Estrogen induces tissue specific changes in the chromatin conformation of the vitellogenin genes in Xenopus.

Nuclei from male Xenopus liver were digested extensively with DNase I and the residual amount of the four vitellogenin genes measured by hybridization with a moderate excess of vitellogenin cDNA. The saturation value was about twofold lower in chromatin isolated from liver cells of estrogen treated than from untreated males or from erythrocytes. Analyzing the disappearance of several defined restriction fragments specific for the A1 and A2 vitellogenin genes, after limited digestion with DNase I, suggested that the entire A1 and A2 vitellogenin genes are about twofold more sensitive to DNase I in chromatin of hepatocytes isolated from estrogen treated than from untreated males. Using the same assay no change in the DNase I sensitivity of the two vitellogenin genes in erythrocyte chromatin was observed. Analysis of the beta 1-globin and an albumin gene demonstrated that the DNase I sensitivity of these genes in both cell types is not altered by estrogen. All these data indicate that estrogen stimulation results in an increased DNase I sensitivity specific for the vitellogenin genes in hepatocytes.     

The human estrogen receptor can regulate exogenous but not endogenous vitellogenin gene promoters in a Xenopus cell line.

Transfection of a human estrogen receptor cDNA expression vector (HEO) into cultured Xenopus kidney cells confers estrogen responsiveness to the recipient cells as demonstrated by the hormone dependent expression of co-transfected Xenopus vitellogenin-CAT chimeric genes. The estrogen stimulation of these vit-CAT genes is dependent upon the presence of the vitellogenin estrogen responsive element (ERE) in their 5' flanking region. Thus, functional human estrogen receptor (hER) can be synthesized in heterologous lower vertebrate cells and can act as a trans-acting regulatory factor that is necessary, together with estradiol, for the induction of the vit-CAT constructs in these cells. In addition, vitellogenin minigenes co-transfected with the HEO expression vector also respond to hormonal stimulation. Their induction is not higher than that of the vit-CAT chimeric genes. It suggests that in the Xenopus kidney cell line B 3.2, the structural parts of the vitellogenin minigenes do not play a role in the induction process. Furthermore, no stabilizing effect of estrogen on vitellogenin mRNA is observed in these cells. In contrast to the transfected genes, the endogenous chromosomal vitellogenin genes remain silent, demonstrating that in spite of the presence of the hER and the hormone, the conditions necessary for their activation are not fulfilled.     

Oogenesis in Xenopus laevis (Daudin)

Summary This study was designed to explore the relationship of estrogen, human chorionic gonadotropin (HCG), and food availability to endocytosis in developing oocytes. When estrogen alone is administered to an animal, large amounts of vitellogenin are synthesized by the liver and secreted into the circulatory system, where it accumulates. Under these conditions there is no evidence of endocytosis at the surface of the oocytes. Other studies have shown that following HCG injection into estrogen-treated animals, vitellogenin is removed from the circulation and the oocyte surface is highly contoured and displays endocytotic activity. Food deprivation has much the same effect on oocyte endocytosis as does estrogen. When animals are given HCG and subsequently starved for 20 days, developing oocytes show little endocytotic activity. We conclude that HCG acts to promote or stimulate endocytosis in developing oocytes while estrogen and/or starvation inhibits this process.Research sponsored by the Energy Research and Development Adiministration under contract with Union Carbide Corporation.Predoctoral fellow supported by Grant GM 1974 from the National Institute of General Medical Sciences, National Institutes of Health.     

Expression of endogenous and transfected apolipoprotein II and vitellogenin II genes in an estrogen responsive chicken liver cell line

A recently described chicken liver cell line, LMH, was characterized to evaluate responsiveness to estrogen. Expression of the endogenous apolipoprotein (apo) II gene was induced by 17 beta-estradiol when LMH cells were cultured with chicken serum. The response was low and yielded apoll mRNA at only 0.3% of the level seen in estrogenized rooster liver. Higher levels of apoll mRNA were achieved when LMH cells were transiently transfected with an expression plasmid for estrogen receptor. A transfected apoll gene was strongly expressed only when cotransfected with receptor. Expression of the endogenous vitellogenin (VTG) II gene was not detected. However, when cotransfected with a receptor expression plasmid, VTG II reporter plasmids were expressed in LMH cells in response to 17 beta-estradiol. These results suggest that estrogen responsiveness of LMH cells is limited by the availability of functional receptor. Low levels of estrogen receptor mRNA were detected in LMH cells, and receptor binding sites and mRNA were greatly increased following transient transfection with a receptor expression plasmid. Using this transient transfection protocol, several VTG II reporter plasmids were compared in LMH cells and chick embryo fibroblasts. A plasmid containing VTG II estrogen response elements linked to a heterologous promoter was regulated by estrogen in both cell types. In contrast, reporter plasmids containing the VTG II promoter were regulated by estrogen in LMH cells but were not expressed at all in chick embryo fibroblasts. These results suggest that regulation of the VTG II gene involves cell type-specific elements in addition to estrogen response elements.(ABSTRACT TRUNCATED AT 250 WORDS)     

