Estradiol Induced Mechanisms Regulating Vitellogenin (VG) and Choriogenin Synthesis and Expression of VG in the Cultured Hepatocytes of Indian Freshwater Murrel Channa punctatus

Abstract

Introduction: Vitellogenin (Vg) and Choriogenin (Chg) are femalespecific proteins, synthesized in the hepatocytes of teleosts. Several in vivo studies have shown that it is difficult to distinguish the primary effects of a compound from those induced secondarily because liver functions are under the influence of multiple endogenous factors. Therefore, in vitro hepatocyte systems present a better experimental model to investigate mechanism(s) by which Vg and chg are synthesized. Primary cultures of fish hepatocytes have been used to study hepatic structure and function, viz. endocrine regulation, Vg synthesis, hormone receptor expression, and toxicology. The Vg and Chg induction in cultured fish hepatocytes is clearly influenced by culture conditions (medium composition, temperature etc.) and culture system (hepatocyte monolayers, aggregates, liver slices etc.). The main advantages of the hepatocyte Vg and Chg assay are considered its ability to detect effects of estrogenic metabolites, since hepatocytes in vitro remain metabolically competent, and its ability to detect both estrogenic and anti-estrogenic effects. The aim of the present study was to understand the induction of Vg & Chg by estradiol (E2) and expression of Vg genes. Methods: Hepatocytes were isolated following the method of Rani et al.[1]. Hepatocytes were exposed to various doses of E2, optimum dose was selected and used for rest of experiments. Vg and Chg concentrations were determined in culture medium using standardized ELISAs [2] with few modifications. Cells exposed to a constant dose of E2 (500nM) in the presence of actinomycin D (3 and 6μg/ml) or cycloheximide (10 μg/ml). After 48 hrs media were collected from all the groups and processed for Vg and Chg estimation. Statistical Significance was calculated in between different doses of same treated groups by Mann-Whitney Wilcoxon test/student t-test; significance is given as p<0.005. Vg was extracted from the medium by PAGE and cut band was characterized by LC-MS/MS peptide mapping. Total RNA was isolated by TRIzol method from treated and non treated hepatocytes and processed for expression of Vg gene(s) by reverse transcription PCR (RT-PCR). E2 exposed hepatocytes were fixed and processed for localization of Vg and Chg in cytoplasm by immunocytochemistry and for ultrastructural analysis by TEM.