Quantitation of vitellogenin messenger RNA in the liver of male xenopus toads during primary and secondary stimulation by estrogen

From livers of estrogen-stimulated female Xenopus toads, large quantities of estrogen-induced, poly(A)-containing RNA could be isolated, showing the same characteristics as vitellogenin mRNA obtained from hormone-treated males.Using cDNA hybridization, vitellogenin mRNA was monitored in the cytoplasmic poly(A)-containing RNA of the liver of male toads during 13 days of primary and the initial phase of secondary stimulation with estrogen.During primary stimulation, low amounts of vitellogenin mRNA, not exceeding 0.18% of the cytoplasmic poly(A)-containing RNA, were first detected after 12 hr of hormone treatment, and vitellogenin mRNA was found to increase on the average to 34% of the cytoplasmic poly(A)-containing RNA on the seventh day of hormone treatment. After 3 days of primary stimulation, accumulation of vitellogenin mRNA leveled off, showing no significant increase in the cytoplasm up to 13 days of hormone treatment. As judged from incorporation of ³²PO₄ into blood plasma proteins of males during primary stimulation, vitellogenin was first detected after 1 day, and its synthesis was found to increase dramatically until the thirteenth day of hormone treatment. This implies that there is a coincidence between appearance and extent of synthesis of vitellogenin and the abundance of vitellogenin mRNA in the cytoplasm, but there is evidence that during later phase of primary stimulation (day 3–13), the increase in synthesis of vitellogenin cannot be attributed anymore to a significant accumulation of vitellogenin mRNA.In male Xenopus, estrogen-induced synthesis of vitellogenin is no more detectable 41 days after hormone injection, and the concentration of vitellogenin mRNA was found to be <0.03% of the cytoplasmic poly(A)-containing RNA. Secondary stimulation by estrogen of these animals results in an at least 30 fold faster accumulation of vitellogenin mRNA in the cytoplasm within the initial 12 hr of hormone treatment. This may explain the faster appearance of vitellogenin in the blood plasma.     

Ribosome loading, but not protein synthesis, is required for estrogen stabilization of Xenopus laevis vitellogenin mRNA.

We have examined the effect of protein synthesis and of ribosome loading on the estrogen-mediated stabilization of hepatic Xenopus laevis vitellogenin mRNA. Removal of estradiol-17 beta from the culture medium, which destabilizes vitellogenin mRNA, does not alter the density of ribosomes on polysomal vitellogenin mRNA, or change the proportion of vitellogenin mRNA associated with the endoplasmic reticulum. Cycloheximide, which inhibits elongation, without changing the density of ribosomes on vitellogenin mRNA, does not block estrogen-mediated stabilization. In contrast, 2-(4-methyl-2,6-dinitroanilino)-N-methylpropionamide, (MDMP), which inhibits initiation, greatly reduces the density of ribosomes on vitellogenin mRNA, and completely blocks estrogen-mediated stabilization. Vitellogenin mRNA in MDMP treated cells is degraded at a rate similar to that seen when untreated cells are transferred from medium containing estrogen to estrogen-free medium. This suggests that a ribosome-associated degradative system may not be responsible for vitellogenin mRNA degradation. The failure of estrogen to stabilize vitellogenin mRNA in MDMP-treated cells is not due to the release of vitellogenin mRNA from the endoplasmic reticulum. Vitellogenin mRNA in MDMP-treated cells remains associated with the endoplasmic reticulum in small polysomes containing 3-5 ribosomes. These data demonstrate that maintaining a high density of ribosomes on vitellogenin mRNA, but not continuing protein synthesis, is necessary for estrogen-mediated stabilization of vitellogenin mRNA.     

Sequence organization of a cloned tDNA1met fragment from xenopus laevis

3.18 kb fragments of X. laevis DNA coding for tRNA₁^met have been inserted into a λ vector via Hind III termini and cloned in E. coli. The organization of one cloned fragment has been analyzed by restriction endonuclease digestion and RNA-DNA hybridization. From the distribution of sites for three enzymes, this fragment appears to be typical of the majority of λ. laevis tandem tDNA₁^met repeat units. Evidence is presented to suggest that it contains two genes coding for tRNA₁^met and at least one gene coding for a second as yet unidentified 4S RNA species. The two tRNA₁^met genes are located on the same DNA strand 0.96 and 1.38 kb from one end of the repeat unit. A detailed restriction map for 19 enzymes reveals that the spacers between these genes are not identical, and it provides no indication of short repetitive sequence elements within the spacers.     