Estradiol and estrogen receptor-dependent stabilization of a minivitellogenin mRNA lacking 5,100 nucleotides of coding sequence.

We have developed a transfection assay to investigate the estrogen-mediated stabilization of cytoplasmic vitellogenin mRNA. A minivitellogenin (MV5) gene containing the 5' and 3' untranslated and coding regions but lacking 5,075 nucleotides of internal coding sequence was constructed. Cotransfection of the MV5 plasmid and a Xenopus estrogen receptor expression plasmid into Xenopus liver tissue culture cells yielded a 529-nucleotide MV5 mRNA, which was specifically stabilized by estrogen. MV5 mRNA exhibited the increased stability indicative of positive regulation when the estradiol-estrogen receptor complex was present and was not destabilized by unliganded estrogen receptor. Transfected estrogen receptor, estradiol, and 529 nucleotides of the 5,604-nucleotide vitellogenin B1 mRNA were sufficient for stabilization.     

Effects of estradiol-17β in the male xenopus laevis: Isolation and translation of cytoplasmic messenger rna populations

Summary Total Xenopus liver cytoplasmic RNA isolated following long-term estrogen administration (14 days) was fractioned using Sepharose 4B chromatography. One of the Sepharose 4B peaks was shown to contain RNA with a molecular weight reported for vitellogenin mRNA (34S). The presence of estrogeninduced vitellogenin mRNA in the peak 5 RNA was determined by translation of the RNA in the oocyte and analysis of the oocyte translational products by immunoprecipitation with anti-vitellogenin.Sepharose 4B peaks 2 and 3 were also observed to contain estrogen induced mRNA populations sedimenting between 9-18S. These findings suggest that Sepharose 4B chromatography might prove useful in separating different mRNA populations following estrogen-induced gene activation.     

Hormonal regulation of vitellogenin genes: an estrogen-responsive element in the Xenopus A2 gene and a multihormonal regulatory region in the chicken II gene

Expression of the vitellogenin genes in avian and amphibian liver is regulated by estrogens. The DNA elements mediating estrogen induction of the various vitellogenin genes of chicken and Xenopus encompass one or more copies of a 13-mer palindromic sequence called the estrogen-responsive element (ERE). Here we show that upon incubation with the purified estrogen receptor (ER) from calf uterus the Xenopus vitellogenin A2 gene yields a DNase-I footprint over the ERE between -331 and -319. This element does not mediate the response to glucocorticoids or progestins in T47D cells. The three guanine residues in each half of the palindrome are protected against methylation by dimethylsulfate after incubation with ER, but not with glucocorticoid (GR) or progesterone (PR) receptors. In contrast, the chicken vitellogenin II gene exhibits multihormonal regulation by estrogens, progestins, and glucocorticoids in T47D and MCF7 cells. Regulation is mediated by the DNA region between -721 and -591 that contains four binding sites for hormone receptors, as demonstrated by DNase-I footprints and methylation protection experiments. The two distal and most proximal binding sites are recognized by ER, GR, and PR, whereas the central binding site is only bound by ER and GR. At suboptimal concentrations, estrogens and progestins or glucocorticoids act synergistically. In experiments using a DNA fragment containing an ERE adjacent to a glucocorticoid-responsive element/progesterone-responsive element, ER and PR bind synergistically to their corresponding sites, perhaps explaining the functional synergism of both hormones. Thus, two very different regulatory elements are used to mediate estrogen induction of related genes in chickens and amphibians.     

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.     

