Expression cloning and regulation of steroid 5 alpha-reductase, an enzyme essential for male sexual differentiation

Abstract

The conversion of testosterone into the more potent androgen, dihydrotestosterone, catalyzed by the enzyme steroid 5 alpha-reductase, is required for the differentiation of male external genitalia. Here, we report the isolation of cDNA clones encoding the rat steroid 5 alpha-reductase using expression cloning in Xenopus oocytes. DNA sequence analysis demonstrates that the liver and ventral prostate forms of steroid 5 alpha-reductases are identical hydrophobic proteins of 29 kDa. The amount of steroid 5 alpha-reductase mRNA in liver increased in response to castration, but remained unchanged in the prostate. Testosterone administration to castrates induced expression of mRNA in the prostate but had no effect on liver. The data suggest that the steroid 5 alpha-reductase gene is differentially regulated by testosterone in androgen-responsive versus non-responsive tissues.

Fig. 1. Expression cloning of steroid 5α-reductase

Female rat liver RNA was size fractionated on 10–25% sucrose gradients and aliquots of RNA were assayed for steroid 5α-reductase activity in Xenopus oocytes. Peak activity fractions were used to construct an oriented cDNA library in a plasmid RNA expression vector. E. coli transformants from this library were pooled in groups of 150–200 clones and assayed for enzyme expression. A thin layer chromatography assay was employed in which the substrate testosterone (T) could be separated from androstenedione (A) and the 5α-reduced forms of these two steroids (DHT and 5αA, respectively). Sibling selection of a positive pool of clones was carried out as described in the text.

Fig. 2. Dilution cloning of a liver steroid 5α-reductase cDNA

Xenopus oocytes were injected with RNA from the indicated source and assayed for steroid 5α-reductase activity by thin-layer chromatography using [¹⁴C]testosterone as a substrate as described under “Experimental Procedures.” Lane 1, H₂O-injected; lane 2, RNA from female rat liver; lane 3, RNA synthesized in vitro from a pool of 150–200 cDNA clones; lane 4, RNA synthesized from cDNAs inoculated in a 96-well microtiter plate; lane 5, RNA synthesized from a pool of 12 clones corresponding to a row from the microtiter plate; lane 6, RNA synthesized from eight clones corresponding to a column from this plate; and lane 7, RNA derived from a cDNA clone corresponding to the intersection of the row and column. Chromatograms from the various experiments were exposed to Kodak XAR-5 film for 16 h. In the chromatographic system employed, hydrophobic steroids migrate further than hydrophilic steroids. The positions of authentic steroid standards are shown on the left of the autoradiograms. T, testosterone, A, androstenedione, DHT, 5α-dihydrotestosterone, 5αA, 5α-androstanedione. An endogenous Xenopus enzyme in the oocyte converts testosterone into androstenedione. Steroids marked with a asterisk are uncharacterized metabolites derived from the 5α-reduce compounds by endogenous Xenopus enzymes (see Fig. 3). The amount of 5α-reduced metabolites in a given experiment varied depending on the batch of oocytes injected and is thus not calculated here.

Fig. 3. Substrate specificity of the cloned steroid 5α-reductase

Xenopus oocytes obtained from a single animal were injected with in vitro synthesized RNA derived from the steroid 5α-reductase cDNA clone and then assayed for enzyme activity using the indicated ¹⁴C-labeled steroid substrates (5 µM) in the absence (−) or presence (+) of the competitive inhibitor 4-MA (5 µm). The various steroids and metabolites are identified on the left and right of the autoradiograms: P, progesterone; 5αP, 5α-dihydroprogesterone; others are as indicated in the legend to Fig. 2. The amount of 5α-reduced metabcolites for each substrate is indicated at the bottom of the figure and was determined by liquid scintillation counting after cutting out appropriate zones from the chromatograms. In lanes 5 and 6, all radioactive derivatives of dihydrotestosterone were counted. In experiments not shown, the pattern of metabolites obtained when dihydrotestosterone was employed as a substrate was identical in both H₂O-injected and steroid 5α-reductase RNA-injected oocytes.