The hatching enzyme from xenopus laevis: Limited proteolysis of the fertilization envelope

Hatching in the amphibian Xenopus laevis involves release of an embryo-secreted hatching enzyme, a protease, which weakens the envelope surrounding the embryo. The envelope is not totally solubilized, which infers that only selected envelope components are hydrolyzed by the enzyme. The susceptibility of the glycoprotein components composing the envelope to hydrolysis by the hatching enzyme was investigated. Isolated envelopes in various physical states, ie, particulate and solubilized, were treated with the hatching enzyme, and the resulting envelope hydrolysis products were characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The susceptibility of the envelope components to proteolysis was not a function of the state of the envelope. The envelope components most susceptible to proteolysis were the 125K and 11 8K components followed by the 60K and 71 – 77K components. These components are minor constituents of the envelope. The major constituents, 33K and 40K, were relatively resistant to hydrolysis by the hatching enzyme. From these observations, we infer that the envelope components hydrolyzed are components that link or bind together the major structural elements of the envelope, eg, the 33K and 40K components. Selective destruction of the components required for maintaining the structural integrity of the envelope, eg, the “nuts and bolts” of the structure, permits a weakening of the envelope that allows the embryo to hatch without having to destroy totally (hydrolyze) the envelope.     

Isolation and characterization of the nuclear matrix from the male xenopus laevis following estrogen administration: Kinetics of [3H] uridine incorporation

At various times following estorgen administration, the nuclear matrix was isolated from the liver of male Xenopus laevis by sucrose gradient centrifugation of nuclei treated with a high-salt buffer and DNase I in the presence of a proteolytic inhibitor (PMSC - phenylmethyl sulfonyl chloride). Electron micrographs of the nuclear matrix demonstrate a sponge-like network attached to a well-defined inner envelope with a ribosome-free outer envelope. Chemical analyses show that the HSB-DNase-treated nuclei consist of 16% DNA, 2% RNA, and 82% protein, a composition that is consistent with that of nuclear matrices isolated from other species. The specific activity of the matrix-associated RNA following estrogen treatment appears to be maximally enhanced after 5 h and decreases until approximately 12 h, when the activity begins to increase again.     

The Xenopus laevis estrogen receptor: sequence homology with human and avian receptors and identification of multiple estrogen receptor messenger ribonucleic acids

We have isolated and sequenced a cDNA clone encompassing the entire protein coding region of the Xenopus laevis estrogen receptor (xER). The Xenopus ER, the first steroid hormone receptor to be sequenced from a cold-blooded organism, exhibits two regions of striking amino acid homology with the human and avian ERs. In the putative DNA binding region, the amino acid sequence of the xER differs from those of the human and avian ERs at only one of 83 amino acids. The putative hormone binding region contains 44 and 46 amino acid blocks in which the sequence is identical in the Xenopus and human ERs. Blot hybridizations of Xenopus liver RNA suggest that the xER is encoded by four mRNAs with lengths of approximately 9, 6.5, 2.8, and 2.5 kilobases. In contrast, hybridization of human RNA to a human ER cDNA clone reveals only a single major ER RNA, approximately 6.7 kilobases in length.     

Swimming suppresses hepatic vitellogenesis in European female silver eels as shown by expression of the estrogen receptor 1, vitellogenin1 and vitellogenin2 in the liver

Background  

When European silver eels (Anguilla anguilla) venture into the Atlantic Ocean for their 6,000 km semelparous spawning run to the Sargasso Sea, they are still in a prepubertal stage. Further sexual development appears to be blocked by dopaminergic inhibition of hypothalamus and pituitary activity. Recently, we found that swimming for several weeks in freshwater stimulated the incorporation of fat droplets in the oocytes. So, it was hypothesized that long term swimming in seawater would release the inhibition further and would also stimulate the production of vitellogenin by the liver.     

