Synthesis of Vitellogenin by Eel ( Anguilla anguilla L.) Hepatocytes in Primary Culture: Requirement of 17β-Estradiol-Priming

Abstract

Control and 17β-estradiol-primed eels were used to investigate the hormonal requirement of vitellogenesis in an immature fish, the eel. A primary culture of isolated liver cells from female silver eels was developed. The hepatocytes were maintained as monolayers on poly- l-lysine coated dishes for up to 12 days in a defined medium alone or supplemented with 17β-estradiol (E 2, from 10 -8 to 10 -5 M ). The amounts of vitellogenin (Vg) in the cells and secreted into the medium were measured at 2-day intervals using a homologous vitellogenin ELISA. Different E 2-priming conditions were determined before hepatocyte isolation (one injection of 250 μg of E 2 21 days, 17 days, or 24 hr). The vitellogenic response of hepatocytes to E 2 stimulation was studied in relation to the duration of the E 2-priming. After 8 days of culture, when hepatocytes from control eels were used, Vg was undetectable both in cells and in culture media, even if the culture was performed in the presence of E 2 10 -5 M. However, Vg was detectable both in cells and in culture media of hepatocytes from E 2-primed eels. If the priming was performed 24 hr before the culture, the Vg synthesis significantly increased ( P < 0.001) in the presence of E 2 10 -5 M after 10 days of culture but remained low. When the culture was performed 17 or 21 days after the priming, the level of the vitellogenic response was higher than after a short priming. In particular, with hepatocytes from 2l-day E 2-primed eel, the concentration of secreted Vg was 1.5 times higher than in control dishes ( P < 0.01). in the presence of E 2 10 -8 M after 12 days of culture. Higher doses of E 2 (10 -5 M ) increased Vg 2.7-fold over control values ( P < 0.01) after 4 days of culture. In control dishes, cultured without steroid, the amounts of secreted and intracellular Vg remained unchanged over 12 days of culture (respectively, 72.8 ± 2.7 ng/106 cells/48 hr and 28.7 ± 2.7 ng/10 6 cells). These results show that cultured hepatocytes retain their functional capacity by synthesizing a specific protein, Vg, in the presence of E 2 and there are dose- and time-related effects of E 2 on in vitro Vg synthesis. The induction of hepatic vitellogenesis in vitro requires a preliminary in vivo E 2-priming.