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 In this paper we explore the biochemical basis of this phenomenon as well as the capacity of the "withdrawn" tubular gland cells to be restimulated with steroid hormones.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 Within 2-3 d of estrogen removal a withdrawn state was established and then maintained, as defined by a 1,000-fold-lower level of ovalbumin mRNA and a 20-fold-lower level of nuclear estrogen receptors,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the content of ovalbumin mRNA, the ovalbumin gene remained preferentially sensitive to digestion by DNase I. When 3-d-withdrawn oviducts were restimulated with either estrogen or progesterone, in situ hybridization revealed that greater than or equal to 98% of the tubular gland cells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.     

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.     

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.     

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.     

In situ hybridization of ovalbumin mRNA in the chick oviduct reveals target cell specificity for estrogen and progesterone

An in situ hybridization method using paraffin-embedded sections was used to characterize the chicken oviduct cells synthesizing ovalbumin mRNA due to the action of estrogen and progesterne. The cytodifferentiation of the oviduct cells was induced by 17β-estradiol administration to newly hatched female chicks. To avoid possible effect of estrogen on the action of progesterone the chicks were withdrawn from the estrogen by six days withdrawal period without hormone treatment. Ovalbumin mRNA was not synthesized after a period of estrogen withdrawal. Administration of estrogen induced ovalbumin mRNA in the tubular gland cells. Administration of progesterone induced the expression of ovalbumin mRNA in the surface epithelial cells. It was also found that progesterone induced mucus producing goblet cells in the surface epithelium. Estrogen did not have an effect on the mucus production, which suggests that progesterone could induce the terminal differentiation of the goblet cells. We conclude that the expression of ovalbumin in the surface epithelial cells and in the tubular gland cells is specific for progesterone and estrogen, respectively.     

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.     

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.     

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.     

Retention of estrogen receptors in vitro requires limited estradiol exposure in vivo

Maintenance of functional estrogen receptors in culture has been accomplished in chick oviduct cells by manipulating the estrogen exposure before tissue dissociation. Tissue from chicks pre-treated with daily 17-beta-estradiol injections for 2 weeks or with 2 weekly diethylstilbestrol implants can be established in culture using a variety of enzymes. Tissue from animals with chronic estrogen stimulation must be withdrawn from hormone in culture at least 4 days before the digestion procedure. When tissue is digested using collagenase and pancreatin buffered by bovine serum albumin (Fraction V), large quantities of virtually fibroblast-free cultures can be established. The estrogen and progesterone receptors remain intact at normal levels using this procedure. The receptors have maintained biological function as evidenced by two hormone-dependent measurements. The first was an increase in the amount of ovalbumin mRNA transcribed in response to estrogen supplementation of the cultures compared to cultures with no estrogen. The second function was an increase in ovalbumin protein secreted into the medium upon estrogen stimulation. The protein increment demonstrated that the hormone-induced levels of mRNA were functional and capable of being translated.     

Inhibition by protease inhibitors of binding of adrenal and sex steroid hormones

Binding of steroid hormones is inhibited by protease inhibitors and substrates. The protease inhibitors phenylmethyl sulphonylfluoride, tosyl-lysine chloromethyl ketone, and tosylamide-phenylethyl-chloromethyl ketone and the protease substrates tosyl arginine methyl ester and tryptophan methyl ester eliminate specific binding of aldosterone, dexamethasone, dihydrotestosterone, estrogen, and progesterone to their respective receptors. These protease inhibitors and substrates also inhibit binding of progesterone to the 20,000 molecular weight mero-receptor formed from the progesterone receptor in chick oviduct. The binding of estradiol to rat alpha-fetoprotein is inhibited by the protease inhibitors and substrates but not by tryptophan or tryptophan amide, indicating the importance of an ester structure in the inhibition of steroid binding. Our results suggest that all steroid hormone receptors have a site with both common structural features and a role in the regulation of steroid hormone binding.