Bert W. O'Malley

Estrogenic Induction of Ornithine Decarboxylase in vivo and in vitro

Injection of estrogens (17β-estradiol or diethylstilbestrol) into immature chicks results in a marked (30- to 50-fold) increase in the ornithine decarboxylase activity of oviductal homogenates within a 4-hour period. Similar stimulations were obtained when estrogen was injected into hypophysectomized or castrated rats and the uterus was examined for decarboxylase activity. An elevation of decarboxylase activity was obtained in vitro when oviducts from immature chicks were incubated in the presence of estrogen. These data indicate a direct action of estrogen on oviduct tissue to promote a rapid increase in the activity of a specific enzyme and represent the first example of a completely in vitro enzyme response to estrogen.

Chromatin of the developing chick oviduct: Changes in the acidic proteins

Abstract 1. The chromatin of the oviducts of immature chicks undergoing estrogen-induced differentiation was studied. Both the species and quantitative level of the histones remained relatively unaltered throughout development. In contrast, the level of the acidic proteins displayed a biphasic pattern, increasing during the first few days of differentiation followed by a decrease during the final stages of maturation. The level of chromatin-associated RNA and the capacities of chromatin to serve as template in in vitro DNA-dependent RNA synthesis showed a similar biphasic pattern. 2. Fractionation of the acidic chromatin proteins into four groups followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated a marked heterogeneity of the acidic chromatin proteins. The majority of protein bands were associated with two of the four groups (AP1 and AP2). Quantitative analysis revealed that variations primarily in the level of one fraction (AP2) were responsible for the changes observed in the total chromatin acidic proteins. Amino acid composition, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunochemical analysis demonstrated that alterations in the molecular species of acidic proteins occurred during development. Using circular dichroism to probe the secondary structure of whole and dehistonized chromatin, a gradual conformational change was observed in both the DNA and protein components during oviduct development. 3. The observed changes in chromatin structure and composition are suspected to be involved in the changing pattern of gene expression during development of the chick oviduct.

Estrogen-induced synthesis of ovalbumin messenger RNA and its translation in a cell-free system

Abstract RNA extracted from estrogen-stimulated oviduct is demonstrated to code for synthesis of 14C-ovalbumin in a cell-free rabbit reticulocyte lysate system. Ovalbumin mRNA is concentrated in an 8–17S fraction isolated from polysomes. Oviduct rRNA or chick liver RNA does not direct ovalbumin synthesis. Hen oviduct contains large quantities of ovalbumin mRNA. Undetectable amounts are present in oviducts of immature chicks but ovalbumin mRNA can be induced in immature animals by estrogen. Finally, hormone withdrawal results in disappearance of ovalbumin mRNA. Readministration of estrogen results in a marked accumulation of ovalbumin mRNA activity. This report constitutes the initial documentation of the hormone-induced synthesis of a specific mRNA and its translation in a heterologous system.

Unified hypothesis for early biochemical sequence of events in steroid hormone action.

Abstract Recent studies using the chick oviduct, a tissue in which progesterone induces synthesis of the protein avidin, have led us to formulate the following sequence of events in progesterone action: steroid permeation of the cell membrane, binding of progesterone to a specific cytoplasmic receptor, transfer of this steroid-receptor complex to the nucleus, binding of the steroid-receptor complex to specific “acceptor sites” on the nuclear chromatin, synthesis of new RNA specific in composition, and synthesis of avidin. These results are discussed in light of evolving experimental information which can now allow us to consider this sequence of events as applicable to the mechanism of action of all steroid hormones.

Changes in chromatin composition and hormone binding during chick oviduct development

Abstract 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.

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

Summary 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.

Mechanism of estrogen action: Early transcriptional and translational events

Abstract This article presents a review of studies concerned with the mechanism by which estrogens stimulate RNA and protein synthesis in target organs. Two model systems are discussed in detail: the rat uterus and the chick oviduct. It appears that the sequence of events is similar in both organs. Based on available evidence, estrogen is bound to a cytoplasmic receptor protein immediately upon entering the cell. This complex subsequently moves into the nucleus where it apparently interacts with specific nonhistone proteins of the chromatin material. This initial interaction results in synthesis of new RNA molecules. These new RNAs are evidently required in the cytoplasm to allow the synthesis of a select complement of new proteins. Some of these proteins may feed back upon the nucleus thus activating the general gene amplification response that results in the overall growth and differentiation of the target organ.

Progesterone receptor components: Identification of subunits binding to the target-cell genome☆

Abstract The chick oviduct cytoplasmic progesterone receptor has been shown to consist of two 4S components, A and B, by ehromatography on DEAE-cellulose. Both A and B are high-affinity, specific progesterone-binding proteins which cannot be interconverted or reeombined. Both appear bound in isolated oviduct nuclei in vitro . The two components differ in their affinity for DNA and chromatin. Component A binds only to pure DNA whereas component B binds only to oviduct chromatin.

Isolation of protein factors from oviduct polysomes which stimulate protein synthesis

Abstract A soluble protein fraction (AvF) was isolated from oviduct polysomes. AvF stimulates by 6–10 fold both rate and extent of protein synthesis on washed ribosomes directed by either natural mRNA or by polyuridylic acid. AvF is inactivated by heat, pronase and N-ethylmaleimide but not by RNase. AvF is distinct from chain elongation factors T1 and T2. The fraction stimulates protein synthesis subsequent to acylation of tRNA since equal stimulations are observed using 14C-val or 14C-leu-tRNA. Addition of AvF results in a distinct lowering of Mg+2 concentration for optimal protein synthesis using natural message or poly U. Finally analysis by affinity chromatography of peptides synthesized in the AvF-dependent natural message system suggests that ovalbumin comprises 14% of the released proteins.

A specific oviduct target-tissue receptor for progesterone: identification characterization partial purification inter-compartmental transfer kinetics and specific interaction with the genome.

Researchers at the Vanderbilt University School of Medicine have used the chick oviduct in many of their efforts to understand the mechanism of steroid hormone action. In this paper they describe their efforts to establish the existence and characteristics of a specific receptor for progesterone in target cells and to relate the initial hormone-receptor interactions to subsequent alterations in RNA synthesis induced in the oviduct cell nucleus. Areas treated include progesterone binding to macromolecules following in vivo administration characterization of macromolecular-progesterone complex formed by oviduct cytosol in vitro partial purification of cytoplasmic complex evidence that binding components mediate progesterone action transfer of steroid-receptor complex from cytoplasm to nucleus and receptor interaction with the genome. OMalley leads a discussion further considering the nature of the progesterone receptor.