Progesterone Receptor Complexes Research Articles

A review of regulation of gene expression by steroid hormone receptors

We have investigated the manner by which progesterone receptors act to induce initiation of RNA synthesis in a cell free system derived from chick oviduct. A method utilizing rifampicin enabled us to measure the formation of binary initiation complexes between RNA polymerase and chick oviduct chromatin and allowed for the quantitative assessment of RNA chain initiation sites. RNA chain propagation rates, and RNA chain size under conditions which prevent secondary chain reinitiations. Purified progesterone-receptor complex stimulated transcription of oviduct chromatin in vitro by promoting an increase of 3000 to 5000 additional sites for RNA chain initiation. These data showed that progesterone receptor can directly increase the number of RNA polymerase initiation sites in the chromatin template in the absence of a detectable change in either the rate of RNA propagation or the size of the RNA product. The stimulatory effect of the purified progesterone receptor was time dependent (T 1 2 of 15 min) and the concentration of receptor for half maximal stimulation of RNA chain initiation was ~5 × 10 −9M, a value which correlates directly with the receptor affinity for binding to chromatin. The stimulatory effect of the purified progesterone receptor appeared to be relatively specific for oviduct chromatin in comparison to non-target tissue chromatins or DNA. Utilizing a copy DNA probe to ovalbumin mRNA, a greater than ten-fold increase in transcription of the ovalbumin gene was detected after the incubation of progesterone receptor with oviduct chromatin. The data presented here show that steroid hormone receptor complexes can directly regulate aene transcription in vitro in a manner which mimics the events observed in vivo in target cells.

Progesterone Binding to Hen Oviduct Genome: Specific Versus Nonspecific Binding

Data are presented to explain discrepancies in the literature involving the in vitro binding of steroid receptor complexes to isolated nuclei and chromatin. The type of binding in vitro of the progesterone-receptor complex to nuclei, chromatin, or DNA of hen organs is largely determined by the ionic strength of the medium. Low ionic conditions (0.01 to 0.05 molar potassium chloride) result in a nonspecific, nonsaturable binding, while high ionic conditions (0.15 to 0.20 molar potassium chloride) create a tissue-specific, saturable binding. Pure DNA binds the steroid receptor complex extensively in low salt but very little in the higher salt conditions.

ATP-PPi exchange activity of progesterone receptor.

Progesterone receptor preparations from avian oviduct catalyze a pyrophosphate (PPi)-exchange reaction between ATP and 32P-labeled PPi. The reaction requires ATP exclusively and is Mn++-dependent. This enzyme activity is detectable in receptor preparations that have been purified extensively by chromatography on ATP-Sepharose and DEAE-Sephadex columns. Polyacrylamide gel electrophoresis of purified preparations reveals a comigration of [3H]progesterone-receptor complex and the enzyme activity. The PPi-exchange reaction is inhibited by both o-phenanthroline and rifamycin AF/013, which also block the nuclear binding of progesterone receptor. These findings indicate that progesterone receptor may be an enzyme or a subunit of an enzyme that is active in nucleotide metabolism.

Progesterone-binding components of chick oviduct. In vitro effects of purified hormone-receptor complexes on the initiation of RNA synthesis in chromatin.

We have investigated the manner by which progesterone receptors act to induce initiation of RNA synthesis in a cell-free system derived from chick oviduct. A method utilizing rifampicin enabled us to measure the formation of binary initiation complexes between RNA polymerase and chick oviduct chromatin (Tsai, M.-J., Schwartz, R.J., Tsai S.Y., and O'Malley, B.W. (1975) J.Biol. Chem. 250, 5165-5174) and allowed for the quantitative assessment of RNA chain initiation sites, RNA chain propagation rates, and RNA chain size under conditions which prevent secondary chain reinitiations. We have measured the available initiation sites for transcription in oviduct chromatin prepared from chicks withdrawn from all hormone and then restimulated with a secondary injection of progesterone. Within 1/2 hour after administration of progesterone, the number of initiation sites increased from 8,700 sites/pg of chromatin DNA for the control to 15,500 sites. After 1 hour, the concentration of RNA polymerase needed to saturate chromatin binding sites was increased 60% in comparison to control values, while the number of initiation sites increased 160%. This rapid increment in transcriptional activity preceded temporally the induction of synthesis of ovalbumin mRNA. To test directly the effect of progesterone receptor on transcription, in vitro, a reconstituted cell-free system was employed which contained purified cytoplasmic progesterone-receptor complexes, Escherichia coli RNA polymerase, and chromatin prepared from hormonally withdrawn chick oviducts. Purified progesterone-receptor complex stimulated transcription of oviduct chromatin in vitro by promoting an increase of 3,000 to 5,000 additional sites for RNA chain initiation. These data showed that progesterone receptor can directly increase the number of RNA polymerase binding and initiation sites in the chromatin template in the absence of a detectable change in either the rate of RNA chain propagation or the size of the RNA product. The kinetics of progesterone-receptor stimulation of RNA synthesis in chromatin revealed a t1/2 of 15 min for this effect to occur. This value was identical with the optimal time required for binding of receptor to chromatin. The concentration of receptor required for half-maximal stimulation of RNA chain initiation was approximately 5 x 10(-9) M. This value agreed closely with our previously reported estimates of the affinity (Kd approximately 5 x 10(-9)M) of the progesterone-receptor complex for oviduct chromatin. The stimulatory effect of purified progesterone receptor appeared to be relatively specific for oviduct chromatin in comparison to nontarget tissue chromatins or chick DNA. The data presented here show that steroid hormone-receptor complex can directly regulate gene transcription in vitro in a manner which mimics the events observed in vivo in target cells.

