Identification of two forms of progesterone receptor from chick oviduct cytosol using non-denaturing gel electrophoresis.

Non-denaturing polyacrylamide gel electrophoresis and non-denaturing agarose gel electrophoresis have been used to resolve [3H]R5020-binding components from chick oviduct cytosol. From both gel systems 2 peaks of bound radioactivity are resolved which display these properties of authentic progesterone receptor: binding of R5020: steroid specificity, saturability, and restriction to target tissues. The two peaks are approximately equal in magnitude, and there is no evidence for interconversion of the 2 peaks. The presence or absence of 10-20 mM sodium molybdate during cytosol preparation had no effect on the magnitude or mobility of either peak. Neither peak contains salt-dissociable components which affect its electrophoretic properties, suggesting a possible alteration of native receptor forms during electrophoresis.

Electrophoretic analysis of the estrogen receptor. Relationship of multiple receptor forms to the molybdate-stabilized form.

The origin of and relationships among multiple forms of the estrogen receptor from rat uteri were investigated using electrophoretic and conventional hydrodynamic methods of analysis. Evidence is presented that the molybdate-stabilized, multimeric receptor (Stokes radius approximately 70A; S20,w approximately 9.5 S; Mr approximately 290,000) corresponds to an acidic form of the receptor that has relatively high electrophoretic mobility. This discrete form, which appears to represent the untransformed state that does not bind to DNA, was converted to a number of derived forms by exposure to conditions that result in receptor transformation and/or subunit dissociation. In crude cytosol, transformation always generated receptor forms that were excluded from polyacrylamide gels, and it was shown that these are large heterogeneous aggregates. This explains previous failed attempts to analyze the receptor by polyacrylamide gel electrophoresis. Transformation of partially purified, molybdate-stabilized receptor never led to aggregate formation, but resulted instead in the generation of two relatively basic estrogen-binding species of low electrophoretic mobility. These components may represent the free or dissociated estrogen-binding subunits. Together, the results suggest a model for the molybdate-stabilized receptor wherein at least one of its components is an acidic, nonestrogen-binding subunit.

Electrophoretic analysis of the estrogen receptor. Molybdate stabilization and identification of the classical estrogen receptor.

Conditions are defined which permit analysis of estrogen receptors from the mammalian uterus by polyacrylamide gel electrophoresis, thereby solving a longstanding problem encountered in previous attempts at such analysis, namely the failure of a large portion of the receptor population to enter such gels. A paramount requirement for entry of the estrogen-receptor complex into polyacrylamide gels is its maintenance in an untransformed state which does not form aggregates that are excluded from these gels. Of the multiple estrogen-binding proteins separated, only one (relative mobility of 0.5-0.6) possessed the definitive characteristics of the classical estrogen receptor. The inclusion of molybdate in extraction buffers selectively enhanced receptor recovery and facilitated its separation. Moreover, the estrogen-receptor complex so resolved is separated from other types of estrogen-binding proteins present in the uterine cytosol. These findings show that the molybdate-stabilized estrogen receptor exists in a single discrete form, but otherwise exhibits multiple forms that are probably artifactual. Electrophoresis in discontinuous buffers, but not in a continuous buffer system, promoted aggregate formation. This finding has implications concerning the subunit structure of the untransformed receptor.

Modulation by estrogen of synthesis of specific uterine proteins.

The contemporary procedure for high resolution two dimensional gel electrophoresis was extended to include an initial nondenaturing dimension of electrophoresis. Use of the resulting three dimensional procedure revealed that the previously described single peak of estrogen-induced protein in the uterus of the rat contains at least three distinct proteins whose rates of synthesis are regulated by estrogen. These proteins were localized within partial protein maps, thereby providing definitive operational definitions for the detection and identification of each. It was unambiguously demonstrated that each of the three proteins is continuously synthesized in control uteri. These findings cast doubt on the simplistic hypothesis that estrogen induces a single key protein that triggers a "cascade" of sequential transcriptional events in the uterus. Our finding that the major uterine protein induced by estrogen is also synthesized in liver and muscle cells is significant in that it points to a more general cellular function for the protein, rather than a unique role within uterine cells. Finally, our procedure for three dimensional gel electrophoresis opens new avenues for the detection of minor proteins in heterogeneous protein mixtures, such as those from the tissues of higher animals.

