Galactoside-binding serum lectin of Xenopus laevis. Estrogen-dependent hepatocyte synthesis and relationship to oocyte lectin.

Xenopus laevis serum contains a lectin which binds alpha- and beta-galactosides. It was purified to homogeneity by affinity chromatography and consists of a single subunit with Mr approximately 69,000, associated in a multimer. The lectin is synthesized and secreted by hepatic parenchymal cells, and its synthesis is increased about 2-fold by estrogen treatment, both in vivo and in primary cell cultures.

Inhibition by estradiol of binding and mitogenic effect of epidermal growth factor in primary cultures of Xenopus hepatocytes.

We demonstrate here the presence of two classes of EGF (epidermal growth factor) binding sites in primary cultures of male Xenopus liver parenchymal cells. One of these corresponds to the high-affinity receptor described in other tissues and species, and which exhibits the property of autophosphorylation. The number of EGF receptors decreased sharply in freshly prepared cultures but recovered to maximum levels within 24 h thereafter.

Deinduction of transcription of Xenopus 74-kDa albumin genes and destabilization of mRNA by estrogen in vivo and in hepatocyte cultures.

The goal of this study is to explain the molecular basis of the marked deinduction of Xenopus albumin synthesis and secretion accompanying the activation of vitellogenin genes by estrogen. We have characterized by restriction analysis, DNA sequencing and hybrid-selected translation of mRNA, a cloned cDNA specifying the two 74-kDa albumins which constitute the predominant circulating form of albumin in Xenopus laevis. Using this recombinant DNA plasmid as a hybridization probe, we have determined the steady-state levels of albumin mRNA, the rate of transcription of the two 74-kDa albumin genes and the stability of the mRNA in male and female Xenopus hepatocytes in vivo and in primary cell cultures following estrogen treatment.

Transient paralysis by heat shock of hormonal regulation of gene expression.

We have investigated the effect of heat shock on primary cultures of male and female Xenopus laevis hepatocytes as a function of estrogen-induced vitellogenin gene expression. Coincident with the induction of heat-shock protein (hsp) synthesis, thermal stress abolishes the estrogen activated transcription and accumulation of vitellogenin mRNA, at the same time causing the destabilization of vitellogenin mRNA accumulated by prior treatment with the hormone. Exposure of the cells to estrogen before heat shock allows an immediate resumption of vitellogenin gene transcription on return to 26 degrees C.

Regulation by estrogen receptor of vitellogenin gene transcription in Xenopus hepatocyte cultures.

We have used primary cell cultures of hepatocytes from male or female Xenopus laevis to study the mechanisms by which estrogen induces vitellogenin gene transcription and how primary exposure to estrogen renders cells more responsive to secondary stimulation. We have characterized the estrogen receptor in hormonally naïve cells and in hepatocytes treated with estrogen under a variety of conditions. Under all conditions the receptor has a Kd congruent to 4 X 10(-10) M.

Primary culture, cellular stress and differentiated function.

Isolation of specialized cell types for the analysis of tissue-specific gene function often results in loss of the differentiated phenotype. Examples of this type of phenotypic change following tissue disaggregation are reviewed together with possible explanations. Close similarities between the effects of cell isolation with those of other cellular stresses such as heat or anoxia point to common biochemical mechanisms being involved.

Culture shock. Synthesis of heat-shock-like proteins in fresh primary cell cultures.

The isolation of Xenopus liver, lung and testis cells by collagenase digestion of the tissue, followed by Percoll density gradient centrifugation, was characterized by the preferential synthesis of two proteins whose size and charge were similar to 70 and 85 kD heat-shock proteins. The synthesis of these two heat-shock-like proteins, relative to that of total protein, declined gradually in the first 3-4 days after the cells were plated out for primary culture. In fresh primary cultures of liver parenchymal cells albumin mRNA concentration declined rapidly and plateaued at 3-4 days of culture.

The thermal denaturation of chromatin core particles.

The thermal denaturation of chicken erythrocyte core particles has been followed using changes in absorption and circular dichroism. The absorption-denaturation curve is biphasic, with transitions centred at 59 degrees C and 74 degrees C. The first of these transitions is reversible, whereas heating into the second transition produces irreversible changes in DNA and histone secondary structure.

Unequal activation by estrogen of individual Xenopus vitellogenin genes during development.

Using a technique of filter hybridization under very stringent conditions to HindIII fragments of complementary DNA cloned in plasmids, we have measured the accumulation in hepatocytes of mRNA specified by each of the four vitellogenin genes (A1, A2, B1, B2) at different stages of development of Xenopus laevis. The ontogenic competence of embryonic liver to respond to the first exposure to estradiol-17 beta, in terms of activation of transcription of this multigene family, is acquired late in metamorphosis at around Nieuwkoop-Faber stage 58. Upon hormonal induction, the four mRNAs accumulate under non-steady-state conditions at different rates and to different extents at all developmental stages in vivo and in cultured adult hepatocytes.

Rapid estrogen metabolism and vitellogenin gene expression in Xenopus hepatocyte cultures.

Male hepatocytes metabolized estradiol-17 beta, 17 alpha-ethinylestradiol and mestranol extremely rapidly (t 1/2 = 40, 60 and 300 min, respectively), whereas these were more stable in cultures of female hepatocytes (t 1/2 = 120, 150 and 640 min, respectively). Vitellogenin mRNA accumulated for only 12 h after a single addition of 10(-6) M estradiol to male hepatocyte cultures; mestranol, but not 17 alpha-ethinylestradiol or diethylstilbestrol, was more potent than the natural hormone. The level and rate of accumulation of vitellogenin mRNA were 5-15 times higher in female than in male hepatocytes, mestranol and estradiol being more potent than 17 alpha-ethinylestradiol and diethylstilbestrol.

Coordinate and non-coordinate estrogen-induced expression of A and B groups of vitellogenin genes in male and female Xenopus Hepatocytes in culture.

The concentration of mRNA transcribed from A and B groups of vitellogenin genes, induced by estrogen added to primary cultures of hepatocytes from male and female adult Xenopus, was measured by a technique of filter disc hybridization with cloned Xenopus vitellogenin cDNA probes. In cells from naive male Xenopus (i.e.

Hormonal regulation and expression of vitellogenin multigene family.

Yolk proteins are the most abundant egg proteins in oviparous animals. They are deposited during oocyte maturation for use after fertilization and are synthesized in the liver or fat body as a common precursor termed vitellogenin. Hybridization with cloned DNA complementary to vitellogenin messenger RNA has revealed a surprisingly high degree of evolutionary conservation of sequence of vitellogenin genes among insects, amphibians and birds.