An in vitro vitellogenin bioassay for oestrogenic substances in the medaka (Oryzias latipes).

ABSTRACT

We developed an in vitro bioassay for oestrogenic compounds based on vitellogenin as a biomarker. To quantify the induction of vitellogenin in medaka (Oryzias latipes) we established a sandwich enzyme-linked immunosorbent assay (ELISA). This ELISA was carried out with two different monoclonal antibodies specific for the yolk precursor protein vitellogenin and the high molecular weight yolk proteins (lipovitellin) of medaka. Purified lipovitellin of medaka was used as a standard for the ELISA and could be quantified down to a concentration of 9 ng/ml. The calibration curve was linear up to at least 600 ng/ml and the intra-assay coefficient of variance was 3.2%. The application of the sandwich ELISA was tested using blood samples of males and females as well as primary hepatocyte cultures of medaka. Vitellogenin synthesis in cultured liver cells of males was induced by 1 and 100 nM of the xenoestrogen 17alpha-ethynylestradiol (EE(2)). The first production of vitellogenin was detected 6 days after hormone application. In contrast, isolated liver cells of sexually mature females, which were treated in the same manner, showed vitellogenin expression from the beginning of cultivation and its synthesis increased and remained high at 100 nM EE(2). However, the induction of vitellogenin synthesis in male hepatocytes in vitro could be maintained for at least one month and this indicated viable and differentiated liver cells. The hepatocyte cultures of male medaka in combination with the sandwich ELISA were shown to be a suitable tool to detect and quantify oestrogenic activity of chemicals.