Xenopus liver: ontogeny of estrogen responsiveness.

Estradiol-17 beta stimulates the synthesis of numerous proteins exported into the culture medium by Xenopus tadpole liver tissue obtained after stage 50 and throughout metamorphosis to stage 66. Although estrogen-induced vitellogenin can be detected as early as stage 54, it is a minor percentage of the exported proteins until after the completion of metamorphosis. In hepatic tissue obtained after metamorphosis, the hormone evokes the synthesis of vitellogenin specifically without affecting the labeling of other secreted proteins.

Regulation by estrogen of the vitellogenin gene.

The vitellogenin gene is inactive in the liver of male Xenopus laevis, unless exogenous estrogen is administered. We have previously shown that conventional doses of estradiol-17beta result in the appearance of new hepatic messenger RNAs, some of which are encoded for vitellogenin. We now report that much higher doses of the hormone (2 mg/frog per day for 4 days) are required to elicit maximal responses. The relative levels of membrane-bound polysomes and vitellogenin mRNA were determined as a function of time and dose of hormone. Translation of total polysomal RNA in a cell-free system derived from wheat germ was used to estimate the relative levels of vitellogenin messenger RNA. Faithful translation of this messenger RNA was indicated by two lines of evidence: labeled cell-free products were immunoprecipitated with antivitellogenin antibody, and the migration of the major labeled product in sodium dodecyl sulfate/acrylamide gels was identical to that of native vitellogenin. Our results establish conditions for maximal estrogen-induced responses in this system, and are compatible with the hypothesis that a major regulatory mechanism of steroid hormones in the control of protein synthesis is that of gene activation and regulation of messenger RNA levels.

Effect of extradiol-17beta on the synthesis of specific uterine nonhistone chromosomal proteins.

The synthesis of specific nonhistone chromosomal proteins in the uterus of the ovariectomized rat was examined as a function of time after treatment with estradiol-17beta. Sequential stimulations in the rates of synthesis of at least five nonhistone chromosomal proteins having molecular weights of 96,000, 70,500, 29,400, 20,700, and 16,400, respectively, were observed. The rate of synthesis of the nonhistone chromosomal protein having a molecular weight of 70,500 was increased at 1 hr after hormone treatment. This was the first nonhistone chromosomal protein to be induced by estrogen, and its induction was blocked by pretreatment with actinomycin D. The data reported suggest but do not prove that this protein is the 4.5 S estrogen receptor, and that it is the induced nuclear acidic protein described earlier by Teng and Hamilton. The rates of synthesis of the nonhistone proteins with molecular weights of 96,000, 29,400, 20,700, and 16,400 were increased at 3,5,24, and 24 hr, respectively, after hormone treatment.

Translation of hormone-induced messenger RNA in amphibian oocytes: I. Induction by estrogen of messenger RNA encoded for vitellogenic protein in the liver of the male African clawed toad (Xenopus laevis).

Induction of the synthesis of the vitellogenic proteins, lipovitellin and phosvitin, in the liver of the male African clawed toad (Xenopus laevis) was investigated as a function of time after treatment with estradiol-17beta [1,3,5(10)-estratriene-3,17beta-diol]. The appearance of mRNAs encoded for lipovitellin and phosvitin in the cytoplasmic fraction of the liver was assayed by microinjections of hepatic mRNA preparation [either polyribosomes or poly(A)-rich RNA] into oocytes obtained from mature female toads. Oocytes were then incubated in the presence of radioactive amino acid(s) at 19 degrees for periods of time varying from 4 to 18 hr after microinjection. The results show that at 2 hr after hormone treatment more mRNA was present in the cytoplasm, and that from 2 to 72 hr after treatment the level of induced mRNA increased almost linearly to 110% above the control values. Experiments employing specific lipovitellin antiserum indicated no radioactive lipovitellin among the proteins synthesized in oocytes microinjected with hepatic mRNAs isolated from 3 to 9 hr after hormone treatment. However, a marked synthesis of immunoprecipitable, radioactive lipovitellin and an enhanced incorporation of [3H]serine occurred in the oocytes microinjected with hepatic mRNA preparations obtained from toads treated with hormone for 12 or more hr. The identities of the proteins encoded by the mRNAs induced early in estrogen action (2-9 hr) in the male amphibian liver are unknown. It is surmised that some of these proteins may function in the regulation of the subsequent synthesis of the vitellogenic proteins.