Partial purification of estradiol receptor from Xenopus laevis liver and levels of receptor in relation to estradiol concentration

We have used ammonium sulphate precipitation followed by affinity chromatography to partially purify the estrogen receptor from Xenopus laevis liver which may control the genes for vitellogenin, the precursor of the egg yolk proteins. The rate at which receptor binds estradiol explains the kinetics of the induction of vitellogenin synthesis by estradiol, and the dissociation constant (0.5 X 10(-9) M) explains the concentration dependence of the response, which has a threshold of 10(-9) M estradiol, when 67% of the receptor is bound to estradiol. The estradiol concentration in male liver, which does not make vitellogenin, is 0.18 X 10(-9) M, sufficient to saturate 26% of the receptor, while in female liver, which makes vitellogenin continuously, the estradiol concentration is 3.5 X 10(-9) M, giving 88% saturation of receptor, suggesting that the proportion of occupied receptor decides whether or not the vitellogenin genes are active. In the physiological concentration range, estradiol modulates the level of receptor, which varies between 100 binding sites per nucleus in males and 440 in females, but artificially high concentrations of estradiol raise the level to approximately 1000 sites per nucleus. This suggests that the small increase in vitellogenin mRNA induced by physiological concentrations of estradiol is due to pre-existing receptor and that the much larger increases induced by very high concentrations depends on newly-synthesized receptor.     

Functional reconstitution of a receptor-activated signal transduction pathway in Xenopus laevis oocytes using the cloned human C5a receptor.

We have used the polymerase chain reaction to isolate and clone the cDNA encoding the human C5a receptor, and have injected the cDNA-derived receptor cRNA into Xenopus laevis oocytes for functional characterization of the receptor protein. Receptor activity was determined either electrophysiologically by measuring the agonist-dependent opening of [Ca2+]i-dependent Cl- channels, or by analysing the agonist-dependent efflux of 45Ca2+ from the oocytes. Using both methodologies, injection of pure C5a receptor cRNA failed to confer C5a sensitivity on the oocytes. In contrast, marked responses to C5a were observed when the receptor cRNA was supplemented with poly(A)+ RNA isolated from undifferentiated HL-60 cells, which is devoid of C5a receptor mRNA. Binding studies using radioiodinated C5a revealed that the C5a receptor polypeptide was in fact synthesized and targeted to the oocyte plasma membrane in oocytes injected with receptor cRNA alone, and that the level of receptor expression was not influenced by coinjection of poly(A)+ RNA from undifferentiated HL-60 cells. These results strongly suggest that the human C5a receptor requires a specific cofactor(s) lacking in Xenopus oocytes but present in undifferentiated HL-60 cells, to generate intracellular signals in oocytes. Identification and characterization of this factor will provide important information about the molecular mechanisms by which G-protein-coupled receptors activate phospholipase C.     

Vitellogenesis in Xenopus laevis and chicken: cognate ligands and oocyte receptors. The binding site for vitellogenin is located on lipovitellin I

Vitellogenesis is the process of yolk formation in rapidly growing oocytes of oviparous species. The transport of yolk precursor proteins from the blood plasma into the oocyte is achieved by receptor-mediated endocytosis. Although the Xenopus oocyte is one of the prime experimental systems for expression of foreign genes and their products, the receptor for the main vitellogenic protein, vitellogenin, from this extensively utilized cell has not been identified. Here we have applied ligand and immunoblotting to visualize the Xenopus laevis oocyte receptor for vitellogenin as a protein with an apparent Mr of 115,000 in sodium dodecyl sulfate-polyacrylamide gels under nonreducing conditions. The receptor from the amphibian oocyte also recognizes chicken vitellogenin, and vice versa; furthermore, the two receptor proteins are immunologically related as revealed by Western blotting with anti-chicken vitellogenin receptor antibodies. The receptors from both species bind the lipovitellin moiety of vitellogenin, as revealed by ligand blotting with radiolabeled lipovitellin polypeptides as well as by a novel reverse ligand blotting procedure utilizing nitrocellulose-immobilized ligand. Since vitellogenins of chicken and Xenopus have been shown to be structurally similar and evolutionarily related (Nardelli, D., van het Schip, F. D., Gerber-Huber, S., Haefliger, J.-A., Gruber, M., AB, G., and Wahli, W. (1987) J. Biol. Chem. 262, 15377-15383), it appears that conservation of key structural elements required for efficient vitellogenesis extends from the ligands to their receptors on the oocyte plasma membrane.