Distribution of Binding Sites for the Progesterone Receptor Within Chick Oviduct Chromatin

Chromatin from the oviducts of estrogen-treated chicks was sheared and fractionated on sucrose gradients. This resulted in the production of several chromatin fractions which differ in their sedimentation properties, protein composition, the number of acceptor sites for the progesterone-receptor complex, and the ability to serve as a template for RNA synthesis. The pellet chromatin fraction shows an enhanced ability to bind the progesterone-receptor complex in vitro and in cell-free systems. Kinetic analysis indicates that the majority of the acceptor sites for the progesterone-receptor complex are located in the pellet chromatin fraction which is lowest in template activity. The sites may be important loci for initiating the changes in RNA synthesis following the exposure of target cells to steroid hormones.

Chromosomal proteins regulate steroid binding to chromatin

STEROID hormones accumulate in their respective target cells due to the presence of “receptor” proteins which bind the steroid with high affinity and specificity1–4. It seems that the steroid–receptor complexes then migrate and bind to nuclear chromatin2–5. Within about 15 min there is an alteration in transcription which is followed after 2 h by the appearance of specific messenger RNAs6–9. At present, the mechanism by which steroids alter gene expression is obscure. The group in our laboratory is attempting to identify the nuclear (or chromatin) component which binds the progesterone-receptor (P–R) complex because it is possible that the macromolecules involved are also involved in the steroid-induced modifications of gene expression. We have demonstrated recently more than one class of binding sites in chick oviduct nuclei in vivo and in hen oviduct nuclei and chromatin in vitro10–12,13. The highest affinity class of sites, representing 5,000–10,000 sites per cell nucleus, is suspected to be the biologically important class, because: (1) it is the only class which binds P–R in vitro in physiological ionic conditions (0.15–0.20 M KCl); (2) it is the only class bound in vivo when the steroid is at physiological levels in the serum; (3) binding to whole chromatin is tissue-specific, found only in oviduct chromatin, and (4) when these sites are saturated in vivo with the P–R, maximal responses of the RNA polymerase I and II activities are observed in vitro. Although additional nuclear binding to the sites of weaker affinity can be achieved in vivo, this causes no further alteration of polymerase activities13. We have also found that the low ionic conditions (0.01–0.05 M KCl), often used in binding assays, result in a pattern of nuclear binding which is non-saturable and shows no tissue specificity. When the ionic strength of the binding assays is increased to 0.15 M or 0.2 M KCl, there is a saturable, tissue-specific binding which measures selectively only the highest affinity class of binding sites10–12. We now present evidence which suggests that one fraction of the non-histone protein (NHP) (called AP3) is involved in the high affinity binding of P–R to the chromatin of the progesterone target tissue—oviduct—while other fractions (AP1 or AP2) are involved in masking these binding sites. We also present data which indicate that the high affinity binding sites are present but completely masked in non-target tissues.

