hen oviduct nuclei, a receptor, of protein nature according to the effects of enzymic treatments, has been identified. It exhibits high affinity for oestradiol with an apparent equilibrium association constant KA = 4 - 109 M-1 at 4 degrees C. The binding of [3H] oestradiol is abolished by 1 muM oestriol, oestrone and diethylstilboestrol, but not by the same concentration of progesterone, testosterone, and cortisol. Sucrose gradient ultracentrifugation studies in the presence of 0.5 M KCl indicate a sedimentation coefficient of 4.3 S, and there is partial aggregation in low-ionic-strength medium. The estimated number of binding sites per nucleus is about 5000, as calculated from DNA content of chick diploid genome. Most of the binding sites were found to be occupied by endogenous oestrogen(s). Oestradiol dissociates from the receptor according to an apparent two-step mechanism. The half-life time for the faster dissociation step is 18 h at 0 degrees C, 25 min at 20 degrees C and 10 min at 30 degrees C, and for the slower one is 180 h, 115 min and 60 min, respectively. In the 0.5 M KCl extract of immature chicken oviduct nuclei, there are approximately 500 receptor sites per nucleus; their affinity for oestradiol is the same as in the case of laying hen soluble nuclear receptor. After repeated extractions of nuclei with 0.5 M KCl medium, a substantial quantity of oestrogen binding sites remains in the residual fraction. Binding characteristics of this insoluble nuclear receptor resemble those of the soluble nuclear receptor: high affinity for oestradiol (KA = 7 - 10(8) M-1 at 37 degrees C) and specificity for oestrogens. The estimated number of binding sites are approximately 2000/cell for laying hen, and approximately 1000/cell for immature chicken. In the high-speed supernatant fraction of laying hen oviduct homogenates, an oestrogen receptor is also present, but its concentration is low (less than or equal to 100 sites/cell) and at the limits of sensitivity of the methods used. In the cytosol of immature chicken oviduct, there are approximately 2500 oestradiol receptor sites per cell.