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The administration of either progesterone or estrogen to withdrawn chicks several hours after a first dose of estrogen affected ovalbumin synthesis differently than its mRNA levels [S. S. Seaver (1981) J. steroid Biochem. 14,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-like effects of combined dexamethasone and tamoxifen in the chick oviduct

The effects of dexamethasone alone on withdrawn chick oviduct weight, DNA, protein content and progesterone receptor concentration were barely detectable, whereas ovalbumin and conalbumin synthesis were increased. When dexamethasone and tamoxifen were combined, a marked increase of total proteins, including egg white proteins, DNA and wet weight occurred. Progesterone receptor also was increased. The most striking result was the stimulation of DNA polymerase-α activity by combined dexamethasone and tamoxifen, whereas either compound was completely ineffective.     

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.     

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.     

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.