Nuclear binding of progesterone in hen oviduct. Role of acidic chromatin proteins in high-affinity binding

The multiple classes of binding sites for the progesterone-receptor complex in hen oviduct muclei were found to be of chromatin origin. The highest-affinity, and presumably most physiologically important class, is localized in oviduct chromatin and contains approx. 6000-10000 sites per nucleus. None of these sites is detected in spleen chromatin. Two new techniques were used for assaying rapidly the binding of steroid-receptor complexes to soluble deoxyribonucleoproteins in vito. The extent of high-affinity binding by the nucleo-acidic protein fraction from spleen chromatin is as great as that by the nucleo-acidic protein from oviduct chromatin. Consequently the tissue-specific nuclear binding of the progesterone receptor is found not to be a consequence of the absence of the nuclear binding sites (acceptors) from chromatin of non-target tissue (spleen), but rather a result of complete masking of these sites. In the target-tissue (oviduct) chromatin, approx. 70% of the high-affinity acceptor sites are also masked. Acidic proteins, and not histones, appear to be responsible for the masking of these acceptor sites. In addition, acidic proteins represent (or at least are an essential part of) these high-affinity sites in the oviduct nucleus. Pure DNA displays a few high-and many low-affinity binding sites. In support of previous work with immature chicks, the acidic protein fraction of the nucleo-acidic results thus support the hypotheis that protein complexed with DNA, and not DNA alone, represent the high-affinity binding sites for the steroid-receptor complexes in nuclear chromatin. The lower-affinity classes of binding sites may represent DNA and/or other nuclear components.

The Stimulatory and Inhibitory Function of Theophylline on Amino Acid-Induced Insulin Release: Studies with the Perfused Rat Pancreas

Although progesterone receptors have been studied in the uterine cytoplasm of many species, relatively little was known about the nuclear content of these binding proteins. In the present study, a nuclear progesterone receptor was deteced in the guinea pig uterus. The binding of progesterone to the nuclear receptor was hormone and tissue specific. Furthermore, the nuclear localization of the progesterone receptor complex in the uterus was both time and temperature dependent. Since the nuclear receptor was extracted in high salt buffer, the effects of KCl on several physical properties of the cytoplasmic and nuclear binders were studied. In the presence of high salt, cytosol and nuclear receptors were virtually indistinguishable. These proteins were clearly distinguished upon removal of the KCl by rapid dialysis: the nuclear receptor had a slower sedimentation rate, a faster rate of dissociation and a higher binding affinity than did the cytosol receptor for progesterone. We conclude that the cytosol and nuclear receptors for progesterone in the guinea pig uterus are distinct macromolecules. These observations are consistent with the postulate that the cytoplasmic receptor is a precursor of that in the nucleus.

An analysis of the binding of the chick oviduct progesterone-receptor to chromatin

The binding of progesterone-receptor complexes to chromatin from target and nontarget tissues was studied in vitro. Chromatin from both target and non-target tissues responds in a similar manner to saly and cofactors and has the same K D (approx. 3·10 −9 M) for the progesterone-receptor complex. The only observed difference in the binding of the progesterone-receptor complex to target and nontarget chromatins is the difference in total number of acceptor sites. Oviduct chromatin has approx. 1300 sites/pg DNA, spleen chromatin has approx. 840 sites/pg DNA, and erythrocyte chromatin has about 330 sites/pg DNA. The K D and number of acceptor sites for progesterone-receptor complex binding to oviduct chromatin remains the same even after extensive purification of the progesterone-receptor complex. Activation of cytosol labeled with [ 3H]progesterone by preincubation at 25°C, analogous to that required for maximal nuclear binding, occurs if the binding studies to chromatin are performed in 0.025 M salt. The absence of an observable temperature effect when the studies are performed at 0.15 M salt is due to the activation of the receptor by salt. The dissociation of the progesterone-receptor complex from chromatin exhibits a single dissociation rate and the initial event is the appearance of free progesterone rather than a progesterone-receptor complex. Lastly, the treatment of chromatin with an antibody prepared against either single-stranded DNA or double-stranded DNA does not alter the extent of binding of the progesterone-receptor complex. Similarly, pretreatment of chromatin with a single-stranded nuclease does not inhibit the capacity of chromatin to bind the hormone-receptor complex.

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.