Early estrogen action. Stimulation of the synthesis of methylated ribosomal and transfer RNAs.

The effects of estrogen on the rates of incorporation in vivo of radioactive uridine and Me-methionine, administered together, into RNA in the uterus of the ovariectomized adult rat have been measured. The ratio of incorporation of methionine to uridine during a 45-min labeling period was increased several-fold by hormone treatment. The increased rate of methylation was apparent in the uterus taken from the rat administered estrogen for 1 h, and the effect was more striking following 2 and 3 h of hormone treatment. This stimulation of methylation of RNA occurred in association with an increase in the whole-organ concentration of RNA. Analysis of the doubly-labeled uterine RNA on sucrose gradients revealed that the methylated species were mainly ribosomal and transfer RNA. These results show that very little methylation of RNA occurs in the atrophied uterus of the ovariectomized rat. During the first 3 h following estrogen administration to the ovariectomized animal, an increasing percentage of the newly synthesized RNA formed by the uterus is methylated ribosomal and transfer RNA. This result is discussed in light of recent studies of the efficiency of processing of ribosomal precursor RNA, as well as the synthesis of high-molecular-weight heterogeneous RNA in the early action of estrogen.

The transformation of the cytoplasmic oestradiol-receptor complex into the nuclear complex in a uterine cell-free system.

Experiments performed with a cell-free system in tris-EDTA buffer, pH 7.4, indicate that the high-speed supernatant fraction of the rat uterus contains all the factors necessary to transform the 8S cytoplasmic oestradiol-receptor complex to the nuclear complex. The transformation is temperature-dependent. This nuclear complex was extracted in the form of a 5S particle with 0.4m-KCl from sediments of either uterine or heart nuclei that had been incubated together with the cytoplasmic soluble fraction of the uterus at 2 degrees C for 30min. This complex can also be obtained similarly from the soluble fraction of the uterus, incubated in the absence of nuclei. Previous warming of the soluble fraction to 37 degrees C for 7min was necessary for the successful extraction of the nuclear particle under these conditions of incubation. After an incubation of the transformed complex with the nuclear sediment at 37 degrees C for 7min, the 5S complex was extractable from the uterine nuclear sediment but not from the heart nuclear sediment, which may indicate the tissue specificity of the nuclear acceptor sites for the transformed complex. The extracted uterine nuclear complex sediments in the 5S region, but whether it is the native complex or a subunit or other part of the native complex resulting from the extraction with salt is unknown.

Early estrogen action: stimulation of the metabolism of high molecular weight and ribosomal RNAs (estradiol-17 -RNA isolation-uterus-ribosomal RNA precursors-actinomycin D).

Samples of RNA, isolated from uteri of ovariectomized adult rats treated with estrogen, have been analyzed on sucrose gradients. Treatment with estrogen either for 20 min or 2 hr increased the specific activity of all classes of uterine RNA, but produced no significant alteration in the distribution of radioactivity in the gradients, when animals received [(3)H]uridine intraperitoneally 15 min before they were killed. After labeling periods of 30 min, 1 hr, or 2 hr, however, the RNAs isolated from animals treated with estrogen had a smaller percentage of rapidly sedimenting (faster than 28S) species of RNA than did RNA from animals not treated with the hormone. The decreased percentage of high molecular weight RNA correlated with increases in both the specific activity of 28S and 18S RNA and the concentration of RNA in the whole organ. The labeled RNA of high molecular weight was also demonstrated, by the use of actinomycin D in vivo, to have a more rapid turnover rate in the estrogen-stimulated uterus. Our results indicate that estrogen increases not only the rate of synthesis of ribosomal RNA in the uterus of the ovariectomized adult rat, but also the rate or efficiency of processing of precursor RNA species of high molecular weight.