Effects of serial hormone treatments on egg white protein synthesis. Further evidence of translational regulation

The administration of either progesterone or estrogen to withdrawn chicks several hours after a first dose of estrogen affected ovalbumin synthesis differently than its mRNA levels [S. S. Seaver (1981) J. steroid Biochem. 14, 949-957]. This suggested that the hormones were regulating the translation of ovalbumin directly. In this paper we report that serial hormone treatments also affect the rates of synthesis of two other egg white proteins, conalbumin and ovomucoid. When progesterone was administered 4 h after estrogen, conalbumin synthesis decreased. When either progesterone or a second dose of estrogen was administered 12 h after the first dose of estrogen, conalbumin synthesis increased. Serial hormone treatments did not always affect all three proteins similarly. At later times, administering progesterone after estrogen decreased ovomucoid synthesis but did not affect conalbumin or ovalbumin synthesis. To determine if the serial hormone treatments affect egg white protein mRNA's in a similar way, changes in ovalbumin and conalbumin mRNA levels were quantified in a rabbit reticulocyte cell-free translation system and were compared to changes in ovalbumin and conalbumin synthesis as measured in chick oviduct tissue minces. When serial hormone treatments were 12 h apart, ovalbumin and conalbumin synthesis was 50-300% higher than that predicted by the changes in ovalbumin or conalbumin mRNA levels. This is further evidence that translation of both conalbumin mRNA and ovalbumin mRNA is directly regulated by steroid hormones.     

Isolation of a nuclear ribonucleoprotein fraction from chick oviduct containing ovalbumin messenger RNA sequences

A method was developed for the isolation of a ribonucleoprotein fraction from chick oviduct nuclei that contains 70% of the pulse-labeled RNA. These fractions also contain about 1% of the nuclear DNA and have an average RNA to DNA ratio of about 4:1. The major nuclear RNP proteins of 32,000 M_r are present along with many additional proteins including histories. However, polysomal proteins and major oviduct cytoplasmic proteins are absent. Nuclei from fully stimulated chick oviduct contain about 3000 copies of ovalbumin messenger RNA sequences of which about 200 are in the RNP complexes: these complexes have sedimentation coefficients of 30 to 350 S and are resistant to disruption by EDTA.The level of ovalbumin mRNA sequences in these complexes reflects the overall rate of synthesis of this RNA. Withdrawal of estrogen leads to a parallel decline of nuclear estrogen receptors and ovalbumin mRNA sequences in the RNP complexes and a subsequent loss of cytoplasmic ovalbumin mRNA about three hours later. The 300-fold decrease in the level of ovalbumin mRNA sequences in these complexes and the eightfold decrease in stability of cytoplasmic ovalbumin mRNA account for the 2500-fold decrease in the level of cytoplasmic ovalbumin mRNA observed during withdrawal. Upon stimulation with estrogen, the kinetics of reappearance of ovalbumin mRNA sequences in the RNP complexes apparently accounts for the accumulation of cytoplasmic ovalbumin mRNA. Thus the nuclear RNP has some of the properties expected of nascent RNP complexes.The levels of ovalbumin and conalbumin mRNA sequences increase in the nuclear RNP with markedly different kinetics: conalbumin mRNA sequences reach half maximum by 1.5 hours, whereas ovalbumin mRNA sequences in these complexes reach half maximum at about eight hours. In the analysis in the accompanying Appendix, we show that the immediate increase of conalbumin mRNA sequences in the nuclear RNP may be accounted for by interaction of the hormone receptor complex with a single regulatory site, whereas the delayed increase of ovalbumin mRNA sequences in the RNP may be due to a requirement for interaction of the hormone receptor complex with multiple regulatory sites.     

Effects of insulin-like growth factor-I,estrogen, glucocorticoid,and transferrin on the mRNA contents of ovalbumin and conalbumin in primary cultures of quail (Coturnix coturnix japonica) oviduct cells

The effects of estrogen, dexamethasone, insulin-like growth factor-I (IGF-I), and transferrin on the messenger RNA (mRNA) contents of ovalbumin and conalbumin in primary cultures of quail oviduct cells were investigated. In the absence of one of the above hormones or factors, a decrease in ovalbumin mRNA was prominent. In particular, removal of IGF-I and transferrin caused a significant effect. Studies using a combination of estrogen, dexamethasone, IGF-I and transferrin indicated that IGF-I cooperates with estrogen or dexamethasone and transferrin works with dexamethasone. Specifically, IGF-I enhanced ovalbumin synthesis or increased cellular ovalbumin mRNA content depending on its concentration in the medium in the presence of estrogen. However, the effects of estrogen, dexamethasone, IGF-I, and transferrin were not similarly observed with conalbumin mRNA. These results show that ovalbumin synthesis is controlled by estrogen or glucocorticoid with IGF-I or transferrin and that cellular ovalbumin mRNA content is also regulated by these hormones or transferrin. In contrast, conalbumin synthesis and cellular content of conalbumin mRNA are not affected by these hormones under the conditions of the present study.     