Studies on Progesterone Receptor in Rabbit Uterine Cytosol

Estrogen priming increases uterine 8S macromolecule which binds progesterone specifically. Progesterone-8S complex in the cytoplasm enters into nucleus and is bound to chromatin finally. In this paper, the mode of nuclear translocation of steroid in exchange assay of receptor introduced by Anderson et al., and the mode of binding to chromatin were studied on the progesterone-receptor complex in the uterus of estrogen primed female rabbit. 1. After intravenous administration of 200 mug progesterone into the estrogen primed immature rabbit, uterine nuclei were prepared by the method in Table 1. These nuclei were incubated with 3H-progesterone and cold steroids at 4 degrees C for 30 minutes, and then washed with buffer A. The radioactivity of the nuclei was counted. This experiment was performed at 4 degrees C because progesterone receptor and chromatin were observed to be degraded at 37 degrees C for 20 minutes. The effect of cold steroids in vitro on the incorporation of 3H-progesterone into the uterine nuclei of rabbit pretreated with progesterone was found to be similar to their effect on progesterone-receptor binding in cytosol or chromatin (Fig. 1). 2. The effect of cold steroids on 3H-progesterone-receptor-chromatin triplex (Table 2 and Fig. 2) was examined. Once 3H-progesterone-receptor-chromatin triplex was formed, it was difficult to exchange 3H-progesterone to other steroids at 4 degrees C. These results (1 & 2) indicate that progesterone-receptor complex enters into nucleus and is bound to chromatin. Exchange of steroid may occur in the nuclear progesterone-receptor complex, which is free from the binding with chromatin. And thus exchange assay cannot represent quantitative data on receptor content. 3. 3H-progesterone-8S or 5S complexes were obtained by 5 approximately 20% sucrose linear gradient centrifugation (Fig. 3). The same molar concentration of these complexes from estrogen primed or castrated rabbit uterus were incubated with primed uterine chromatin for 30 minutes. Then the chromatin was washed with buffer A and the radioactivity was counted. It was shown in Fig. 4 that 3H-progesterone-8S complex was bound to chromatin much more tightly than 3H-progesterone 5-S complex in preparations obtained from both castrated or primed uterine cytosols. All these results indicate that 8S may be the biologically active form of the receptor. 4.3H-progesterone uptake into uterine nuclei was observed in very limited amount following the injection into uterine artery. The radioactivity in nuclei decreased easily by washing with buffer A as in Fig. 5. The small amount of residual radioactivity after washing, that is, very limited number of binding sites with high affinity is considered to be indicative of biologically active binding.

Molecular mechanisms of steroid hormone action

The differentiated chick oviduct is a target tissue for progesterone. Administration of the hormone to estrogen-primed chicks causes a rapid induction of new messenger RNA, resulting in the appearance in cytoplasm of specific mRNA species. One of these species is the mRNA coding for the egg white protein avidin, which is produced only in response to progesterone. The cytoplasm contains receptors which bind progesterone specifically and transport it as a complex into oviduct nuclei. Only a small fraction of the receptors are active in vitro; this fraction is increased to 30% by brief warming of the receptor-hormone complexes before incubation with the nuclei. The binding reaction is slow, and the receptors cannot be released from nuclei by DNase treatment. The reaction occurs preferentially with oviduct nuclei, which contain at least twice as many acceptor sites (8000/nucleus) as other chick tissues (< 2000/nucleus). The binding constants are the same in all tissues ( K d ~ 10 −8 M), and are identical to the constant for receptor binding to isolated chromatin. We have isolated the progesterone receptor component which binds to the chromatin. Oviduct cytosol from laying hens was prepared and progesterone receptors were precipitated with ammonium sulfate (30% sat.). The re-dissolved pellet was eluted from a steroid-affinity column (Sepharose 4B-BSA-deoxycorticosterone) with 3M urea. The receptors were reconstituted by dialysis and labeled with [ 3H]-progesterone. The [ 3H]-progesterone-receptor complexes were then purified by sequential chromatography and elution with the indicated slats at pH 74 from DEAE-cellulose (0.2 M KCl), phosphocellulose (0.26 M KCl), and hydroxylapatite (0.15 M K x PO 4). The peak fraction was finally chromatographed on an agarose A-1.5 M column ( K av = 0.28). Yield was 1 %, and purity approached the theoretical maximum specific activity, 10 9 d.p.m./mg protein.

