An estrogen-dependent polysomal protein binds to the 5'' untranslated region of the chicken vitellogenin mRNA.

An estrogen-dependent protein present in chicken liver polysomes binds to the 5' untranslated region of the chicken vitellogenin II mRNA. Competition binding assays with different RNAs indicate that the binding of the polysomal protein to this region is sequence specific. Of the tissues tested, this RNA binding activity is liver specific. In vivo kinetics of appearance of the binding activity following a single injection of estrogen to immature chicks are similar to the rate of accumulation of vitellogenin mRNA. The molecular weight of the polysomal protein has been estimated to be 66,000 on the basis of UV crosslinking and subsequent SDS polyacrylamide gel electrophoresis. In vitro RNA decay assays carried out with a minivitellogenin mRNA suggest that the estrogen-dependent polysomal protein may be involved in the estrogen-mediated stabilization of the chicken vitellogenin II mRNA.     

Estrogen-dependent expression of the chicken very low density apolipoprotein II gene in serum-free cultures of LMH cells

Summary The estrogen-responsive Leghorn strain M chicken hepatoma (LMH) cell line provides a model system for studying the estrogen-dependent, liver-specific expression of avian genes. Serum-free culture conditions have been established that allow expression of apolipoprotein B, very low density apolipoprotein II (apoVLDLII), serum albumin, and transferrin at levels detectable by Northern blot analysis. Regulation of apoVLDLII mRNA by estrogen occurred in an appropriate time-and dose-dependent manner in serum-free cultures of the LMH cells. The expression of apoVLDLII mRNA in serum-free culture was at least 100-fold higher than that expressed in cultures containing 10% serum. The level of estrogen receptors in LMH cells cultured with 10% serum was approximately 2000 receptors per cell, and in serum-free culture approximately 1000 receptors per cell. When these cells were transfected with estrogen receptor DNA and cultured in serum-free medium, apoVLDLII mRNA was decreased relative to that expressed in cells transfected with a control plasmid. These results indicate that when the LMH cells are cultured without serum, estrogen receptors are not the limiting factor for the expression of the apoVLDLII gene.     

Long-term effects of estrogen on avian liver: estrogen-inducible switch in expression of nuclear, hormone-binding proteins.

The stimulation of chicks or embryos with estrogen results in transient, hepatic expression of the vitellogenin gene, as well as long-term, propagatable alterations in the rapidity with which the gene can be reactivated. We examined the possibility that nuclear, type II estrogen-binding sites are involved in this long-term change in response characteristics. We demonstrate that the primary induction kinetics of type II sites in embryos and chicks correlated with the expression of the vitellogenin gene and that once their induction was triggered by estrogen, they accumulated, were propagated, and persisted for months after withdrawal of the hormone. We also show that their accumulation in the embryo was accompanied by prolonged expression of both the vitellogenin and very low-density apolipoprotein II genes, in the absence of elevated levels of type I receptor, and that the type II sites, like the classical receptor, appear to be preferentially associated with active or potentially active chromatin. Finally, we describe a regulatory mechanism, tested by computer modelling, that simulated the behavioral characteristics of these nuclear estrogen-binding sites and which may explain their role in mediating the long-term effects of estrogen.     

An estrogen-inducible protein binds specifically to a sequence in the 3'' untranslated region of estrogen-stabilized vitellogenin mRNA.

The 3' untranslated region (3'-UTR) has been implicated in the estrogen stabilization of hepatic Xenopus laevis vitellogenin mRNA. We used RNA gel mobility shift assays to demonstrate that Xenopus liver contains a factor which binds with very high specificity to a segment of the 3'-UTR of vitellogenin B1 and B2 mRNAs. We detected a single high-affinity binding site in the vitellogenin mRNA 3'-UTR and localized the binding site to a 27-nucleotide region. Since binding was abolished by proteinase K digestion, at least a component of the factor is a protein. Following estrogen administration, binding was induced approximately four- to fivefold in extracts from liver polysomes. The hepatic vitellogenin mRNA-binding protein was found in both polysomes and cytosol. Since the protein was also estrogen inducible in cytosol, this represents a genuine induction, not simply recruitment of the cytosolic protein into polysomes. UV cross-linking studies with the 27-nucleotide recognition sequence revealed bands corresponding to bound proteins with apparent molecular weights of 71,000 and 141,000. This appears to be the first example of steroid hormone-inducible proteins binding to an mRNA 3'-UTR. Its induction by estrogen and its sequence-specific binding to a region of vitellogenin mRNA important in estrogen-mediated stabilization suggest that the protein may play a role in the regulation of mRNA stability.     

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.     

Detection and characterization of degradative intermediates of avian apo very low density lipoprotein II mRNA present in estrogen-treated birds and following destabilization by hormone withdrawal

The apo very low density lipoprotein II (apoVLDLII) gene is dormant in embryos, chicks, and roosters but can be activated by estrogen. ApoVLDLII mRNA is relatively stable in estrogen-treated birds. However, its stability decreases 4-5-fold following withdrawal of hormone. We have characterized degradative intermediates of apoVLDLII mRNA detected in liver total RNA from estrogen-treated birds and searched for alterations in the pattern of intermediates that occur upon hormone-withdrawal. Primer extension and S1 nuclease analyses have demonstrated that these intermediates consist of fragments of the molecule with intact 5' ends but which lack various 3' regions. Estrogen withdrawal results in a decrease in the steady state levels of several of these intermediates and the detection of two new species. The end points of the major fragments present in RNA from both estrogen-treated and withdrawn birds all map in, or within four nucleotides of, the tetranucleotide, GAUG. The two fragments detected only in RNA from withdrawn birds have 3' ends that immediately precede the sequence, CAGU. Based on secondary structures predicted by a global folding program, the end points also appear to be preferentially located in, or at the base of, internal "bulge-loops".     

Regulation of reproduction- and biomarker-related gene expression by sex steroids in the livers and ovaries of adult female western mosquitofish (Gambusia affinis)

To assess the adverse toxicological effects of steroid hormones on western mosquitofish (Gambusia affinis), 180 adult females were exposed to individual or binary combinations of progesterone (1μg/L), testosterone (1μg/L) and 17β-estradiol (1μg/L) for eight days. The expression patterns of vitellogenin, estrogen receptor, androgen receptor, metallothionein, and cytochrome P450 1A genes in mosquitofish varied according to tissue as well as the specificity of steroids. Treatment by progesterone or testosterone alone inhibited target gene expression in the livers. The expression levels of both vitellogenin A and vitellogenin B mRNAs were up-regulated by17β-estradiol, and a parallel induction of estrogen receptor α mRNA expression was also observed in the livers. In addition, 17β-estradiol treatment alone suppressed androgen receptor α, metallothionein and cytochrome P450 1A mRNA expression in the livers. In general, multiple hormone treatments had different effects on target gene expression compared with corresponding hormone alone. The results demonstrate that steroid hormones cause multiple biological responses including the expression of vitellogenin, estrogen receptor and androgen receptor mRNA in the hormone signaling pathways and the expression of metallothionein and cytochrome P450 1A mRNA in the xenobiotic signaling pathway.