Oestrogen reporter transgenic medaka for non-invasive evaluation of aromatase activity

Abstract

Vertebrate reproduction involves complex steroid hormone interplay and inter-conversion. A critical element in maintaining sex steroid levels is the enzyme aromatase (cytochrome P450 19A1) which converts androgens to oestrogens. In turn oestrogen signalling is targeted by numerous chemicals, from pharmaceuticals to agricultural chemicals, both frequent sources of contamination in waste waters and consequently rivers. Although many models are now available to address disruption of oestrogen signalling, there are currently no published protocols allowing discrimination between alterations in testosterone metabolism and in oestrogenic signalling. It was with this limitation in mind that we optimised this protocol. We show using a 48h protocol that pre-feeding fry of the choriogenin h-gfp (chgh-gfp) medaka line are sensitive to 0.05nM EE2 (15ng/L), within the range of the lowest published observable physiological effect concentrations for medaka. In addition, co-treatment with testosterone can reveal potential effects of test substances on aromatase enzymatic activity. As the measurements are visualised in real-time without affecting embryo viability, repeated measures are possible. We demonstrate the ability of this model to detect oestrogen receptor agonists, aromatisable androgens, P450 aromatase activity modulators and selective oestrogen response modulators. Importantly, the range of this assay is physiologically relevant.