Receptor progesterone complex in the nuclear fraction of the rat uterus: Demonstration by 3H-progesterone exchange

We have previously shown that 3H-estradiol exchange can be used to measure the quantity of estrogen receptor complex in the nuclear fraction of target tissue cells. This method has been modified for the measurement of the progesterone receptor complex (R n·P) in the nuclear fraction of uterine cells. Nuclear fractions were incubated for 5 hrs. at 15°C in the presence of varying concentrations of 3H-progesterone ( 3H-P) with or without a 250-fold excess of non-labeled progesterone (P). R n·P was determined by subtracting the 3H-P bound in the presence of excess P (non-specific binding) from 3H-P bound in the absence of excess P (total binding). All R n·P studies were done in adult castrate female rats that had received estradiol benzoate (0.4 mg) one week before use. The quantity of R n·P increased in the uterine nuclear fractions by 280% 30 min. after injection of 5 mg of P. R n·P was not increased in muscle or fat pad by this treatment. Injections of corticosterone (B), cortisol (F), dexamethasone (Dx) or testosterone (T) failed to increase R n·P. The exchange reaction was specific for P; B, F, Dx or T did not compete. These results demonstrate the existence of a low capacity, high affinity, stereospecific progesterone binding site in the nuclear fraction of the uterus.

Hormone-induced Maturation of Xenopus laevis oocytes: Effects of Different Steroids and Study of the Properties of a Progesterone Receptor

The ability of various steroids to induce maturation of Xenopus laevis oocytes after incubation or injection has been studied. Only two of these hormones, hydrocortisone and testosterone, are active by injection as well as by incubation. Progesterone can induce maturation only when administered from the outside medium, which suggests the presence of a receptor in or near the membrane. This progesterone-receptor has been localized in the melanosomes, and its properties and affinity for other steroids have been studied. The morphological and biochemical effects of the injection of the progesterone-receptor complex have been studied.

The use of deoxyribonucleic acid-cellulose chromatography and isoelectric focusing for the characterization and partial purification of steroid-receptor complexes.

1. Two characteristic properties of the specific high-affinity steroid-binding proteins or receptors, their ability to bind to DNA-cellulose and their relatively acidic isoelectric point, have been exploited as a means of purification. These two fundamental properties distinguish the receptors from the steroid-binding proteins in serum and the non-specific low-affinity steroid-binding proteins in hormone-responsive cells. 2. A significant degree of purification of both cytoplasmic and nuclear steroid-receptor complexes can be achieved with practical facility by these procedures. The purity of the receptor complexes is sufficient to enable studies on their possible control of metabolic processes to be investigated in the future. 3. After extensive purification the physicochemical properties of the cytoplasmic androgen-receptor complex, such as sedimentation coefficient, were unchanged. Further, the purified complex fully retained at least one of its fundamental physiological properties, namely the ability to transfer 5alpha-dihydrotestosterone (17beta-hydroxy-5alpha-androstan-3-one) into chromatin in vitro. 4. The methods may also be employed for studying the changes in the structure and properties of the receptor complexes that are an essential prerequisite for the transfer of cytoplasmic receptor complexes into nuclear chromatin. The temperature-dependence of the binding of androgen-receptor complexes into chromatin is essentially due to a major change in cytoplasmic receptor complex before its attachment to nuclear chromatin. 5. The resolution of these analytical procedures was sufficient to enable a critical comparison of the receptor proteins from different male accessory glands to be undertaken. From these studies, no substantial evidence in support of the tissue specificity of androgen receptors could be established; rather the receptors from different androgen-dependent glands were remarkably similar in physicochemical properties. 6. Although the methods were initially developed for the partial purification of androgen-receptor complexes, they are equally suitable for the prompt and extensive purification of oestrogen-receptor and progesterone-receptor complexes.

Target tissue receptors for progesterone: the influence of estrogen treatment.

The effects of estrogen treatment on the specific progesterone binding protein (receptor) in the chick oviduct were investigated. The progesterone receptor is present in non-estrogen treated oviducts but the tissue concentration of this protein increases several fold during a 2- week-period of estrogen treatment. In addition, qualitative changes in the receptor protein are indicated by its sedimentation properties on sucrose gradients. Following estrogen treatment, the 4-5S progesterone-receptor complex is apparently altered to a form which sediments in the 6-8S region on sucrose gradients. These results suggest that the enhanced responsiveness of the oviduct to progesterone following estrogen treatment may arise through quantitative and qualitative changes in progesterone receptor molecules. (Endocrinology 90: 1041, 1972)

Progesterone-binding Components of Chick Oviduct: V. EXCHANGE OF PROGESTERONE-BINDING CAPACITY FROM TARGET TO NONTARGET TISSUE CHROMATINS

