Determination of oestrogen concentrations in bovine plasma by a recombinant oestrogen receptor-reporter gene yeast bioassay

A recombinant cell yeast bioassay (RCBA) was applied to the generic measure ment of bovine plasma oestrogen concentration. Samples were prepared by die thyl ether extraction of plasma following addition of [H-3]17 beta-oestmdio l as internal standard; organic and aqueous phases were separated by freezi ng (recovery 97.1 +/- 0.7%) and dried extract reconstituted in culture medi um (recovery 31.4 +/- 4.5%). Plasma oestrogen concentrations were measured by incubation of extracts with yeast containing a stable human oestrogen re ceptor (hER) and a reporter construct comprising an hER response element re gulating beta-galactosidase expression. The linearity of response for the a nalysis of spiked plasma samples using the RCBA, following corrections, is described by y = 0.8994x - 0.111 (r(2) = 0.9776, P < 0.0001). Inter-assay v ariation for endogenous oestrogen was 11.5% for >1 pg ml(-1). Plasma oestro gen concentrations for intact (n = 5) and castrated (n = 3) males were < 0. 5 pg ml(-1), and 3.7 +/- 2.6 pg ml(-1) for luteal phase females (n = 10). A nalysis by RCBA of sequential samples from heifers during the reproductive cycle failed to detect the pre-ovulatory increase in plasma 17 beta-oestrad iol as determined by radioimmunoassay (RIA) (maximal concentrations 2.09 +/ - 2.1 pg ml(-1) and 32.6 +/- 14.6 pg ml(-1), respectively). Interestingly, when samples were hydrolysed using Helix pomatia glucuronidase the RCBA gav e concentrations (29.5 +/- 8.9 pg ml(-1)) not significantly different to th ose obtained by RIA. These preliminary findings suggest that a substantial proportion of plasma oestrogen during the pre-ovulatory period may be conju gated. These data indicate the potential of the RCBA to measure biologicall y active and physiological levels of plasma oestrogens in cattle. One poten tially valuable application of this generic oestrogen assay could be in sur veillance programmes to detect illegal use of anabolic oestrogens in live-s tock where the identity of the analyte may be unknown.