A significant lag in the induction of ovalbumin messenger RNA by steroid hormones: a receptor translocation hypothesis.

Although ovalbumin and conalbumin mRNA accumulate in the same tubular gland cells of the chick oviduct in response to estrogen or progesterone treatment, the kinetics of induction are markedly different. Conalbumin mRNA begins to accumulate within 30 min after estrogen administration, whereas there is a lag of approximately 3 hr before ovalbumin mRNA begins to accumulate, as measured by three independent assays. The kinetics of estrogen-receptor binding to chromatin indicate that these sites are saturated within 15 min of estrogen administration to the chicks, demonstrating that the lag is not due to slow uptake of the steroid. Suboptimal doses of estrogen produce the same lag, but the resultant rate of ovalbumin mRNA accumulation is lower than with an optimal dose. Partial induction of ovalbumin mRNA by a low dose of estrogen does not shorten the lag with an optimal dose. With progesteone, there is a lag of about 2 hr before either ovalbumin or conalbumin mRNA begins to accumulate. Treatment of chicks with hydroxyurea shortens the lag for ovalbumin induction with either hormone. Inhibition of protein synthesis with emetine does not prevent the accumulation of either ovalbumin or conalbumin mRNA. With cycloheximide, however, ovalbumin mRNA accumulation can be prevented. The existence of a lag suggests that there are intermediate steps between the binding of steroid receptors to chromatin and the induction of ovalbumin mRNA. There are basically two models to explain these delays in response: one involving the accumulation of an essential intermediate, and the other involving a rate-limiting translocation of steroid receptors from initial nonproductive chromatin-binding sites to productive sites. Several aspects of the kinetics of ovalbumin mRNA induction are more consistent with the latter model.     

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The administration of either progesterone or estrogen to withdrawn chicks several hours after a first dose of estrogen affected ovalbumin synthesis differently than its mRNA levels [S. S. Seaver (1981) J. steroid Biochem. 14,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.     

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.     

Estrogen Receptor Is Not Primarily Responsible for Altered Responsiveness of Ovalbumin mRNA Induction in the Oviduct From Genetically Selected High- and Low-Albumen Chicken Lines

The role of estrogen receptor on ovalbumin mRNA induction by steroid hormones was investigated in primary cultures of oviduct cells from estrogen-stimulated immature chicks of genetically selected high- and low-albumen egg laying lines (H- and L-lines). In experiment 1,the extent of ovalbumin mRNA induction and changes in estrogen and progesterone receptors were compared between the oviduct cells from H- and L-lines with or without steroid hormones in the culture medium. In experiment 2, the effect of estrogen receptor gene transfection on the induction of ovalbumin mRNA was studied in the oviduct cells from the L-line chicks. The results showed a close correlation of the changes in ovalbumin mRNA with the numbers of nuclear and total estrogen receptors in the oviduct cells but not with the numbers of nuclear and total progesterone receptors. Estrogen receptor gene transfection induced ovalbumin mRNA to a moderate extent in the absence of the steroid hormones. To our surprise, however, estrogen receptor gene transfection apparently suppressed the ovalbumin mRNA responsiveness to estrogen to a considerable extent. It was concluded, therefore, that the extent of estrogen receptor expression might not be primarily responsible for the differences in responsiveness to steroid hormones of oviduct cells from genetically selected H- and L-line chickens.     

Stability of histone mRNAs is related to their location in polysomes

Synthesis of histone mRNAs is closely coupled to DNA synthesis. Following inhibition of DNA synthesis in L6 myoblasts with cytosine arabinoside, a coordinate and exaggerated rate of degradation of histone mRNAs occurs while other mRNAs, encoding ribosomal protein L32 and actin, are unaffected. Inhibition of protein synthesis by puromycin, emetine, or cycloheximide stabilizes histone mRNAs and results in their accumulation. When inhibition of DNA synthesis was followed immediately by inhibition of protein synthesis, the exaggerated rate of decay of the existing subspecies of histone H4 mRNAs was prevented and histone mRNA accumulated. If inhibition of protein synthesis was delayed longer than 3 minutes following inhibition of DNA synthesis, the ability to accumulate H4 mRNAs was lost. Furthermore, new protein synthesis was required to activate the mechanism which specifically destabilized histone mRNA. Puromycin was able to prevent the exaggerated rate of degradation of the various subspecies of H4 mRNA when added up to 15 min after inhibition of DNA synthesis, whereas emetine was effective only when added up to 5 min following inhibition of DNA synthesis. These data suggest that histone H4 mRNAs in polysomes are better targets than those released from polysomes for the specific mechanism which destabilizes histone mRNAs upon inhibition of DNA synthesis.     