Abstract Progesterone, when forming a complex with a specific protein receptor molecule, interacts in vitro with intact chick oviduct chromatin. In the present study, a series of experiments was performed to elucidate the nature of the chromatin sites of oviduct responsible for binding the progesterone receptor. The proteins of chromatin from various chick tissues were dissociated from DNA and reconstituted back to the DNA of either the same or different tissue. The chemical analysis and polyacrylamide gel electrophoresis demonstrate that the histones are quantitatively reintegrated into the chromatin without detectable degradation when protease inhibitors are present during the reconstitution. Comparison of the chemical composition and immunochemical analysis of the untreated and reconstituted chromatins indicates that non-histone (acidic) proteins also can be switched from oviduct chromatin to the DNA of other tissues to form chromatins. The transfer of the acidic proteins from the chromatin of one tissue to that of another results in a corresponding exchange of the template capacity so that the hybrid chromatin resembles the chromatin serving as the source of the acidic proteins. These data suggest that the acidic proteins are involved in maintaining open template. Reconstituted chick oviduct chromatin maintains the capacity to bind the progesterone-receptor complex. This tissue-specific binding is transferred to chromatins of non-target organs by exchanging the acidic chromatin proteins during reconstitution. Moreover, quantitative microcomplement fixation assays using antisera specific for the oviduct DNA-acidic protein complex indicate that the conformation of acidic proteins following transfer is similar to that found in the native oviduct chromatin. A subfraction of the acidic proteins of oviduct chromatin has been found to contain the acceptor molecules which are responsible for the binding with the progesterone-receptor complex. These tissue-specific acceptor proteins thus appear to be the molecules responsible for the binding of progesterone-receptor complex to oviduct chromatin.

Changes in chromatin composition and hormone binding during chick oviduct development

1. 1. Previous studies have demonstrated that progesterone, complexed with the oviduct receptor protein, is extensively bound by the deoxyribonucleoproteins (chromatin) of the mature chick oviduct. The chromatin of other tissues of the chick did not display this extensive binding. Results of studies presented here show that the progesterone receptor complex is extensively bound by the chromatin of the chick oviduct at all stages of development. 2. 2. Analyses of the chromatins indicate that the levels of nonhistone (acidic) protein and RNA as well as the ability of the chromatins to serve as a template for DNA-dependent RNA synthesis are altered in a specific pattern during differentiation of the chick oviduct. Differences in the levels of histones were less and showed no specific pattern. The capacity of the oviduct chromatins to bind the progesterone-receptor complex also varies throughout the development of the oviduct. The extent of binding the hormone receptor complex appears to be correlated with the levels of acidic proteins and RNA of the oviduct chromatins. 3. 3. Since chromatin of the undifferentiated oviduct binds the progesterone-receptor complex more extensively than the chromatins of other tissues, e.g., spleen or erythrocyte, it is speculated that the hormone binding capacity is not established during the estrogen induced maturation of the oviduct but must be established earlier at some stage of organogenesis of the chick oviduct.

Soluble complexes between steroid hormones and target-tissue receptors bind specifically to target-tissue chromatin.

Cytoplasmic fractions containing steroid hormone receptor were prepared from rat prostate and uterine tissues, incubated first with [(3)H]dihydrotestosterone or [(3)H]estradiol, and then with their respective target and non-target tissue chromatins. Only prostate and testis chromatin bound the dihydrotestosterone-receptor complex from prostate cytosol extensively. Similarly, uterine chromatin bound more estradiol-receptor complex from uterus than did liver, spleen, or lung chromatin. Complexes between dihydrotestosterone or estradiol with cytosols prepared from liver and spleen bound less extensively, and similarly, to all chromatins. Analogous results are described for the [(3)H]progesterone-receptor complex from chick oviduct cytosol binding to oviduct chromatin. These studies suggest that the chromatin of all steroid hormone target tissues may contain "acceptor sites" for their respective hormone-receptor complexes, and are thus programmed to receive the complex as it is transferred into the nucleus from the cytoplasm of the cell.

Tissue specific binding invitro of progesterone-receptor to the chromatins of chick tissues

When H 3 -progesterone is incubated with chick oviduct cytosol, it binds to a specific receptor protein. The subsequent binding of this complex to oviduct chromatin is greater than that of free progesterone alone. This specific affinity for the progesterone-receptor complex is not observed with spleen, heart, liver or erythrocyte chromatin. Progesterone incubated with the cytosols of non-target tissues such as liver or spleen shows no specific binding to chromatin from any source. Artificial complexes of chick histone and DNA display a low degree of non-specific binding. The results suggest that the chromatin of target tissue (oviduct) may contain “acceptor” sites for the hormone-receptor complex contained in the cytoplasm of the cell. The genome may thus be preprogrammed to receive the hormone receptor complex as it enters the nucleus of the target cell.