Metabolic formation of nucleoside-modified analogues of S-adenosylmethionine

A Pseudo-ovalbumin gene, bearing significant nucleotide sequence homology to the ovalbumin gene, has been cloned from genomic chick DNA. Similar to the authentic ovalbumin gene, the pseudo-gene is a unique sequence gene in the chick genome and is expressed at a low level in the immature chick oviduct. In contrast to the ovalbumin gene, expression of the pseudo-gene in the oviduct is not inducible by estrogen. The concentration of pseudo-gene RNA is only ～0.01% of that of authentic ovalbumin mRNA in estrogen-stimulated oviduct cells. Nucleotide sequence analysis of the two sequence related genes may reveal the molecular basis of differential response to steroid hormone induction in the same tissue.     

INTERACTION OF ESTROGEN AND PROGESTERONE IN CHICK OVIDUCT DEVELOPMENT : II. Effects of Estrogen and Progesterone on Tubular Gland Cell Function

The effects of estrogen and progesterone on the function of chick oviduct tubular gland cells have been studied. Such function, as measured by the increase in specific cell products such as lysozyme and ovalbumin, requires the continuous presence of estrogen or progesterone. Withdrawal of hormone results in a rapid cessation of function and an involution of the oviduct accompanied by rapid decreases in total weight, lysozyme, and RNA. During such involution, tubular gland cells per se persist, as evidenced by a lack of comparable decrease in total DNA content and by histological demonstration of tubular gland cells. When estrogen administration is reinstituted, preexisting tubular gland cells rapidly synthesize ovalbumin and lysozyme without requiring new DNA synthesis. Administration of progesterone also stimulates the function of such cells. Furthermore, the effects of estrogen and progesterone are synergistic on the synthesis of lysozyme and ovalbumin, whereas progesterone antagonizes the estrogen-evoked formation of tubular gland cells. It is suggested that such complex interactions of estrogen and progesterone on oviduct development and function result from differences in responsiveness of the various cell types present in the tissue.     

The induction of ovalbumin and conalbumin mRNA by estrogen and progesterone in chick oviduct explant cultures

Estrogen pretreated chick oviduct tissue can be restimulated in vitro by physiological concentrations of estrogen and progesterone. The rates of synthesis of the major egg white proteins, ovalbumin and conalbumin, as well as the cellular levels of their respective mRNAs, increase after characteristic lag periods; this confirms previously reported results in vivo and demonstrates that both the lag phenomena and the mRNA induction are a function of the direct interaction of steroids with oviduct cells.The antagonistic action of progesterone on an estrogen-mediated induction of conalbumin mRNA also occurs in vitro, and the kinetics of this response are examined. Progesterone terminates the estradiol-induced accumulation of conalbumin mRNA within 30 min after addition to the medium; progesterone alone or in combination with estrogen, however, is capable of inducing conalbumin mRNA after a 4 hr lag period. The temporary nature of this antagonism and the fact that it does not occur with ovalbumin induction indicate the complexity of the oviduct's response to steroids.The role of protein synthesis in the induction of both ovalbumin and conalbumin was examined by including protein synthesis inhibitors in the culture medium. Puromycin, cycloheximide, emetine, pactamycin and high salt all block the induction of both ovalbumin and conalbumin mRNA when added together with either estrogen or progesterone. The effect of puromycin is reversible. After the drug is removed from the medium, the mRNA accumulation begins with the same characteristic lag period seen when no inhibitors are added. When given 2 hr after estrogen, puromycin stops the accumulation of conalbumin mRNA within 30 min, whereas cycloheximide and emetine allow the mRNA to accumulate for another 2 hr before causing complete inhibition. There is no effect of protein synthesis inhibitors on the number of estrogen receptors localized in the nucleus. The data suggest a direct link between protein synthesis and the steroid-induced accumulation of specific mRNAs in this system.     

Expression of the avian c-erb B (EGF receptor) protooncogene during estrogen-promoted oviduct growth

Expression of cellular erb B protooncogene messenger RNAs has been analyzed in the oviducts of immature chicks during estrogen-promoted growth. Hybridization of oviduct total cellular RNA with viral-derived erb B oncogene probes demonstrated significant expression of c-erb B mRNA in oviduct cells of untreated chicks. Daily administration of estrogen (diethylstilbestrol) to chicks results in marked oviduct growth but did not appreciably affect expression levels of c-erb B messenger RNA in oviducts after 2, 4 or 6 days of treatment. Withdrawal of chicks from estrogen treatment resulted in termination of oviduct growth. However, c-erb B messenger RNAs were detectable in the nonproliferative tissue at 5 days after hormone withdrawal. Readministration of diethylstilbestrol, progesterone or diethylstilbestrol plus progesterone to hormone-withdrawn birds (secondary stimulation) also did not affect c-erb B messenger RNA levels in the oviduct. These results demonstrate significant expression of the cellular erb B (epidermal growth factor receptor) gene in the avian oviduct. However, EGF receptor messenger RNA synthesis is not modulated in the oviduct by steroid hormones.     

