Biosynthesis of catalyticallyt active rat testosterone 5α-reductase in microinjected Xenopus oocytes: Evidence for tissue-specific differences in translatable mRNA

The enzyme 4-ene-3-ketosteroid-5α-oxidoreductase [5α-reductase; 3-oxo-5α-steroid Δ⁴-dehydrogenase, 3-oxo-5-steroid: (acceptor) Δ⁴-oxidoreductase, EC 1.3.99.5] plays a key role in androgen-dependent target tissues, where it catalyzes the conversion of testosterone to the biologically active dihydrotestosterone. The regulation of 5α-reductase expression has not been studied at the molecular level as the enzyme is a membrane protein that is labile in cell-free homogenates. We developed a sensitive bioassay of the enzyme activity expressed in Xenopus oocytes microinjected with rat liver and prostate mRNA. After microinjection, incubation of intact oocytes in the presence of [³H]testosterone revealed the in ovo appearance of active 5α-reductase. Polyadenylylated RNA was fractionated by sucrose gradient centrifugation, and the enzymatic activity was shown to be encoded by a 1600- to 2000-base-pair fraction of hepatic poly(A)⁺ RNA. 5α-Reductase mRNA was most efficiently translated when up to 80 ng of RNA was injected per oocyte. In the injected oocytes, 5α-reductase mRNA was found to be a short-lived molecule (t( 1/2 ) = 2 hr), whereas its in vivo translatable 5α-reductase protein exhibited stable enzymatic activity for over 40 hr. Moreover, the levels of translatable tissue-specific 5α-reductase mRNAs as monitored in the Xenopus oocytes correlated with the variable 5α-reductase activities in female rat liver, male rat liver, and prostate homogenates; the ratio of their specific activities was of 2500:630:1, respectively. Altogether, these results provide supporting evidence in favor of the transcriptional control of 5α-reductase expression in rat tissues.