An immunocytochemical localization of the cortical granule lectin in fertilized and unfertilized eggs of xenopus laevis

At fertilization, the vitelline envelope surrounding the egg of Xenopus laevis is modified by the addition of an electron-dense component termed the “F layer.” The F layer functions as a block to polyspermy and as a block to the escape of macromolecules from the perivitelline space, thereby causing an osmotically driven envelope elevation. F-layer formation has been hypothesized to result from interaction between a cortical-granule lectin, released in the cortical reaction, and a jelly-coat ligand. Evidence for this hypothesis was sought by determining the location of the cortical-granule lectin both before and after fertilization, using a specific antibody conjugated to horseradish peroxidase. The cortical-granule lectin was localized only in the cortical granules of the unfertilized egg and was located predominantly in the perivitelline space and the F layer of a fertilized egg. These observations support the hypothesis that the F layer is formed by a cortical-granule-Iectin–jelly layer-ligand interaction.     

Differential responsiveness of avian vitellogenin I and vitellogenin II during primary and secondary stimulation with estrogen

Avian vitellogenin consists of two major species, VTG I and VTG II, which show major differences in structure and immunological properties suggesting that VTG I and VTG II are distinct gene products. During primary stimulation with estrogen, VTG I was found to accumulate in plasma much more slowly than VTG II. At 1 day after hormone treatment VTG I was only 1–3% of VTG II, but by day 5 VTG I increased to approximately 25% of VTG II. Measurements of hepatic vitellogenin synthesis confirmed the slower induction and reduced expression of VTG I. A further difference was noted in the amnestic or memory response to secondary estrogen treatment. Measurements of VTG I and VTG II accumulation and synthesis after primary and secondary estrogen treatment showed that the memory response occurs to a much greater extent for VTG I than VTG II. These differences indicate that the inductions of VTG I and VTG II are not tightly coupled.     

Cloning and characterization of synthetic sequences from the Xenopus laevis vitellogenin structural gene

The mRNA coding for vitellogenin, the yolk protein precursor, has been isolated from the liver of estrogen-stimulated Xenopus laevis. The mRNA has a size of 6.3 kilobases (kb). Optimal conditions were investigated for the synthesis of long complementary DNA (cDNA, referring to DNA synthesized in vitro) copies of the mRNA. Temperature, salt concentration, and enzyme-to-RNA ratio were important factors. Double-stranded cDNA with an average size of 2 to 3 kb was inserted into the vector pMB9 by the poly(dA:dT) method, and the recombinant plasmids were amplified in E. coli. Twenty-one clones with vitellogenin inserts ranging from 1 to 3.7 kb were studied. The regions in the RNA from which these clones had been derived were mapped by R-loop analysis in the electron microscope and by hybridization of the cloned DNAs with specific fractions of mRNA. Slightly more than half of the clones were derived from the 3′-terminal portions of the mRNA while the remaining clones are located internally.     

Multiple ISWI ATPase complexes from xenopus laevis. Functional conservation of an ACF/CHRAC homolog

The nucleosomal ATPase ISWI is the catalytic subunit of several protein complexes that either organize or perturb chromatin structure in vitro. This work reports the cloning and biochemical characterization of a Xenopus ISWI homolog. Surprisingly, whereas we find four complex forms of ISWI in egg extracts, we find no functional homolog of NURF. One of these complexes, xACF, consists of ISWI, Acf1, and a previously uncharacterized protein of 175 kDa. Like both ACF and CHRAC, this complex organizes randomly deposited histones into a regularly spaced array. The remaining three forms include two novel ISWI complexes distinct from known ISWI complexes plus a histone-dependent ATPase complex. This comprehensive biochemical characterization of ISWI underscores the evolutionary conservation of the ACF/CHRAC family.     

Thyroid hormone directly induces hepatocyte competence for estrogen-dependent vitellogenin synthesis during the metamorphosis of Xenopus laevis

Hepatocytes competent for estrogen-dependent vitellogenin synthesis appeared and increased in number in the liver at the metamorphic climax of Xenopus laevis (A. Kawahara, S. Kohara, Y. Sugimoto, and M. Amano, 1987, Dev. Biol. 122, 139-145). The present study was conducted to determine whether cells competent for vitellogenin synthesis could be induced by thyroid hormone in a primary culture of larval hepatocytes. The thyroid hormone, triiodothyronine (T3), directly induced the competent cells in a primary culture of premetamorphic larval hepatocytes in a dose- and duration-dependent manner. The competency acquired in response to T3 persisted after removal of the hormone. Aphidicholin, an inhibitor of DNA synthesis, failed to block this induction, suggesting the presence of a "precursor cell fraction." This cell fraction in the hepatocyte population increased with the progress of metamorphosis. The thyroid hormone is thus considered the cause of competent cell formation at metamorphic climax.