INTERACTION OF ESTROGEN AND PROGESTERONE IN CHICK OVIDUCT DEVELOPMENT : III. Tubular Gland Cell Cytodifferentiation

Administration of estrogen (E) to immature chicks triggers the cytodifferentiation of tubular gland cells in the magnum portion of the oviduct epithelium; these cells synthesize the major egg-white protein, ovalbumin. Electron microscopy and immunoprecipitation of ovalbumin from oviduct explants labeled with radioactive amino acids in tissue culture were used to follow and measure the degree of tubular gland cell cytodifferentiation. Ovalbumin is undetectable in the unstimulated chick oviduct and in oviducts of chicks treated with progesterone (P) for up to 5 days. Ovalbumin synthesis is first detected 24 hr after E administration, and by 5 days it accounts for 35% of the soluble protein being synthesized. Tubular gland cells begin to synthesize ovalbumin before gland formation which commences after 36 hr of E treatment. When E + P are administered together there is initially a synergistic effect on ovalbumin synthesis, however, after 2 days ovalbumin synthesis slows and by 5 days there is only 1/20th as much ovalbumin per magnum as in the E-treated controls. Whereas the magnum wet weight doubles about every 21 hr with E alone, growth stops after 3 days of E + P treatment. Histological and ultrastructural observations show that the partially differentiated tubular gland cells resulting from E + P treatment never invade the stroma and form definitive glands, as they would with E alone. Instead, these cells appear to transform into other cell types—some with cilia and some with unusual flocculent granules. We present a model of tubular gland cell cytodifferentiation and suggest that a distinct protodifferentiated stage exists. P appears to interfere with the normal transition from the protodifferentiated state to the mature tubular gland cell.     

The Relationship of the Estrogen Receptor to the Induction of Vitellogenin in Chicken and Xenopus Liver

The mechanisms involved in the regulation of gene expression in eukaryote cells, although an area of active research, are still largely unknown. This is at least partly due to the lack of good experimental model systems. One type of system which is being exploited with some considerable success is the induction of proteins by steroid hormones. Studies on the effects of estrogen and progesterone on the synthesis of the egg white proteins in the chick oviduct, for instance, have yielded substantial insight into both the regulation of protein synthesis by steroid hormones [1] and the arrangement of the DNA sequences coding for these proteins [2, 3].
The need for other good inducible systems clearly exists and the induction of vitellogenin, the precursor of the major egg yolk proteins, by estrogen in the livers of the chicken and frog ( Xenopus laevis ) is one that is attracting increasing interest. In common with the chick oviduct, large amounts of a specific protein are synthesised in response to a well defined hormonal stimulus. However, the induction of vitellogenin also has the advantage that the response is not complicated by the extensive hyperplasia that follows estrogen treatment in the chick oviduct [4, 5] and that vitellogenin may be induced in vitro [6–11].
The aims of this review are first to discuss recent data on the induction of vitellogenin and vitellogenin mRNA both in vivo and in vitro and then to relate this data to the properties of the estrogen receptor, present in chicken and Xenopus liver, which is thought to mediate the induction of vitellogenin by estrogen.     

INTERACTION OF ESTROGEN AND PROGESTERONE IN CHICK OVIDUCT DEVELOPMENT : I. Antagonistic Effect of Progesterone on Estrogen-Induced Proliferation and Differentiation of Tubular Gland Cells

Daily administration of estrogen to immature female chicks results in marked oviduct growth and appearance of characteristic tubular gland cells which contain lysozyme. Although a rapid increase in total DNA and RNA content begins within 24 hr, cell specific protein, lysozyme, is first detectable after 3 days of estrogen. Progesterone administered concomitantly with estrogen antagonizes the estrogen-induced tissue growth as well as appearance of tubular gland cells and their specific products, lysozyme and ovalbumin. When the initiation of progesterone administration is delayed for progressively longer periods (days) during estrogen treatment, proportionally greater growth occurs with more lysozyme and tubular gland cells after 5 days of total treatment. Progesterone does not inhibit the estrogen-stimulated increase in uptake of α-aminoisobutyric acid and water by oviduct occurring within 24 hr or the estrogen-induced increase in total lipid, phospholipid, and phosphoprotein content of serum. The above results of progesterone antagonism can best be explained by the hypothesis that progesterone inhibits the initial proliferation of cells which become tubular gland cells but does not antagonize the subsequent cytodifferentiation leading to the synthesis of lysozyme and ovalbumin once such cell proliferation has occurred.     

Non-parallel kinetics and the role of tissue-specific factors in the secretion of chicken ovalbumin and lysozyme from Xenopus oocytes

Ovalbumin and lysozyme made in Xenopus oocytes under the direction of injected chicken oviduct messenger RNA accumulate at different rates in the surrounding culture medium. Pulse-chase experiments confirm that the intrinsic rate of lysozyme secretion from oocytes is 12 times that of ovalbumin. This slower rate of ovalbumin export is maintained following injection of either diluted oviduct RNA or purified ovalbumin messenger, the latter having been obtained by hybridization to cloned ovalbumin complementary DNA. These results suggest that the differential rates of transport observed in oocytes are not the consequence of competition for amphibian or avian factors and show that oviduct-specific proteins are not required for ovalbumin secretion.