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.     

Commitment of chick oviduct tubular gland cells to produce ovalbumin mRNA during hormonal withdrawal and restimulation

Acute withdrawal of estrogen from chicks leads to a precipitous decline in egg white protein synthesis and egg white mRNAs in the oviduct. In this paper we explore the biochemical basis of this phenomenon as well as the capacity of the "withdrawn" tubular gland cells to be restimulated with steroid hormones. During withdrawal, the decline in ovalbumin mRNA was closely correlated with the decline in nuclear estrogen receptors. Within 2-3 d of estrogen removal a withdrawn state was established and then maintained, as defined by a 1,000-fold-lower level of ovalbumin mRNA and a 20-fold-lower level of nuclear estrogen receptors, relative to the estrogen-stimulated state. The number of active forms I and II RNA polymerases declined by 50% during this time. Histological examination of oviduct sections and cell suspensions, combined with measurements of DNA content, revealed that tubular gland cells persisted as a constant proportion of the cell population for 3 d after estrogen removal. Despite a 1,000-fold decrease in the content of ovalbumin mRNA, the ovalbumin gene remained preferentially sensitive to digestion by DNase I. When 3-d-withdrawn oviducts were restimulated with either estrogen or progesterone, in situ hybridization revealed that greater than or equal to 98% of the tubular gland cells contained ovalbumin mRNA. Induction by a suboptimal concentration of estrogen was correlated with a lower concentration of ovalbumin mRNA in all cells rather than fewer responsive cells.     

Steroid hormones induce cell proliferation and specific protein synthesis in primary chick oviduct cultures

A rapid method to obtain large amounts of tubular gland cells from chick oviduct was developed. Combined collagenase and trypsin treatment allowed within 1.5 h complete dissociation of the magnum portion of the oviduct. By differential attachment of cells, fibroblasts were separated from tubular gland- and ciliated cells. Tubular gland cells attached within 18 h to plastic Petri dishes, had large secretory granules and grew very actively. The responsiveness of cells to hormones and/or antihormone was tested by measurement of cell proliferation and specific protein synthesis. After 7 days of culture in the presence of estradiol (50 nM) or progesterone (100 nM), cell growth was increased by approximately 50 and 35% respectively. Tamoxifen (100 nM) inhibited the estradiol induced growth stimulation, but had also negative effects of its own. The anti-progesterone (in mammals) RU 486, inactive per se, did not antagonize progesterone induced growth. Ovalbumin- and conalbumin synthesis after 4-5 days of cultures under different hormonal conditions was assessed after immunoprecipitation of newly synthesized [35S]methionine labelled proteins. In the presence of estradiol (50 and 100 nM), progesterone (50 nM), and both estradiol and progesterone together (50 nM of each), ovalbumin and conalbumin synthesis was increased, when compared to control cultures without hormones, or to oviduct fibroblasts. Hormonal stimulation of ovalbumin synthesis was also shown in cell supernatant and culture medium after gel electrophoresis.     

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.     