Fig. 4. Nucleotide sequence of the cDNA corresponding to the rat steroid 5α-reductase mRNA, predicted amino acid sequence, and hydropathy profile of the protein

A, nucleotides are numbered on the right-hand side. The amino acids are numbered above the sequence with position 1 arbitrarily assigned to the first methionine codon in the nucleotide sequence. Two polyadenylation signals are overlined. B, the sequence of the steroid 5α-reductase protein was subjected to a hydropathy analysis using the algorithm of Kyte and Doolittle (43). Sequences above the central dividing line are hydrophilic, and those below the line are hydrophobic.

Fig. 5. In vitro translation analysis of steroid 5α-reductase RNA

In vitro synthesized steroid 5α-reductase RNA was translated in a reticulocyte lysate as described under “Experimental Procedures.” Additions to individual tubes are indicated above the autoradiogram. Approximately 8% of each translation reaction was analyzed by electrophoresis on 7–15% gradient polyacrylamide-sodium dodecyl sulfate gels. Size standards are indicated on the left. The band at M_r 45,000 represents an endogenous methionine binding protein in the reticulocyte lysate. The band corresponding to steroid 5α-reductase is indicated on the right of the autoradiogram.

Fig. 6. Characterization of the 5′ and 3′ ends of the steroid 5α-reductase cDNA and mRNA

A, expression of 3′-truncated RNAs in Xenopus oocytes. The steroid 5α-reductase cDNA plasmid was linearized with the indicated restriction enzyme and the resulting template was used to synthesize RNA in vitro. Oocytes were injected with the RNA and assayed for activity using testosterone as a substrate. The amount of 5α-reduced steroid metabolites was determined as described in the legend to Fig. 3. The values shown are the average of two or three separate experiments for each RNA. B, primer extension analysis of the 5′ end of liver steroid 5α-reductase mRNA. Ten µg of poly(A⁺) mRNA from the indicated source was subjected to primer extension analysis as described under “Experimental Procedures.” Size standards (STDS) are indicated on the left of the autoradiogram. Exposure times at −70 °C with an intensifying screen were 13 h for lanes 1, 3, and 4, and 1 h for lane 2. nt, nucleotides.

Fig. 7. Blot hybridization of rat RNA and DNA with steroid 5α-reductase cDNA probes

A, poly(A⁺) RNA was isolated from female liver, and male liver and ventral prostate, and amounts as indicated were subjected to electrophoresis and blotting as described previously (44). The probe used corresponded to the complete cDNA insert shown in Fig. 4A. The positions to which standards migrated in adjacent lanes on the gel are indicated on the left of the autoradiogram. The filter was exposed to x-ray film for 16 h at −70 °C with an intensifying screen. B, genomic DNA was isolated from liver using an Applied Biosystems model 340A DNA extractor. Ten-µg aliquots were digested with the indicated restriction enzymes (B, BamHI; Bg, BglII; R, EcoRI; and H, HindIII) and subjected to Southern blotting as described (45). DNA size standards are indicated on the left of the autoradiogram. The entire steroid 5α-reductase cDNA insert was used as a probe, and the washed filter was exposed to film for 5 days.

Fig. 8. Expression of steroid 5α-reductase mRNA in rat ventral prostate and liver

Orchiectomy, testosterone administration, RNA isolation, and blot hybridization were carried out as described under “Experimental Procedures.” Thirty µg of total RNA were electrophoresed in each lane. Lane 1, liver RNA from normal male rat; lane 2, liver RNA from 7-day castrates; lane 3, liver RNA from 10-day castrates given testosterone acetate on days 7–9; lane 4, liver RNA from 10-day castrates; lane 5, liver RNA from normal animals given testosterone acetate on days 7–9; lanes 6–10, prostate RNAs from the same animals. A combination of three single-stranded DNA probes generated from bacteriophage M13 clones (46) were used as a probe. RNA size standards are indicated on the left of the autoradiogram. Exposure times were 16 h for lanes 1–5, and 3 days for lanes 6–10. The weakly hybridizing mRNA of about 1.7 kb in the liver samples may represent the use of a polyadenylation signal at nucleotide position 1549 of Fig. 4A.