Metabolism of [ 3H]-testosterone and [1,2- 3H]-dihydrotestosterone by prolactin secreting rat pituitary tumour cells in culture

Abstract

Rat pituitary tumour cells in monolayer culture (GH 3 cells) respond to oestradiol-17β and testosterone with increased synthesis of prolactin. The present study was undertaken to establish whether this effect of testosterone could be mediated via conversion to oestradiol-17β. Cell cultures were incubated with 400 μCi of [7α- 3H]-testosterone at a concentration of 10 −6 M for 96 h. The metabolites were isolated and identified by repeated paper chromatography, derivative formation and final crystallization to constant specific activity from different solvents. Testosterone was metabolized to a great extent, 88–96%, but no conversion (<0.0001%) of testosterone to oestradiol-17β or oestrone could be detected. 5α-Dihydrotestosterone and 5α-androstane-3β,17β-diol were found as the major metabolites, isolated in a radiochemically pure form. 5α-Androstane-3α,17β-diol and 4-androstene-3,17-dione were also isolated and identified, and they constituted a minor fraction, 5 and 0.2%, respectively. The kinetics of the metabolism of [ 3H]- testosterone were studied by harvesting the cell cultures after 6, 12, 24, 48, and 96 h. The data suggested that 5α-androstane-3β,17β-diol was formed via 5α-dihydro-testosterone, that was subsequently shown to be a good substrate for the formation of the former. It is concluded that the stimulatory effect of testosterone on prolactin secretion by these cells is probably not mediated via conversion to oestradiol-17β. The extensive metabolism of testosterone opens the possibility that the effect of testosterone is in some way related to the formation of a metabolite.