Improvements in immunoprecipitation of specific messenger RNA. Isolation of highly purified conalbumin mRNA in high yield

We have described previously procedures for the isolation of specific mRNA employing immunoprecipitation of polysomes. In spite of our success with ovalbumin mRNA in the chicken oviduct, we have had considerable difficulties in applying these same published techniques to the immunopurification of conalbumin mRNA, despite the fact that the chicken oviduct synthesizes up to 10% of protein as conalbumin. Here we describe a number of modifications and refinements which have proved essential in obtaining intact conalbumin mRNA in high purity and high yields. These refinements include: (a) improved purification of conalbumin in order to remove contaminating proteins that result in impure antibodies; (b) improved isolation of specific conalbumin antibody in high yields; (c) improved methods for reducing contamination by non-specific polysomes; (d) improved techniques for isolation of RNA from immunoprecipitates resulting in less degradation and higher recovery of conalbumin mRNA; (E) improved techniques for efficient translation of conalbumin mRNA involving treatment of the RNA with methylmercury prior to translation. We conclude that problems involved in the immunoprecipitation of different mRNAs may differ, and that various refinements in techniques may be required for obtaining highly purified preparations of intact mRNA in high yields.     

Role of protein synthesis in the activation of cytotoxic mouse macrophages by lymphokines

The role of protein synthesis during the activation of macrophages (M phi) by lymphokines (LK) was studied. Peritoneal murine macrophages elicited by proteose-peptone (pM phi) were activated with LK (supernatants from normal mouse spleen cells pulsed with concanavalin A) and tested for cytotoxicity in an 18 hr assay against 111In-labeled L5178Y lymphoma target cells. Reversible (cycloheximide and puromycin) or poorly reversible (emetine and pactamycin) inhibitors of protein synthesis were added during activation, and their effects on pM phi-mediated cytotoxicity and pM phi protein synthesis were measured. Minimal concentrations of inhibitors, reducing the rate of protein synthesis by more than 90% without toxic effects on macrophages, were chosen. Exposure of pM phi to LK for 2 to 18 hr in the presence of reversible inhibitors of protein synthesis did not affect the induction of cytolytic activity, indicating that protein synthesis was not required during the activation period. In contrast, activation of macrophages for 2 hr in the presence of poorly reversible inhibitors of protein synthesis resulted in a considerable reduction of cytolytic activity. The impairment of cytotoxic activity was also evident when pM phi were treated with such drugs during the first 2 hr of an 18 hr exposure to LK or when LK-activated macrophages were treated for 2 hr with the drugs before the addition of the targets. These results demonstrate that active protein synthesis is not required during the exposure of pM phi to LK, but that new proteins have to be synthesized to allow the expression of the cytotoxic activity in LK-activated pM phi.     

Detection of inhibitors of protein and nucleic acid synthesis using oocytes of Xenopus laevis microinjected with the herpes thymidine kinase gene

We have developed an assay that measures the inhibition of protein synthesis and can be used in conjunction with a whole embryo bioassay that detects the ability of a chemical to cause fetotoxicity, malformation and abnormal growth. The assay involves microinjecting the herpes thymidine kinase gene into stage 6 oocytes of Xenopus laevis then exposing the oocytes to a test compound for 18-24 h. The inhibition of thymidine kinase (TK) expression caused by an inhibitor is then measured by simple enzyme assay. Protein synthesis inhibitors such as cycloheximide, puromycin and emetine all inhibited TK synthesis. Concentrations of cycloheximide (1.4 X 10(-4) mg/ml) and puromycin (0.04 mg/ml) near the 96 h embryo LC50 inhibited thymidine kinase expression by 78% and 97%, respectively but emetine (0.01 mg/ml) had no effect. However, 0.1 mg/ml emetine inhibited TK synthesis by almost 50%. The RNA synthesis inhibitor, actinomycin D (0.013 mg/ml) inhibited TK expression by 61%. DNA synthesis inhibitors hydroxyurea (2.0 mg/ml), cytosine arabinoside (2.0 mg/ml) and ethidium bromide (0.02 mg/ml) failed to inhibit the expression of the TK gene even though these concentrations were near the 96 h embryo LC50. The whole embryo bioassay cannot differentiate the DNA synthesis inhibitors from the RNA and protein synthesis inhibitors but the oocyte assay can. This type of molecular test data can help separate classes of teratogens such as DNA synthesis inhibitors from nonteratogenic compounds such as protein synthesis inhibitors and allow the extrapolation of test data to other species.