Primary induction of vitellogenin synthesis in monolayer cultures of amphibian hepatocytes.

Abstract

Direct induction of vitellogenin production in cultured male amphibian hepatocytes by estradiol-l7ß has been accomplished. Liver cells were isolated from adult male bullfrogs by collagenase perfusion and maintained as monolayers in serumfree medium containing insulin and estradiol . Vitellogenin production was measured by direct immunoprecipitation from radioactively labeled secreted protein with a specific antiserum against vitellogenin . Significant quantities of vitellogenin were detected in the exported protein on the second day of hormone treatment . Vitellogenin production increased with duration of culture in the presence of estradiol until by the eighth day -90% of secreted protein was vitellogenin . This response is largely comparable to that obtainable in vivo .Indirect immunofluorescence microscopy was used to identify cells synthesizing vitellogenin in response to estradiol . An increase in cytoplasmic fluorescence could be seen in cells throughout the cultures, with increasing time in the presence of estradiol . By the sixth day of treatment, the majority of cells showed significant fluorescence labeling . The results suggest that studies on the mechanisms underlying the primary activation of the vitellogenin gene may now be conducted under well defined conditions in a monolayer liver cell culture system .

Vitellogenin synthesis in cultured hepatocytes; an in vitro test for the estrogenic potency of chemicals

“…In oviparous animals, the synthesis of VTG by hepatocytes represent a good system with which to study the mechanism of action of steroid hormones [31][32][33][34]. Most of these studies have used hepatocytes of either amphibians (especially Xenopus laevis) or birds (especially chickens), though some have been based on hepatocytes from fish [24,[35][36][37]. Various techniques have been used to culture fish hepatocytes [35,37].…”

Quantitative Analysis of Protein Synthesis Altered by Estrogen in Cultured Xenopus Liver Parenchymal Cell

“…In the present study, using insulin-containing medium (10 mU/ml) in the primary culture, the synthesis of overall protein, especially secretory proteins, increased with culture time. De-induction of albumin synthesis reported in other primary culture systems (17,19) was not observed under the present conditions. Since albumin synthesis was reduced by estradiol treatment even in the presence of insulin, it is strongly suggested that albumin production is negatively controled by estradiol.…”

“…The present procedures for cell culture were similar to those in Rana cutesbeiuna system (17), in which, however, a solution for dispersion of liver cells contained no pyruvate and perfusion was done with a collagenase solution at a higher temperature (40°C). The temperature used for collagenase-digestion was set at 30°C, since the ability of cells become attached to substrutum of the culture dish was reduced by the treatment at a higher temperature.…”

In vitroeffect of pituitary, interrenal and gonadal hormones on vitellogenin synthesis in primary hepatocyte cultures ofChalcalburnus tarichi

“…This number is consistent with the literature mentioned above. It was shown that when E2 was applied to the frog hepatocyte culture, the Vtg fluorescent marking and the Vtg synthesis in the hepatocytes increased over the course of the experiment (Stanchfield & Yager 1980). Van Bohemen, Lambert, Goos and Van Oordt (1982) reported that when E2 and estrone were injected to female rainbow trout (Onchorynchus mykiss), small amounts of Vtg was found to be stored in the liver, and given rapidly to blood 7 days after the injection.…”