Unexpected virilization in male mice lacking steroid 5 alpha-reductase enzymes

Abstract

Mice lacking steroid 5 alpha-reductase 1 and 2 were produced by gene targeting and breeding. Male mice without 5 alpha-reductase 2 or without both enzymes had fully formed internal and external genitalia and were fertile, but had smaller prostates and seminal vesicles than controls. T accumulated to high levels in the reproductive tissues of the mutant mice. DHT administration increased seminal vesicle and coagulating gland weights in mice deficient in 5 alpha-reductase 2 and increased the weights of the prostate, seminal vesicle, and coagulating gland in animals deficient in both enzymes. An inhibitor of both 5 alpha-reductases (GI 208335X) decreased prostate and coagulating gland weights of control mice, but had no effect in those lacking 5 alpha-reductase 1 and 2. Castration reduced the sizes of these tissues in animals of all genotypes. Androgen-dependent gene expression was decreased in the seminal vesicles of mice lacking one or more 5 alpha-reductases and was restored by administration of T or DHT. Female mice missing both enzymes exhibited parturition and fecundity defects similar to those of animals without 5 alpha-reductase 1. We conclude that T is the only androgen required for differentiation of the male urogenital tract in mice and that the synthesis of DHT serves largely as a signal amplification mechanism.

Fig. 1

Mutation of mouse 5α-reductase 2 gene. A, Structures of the normal 5α-reductase type 2 gene, the targeting vector used to remove a portion of the gene extending from intervening sequence 1 through exon 3 and the mutant allele. The positions at which the restriction enzymes XhoI (X) and BamHI (B) cleave the DNAs are indicated. Exons (Ex) are indicated by black boxes, and intervening sequences (IVS) by connecting lines. Two viral thymidine kinase (TK) genes and a gene encoding neomycin resistance (Neo) are indicated by boxes, which are not drawn to scale. B, RNA blotting in wild-type and 5α-reductase type 2 knockout mice. Poly(A)⁺ RNA was isolated from the epididymes of animals (n = 2) of the indicated type 2 genotype. Aliquots (5 μg) of RNA were size-fractionated by agarose gel electrophoresis in the presence of glyoxal, transferred to a nylon membrane, and subjected to blot hybridization using a radiolabeled probe derived from exon 1 of the mouse type 2 gene. After washing, the filter was exposed to x-ray film for 18 h. The locations to which standards of known size migrated to in the gel are indicated on the left of the autoradiogram (upper panel). The exon structures of the 5α-reductase type 2 mRNAs deduced in RT-PCR experiments are shown on the right of the autoradiogram. The structure of the mRNA marked X was not elucidated. The filter was stripped of bound radioactivity by washing at high temperature and then subjected to a second round of hybridization using a radiolabeled probe made from a cyclophilin cDNA. As indicated by the autoradiogram shown in the lower panel, equal amounts of mRNA were analyzed in each lane.

Fig. 2

5α-Reductase enzyme activity in wild-type and knockout mice. A and B, Extracts were prepared from different adult (3 months of age) or fetal (embryonic d 18) tissues of the indicated 5α-reductase genotypes (WT, wild-type; 1KO, type 1 knockout; 2KO, type 2 knockout; DKO, type 1 and 2 double knockout). Aliquots containing 150 μg protein were incubated for 60–120 min at 37 C in the presence of 5.0 μM [¹⁴C]T and 5 mM NADPH in buffers of the indicated pH. 5α-Reductase type 1 activity predominates at pH 7.0, whereas type 2 activity predominates at pH 5.0 (24, 25). Steroids were isolated by extraction with organic solvents, separated by TLC, and detected by autoradiography. The positions to which T substrate and DHT product migrated to on the plates are indicated on the left. Lanes are numbered below the autoradiograms. UGT, Urogenital tract.

Fig. 3

Androgen-responsive tissue weights in wild-type and knockout mice. Tissues were dissected from male mice of the indicated ages and 5α-reductase genotypes (WT, wild-type; 2KO, type 2 knockout; DKO, type 1 and 2 double knockout) and weighed. Plotted values are expressed as milligrams of tissue per g BW and represent the mean ± SEM of values obtained from 7–12 animals for each time point and genotype. Asterisks above the histogram bars indicate organ weights that were significantly different (P ≤ 0.05) from those of wild-type mice. A, Prostate; B, seminal vesicles plus coagulating glands; C, testes.

Fig. 4

Effects of castration on androgen-responsive tissue weights in wild-type and 5α-reductase knockout mice. Male mice, 2.5–3.0 months of age and of the indicated 5α-reductase genotypes (WT, wild-type; 2KO, type 2 knockout; DKO, type 1 and 2 double knockout), were subjected to sham operation (−) or castration (± ) and then maintained for 9–10 d. Tissues were thereafter dissected and weighed. Plotted values are expressed as milligrams of tissue per g BW and represent the mean ± SEM of values obtained from five or six animals for each surgical group. Asterisks above the histogram bars indicate castrate organ weights that were significantly different (P ≤ 0.05) from those of sham-operated mice. A, Prostate; B, seminal vesicles plus coagulating glands.

FIG. 5

Effects of DHT administration on androgen-responsive tissue weights in wild-type and 5α-reductase knockout mice. Time release pellets containing vehicle alone (−) or DHT (+) were implanted sc in male mice, 2.5 months of age and of the indicated 5α-reductase genotypes (WT, wild-type; 2KO, type 2 knockout; DKO, type 1 and 2 double knockout). After 20 d, tissues were dissected and weighed. Plotted values are expressed as milligrams of tissue per g BW and represent the mean ± SEM of values obtained from 9–13 animals for each treatment group. Asterisks above the histogram bars indicate organ weights that were significantly different (P ≤ 0.05) between the groups. A, Prostate; B, seminal vesicles plus coagulating glands.

Fig. 6

Effect of GI 208335X administration on androgen-responsive tissue weights in wild-type and 5α-reductase knockout mice. Male animals, 2.5 months of age and of the indicated 5α-reductase genotypes (WT, wild-type; DKO, type 1 and 2 double knockout), were administered vehicle alone (−) or GI 208335X (+) as described in Materials and Methods. The GI compound inhibits both 5α-reductase enzymes. After 9 d, tissues were dissected and weighed. Plotted values are expressed as milligrams of tissue per g BW and represent the mean ± SEM of values obtained from seven to nine animals for each treatment group. Asterisks above the histogram bars indicate organ weights that were significantly different (P ≤ 0.05) between the groups. A, Prostate, coagulating glands; B, seminal vesicles, penis.

Fig. 7

Gene expression in mice lacking 5α-reductase. Poly(A)⁺-enriched RNA was purified from the seminal vesicles/coagulating glands of 3-month-old male mice of the indicated 5α-reductase genotypes (WT, wild-type; 2KO, type 2 knockout; DKO, type 1 and 2 double knockout) and treatment groups (DKO + DHT, double knockout mice treated for 20 d with pellets containing DHT; DKO + T, double knockout mice treated for 20 d with pellets containing T; WT + inhibitor, wild-type mice treated for 9 d with the 5α-reductase inhibitor GI 208335X). Aliquots (5 μg) were separated by electrophoresis through agarose gels and then subjected to blot hybridization with probes derived from cDNAs encoding metallothionein 1 (top panel), transglutaminase (middle panel), and EST AA124355 (bottom panel). Exposure times for autoradiography were 6, 18, and 96 h, respectively, for the top, middle, and bottom panels. The sizes of the mRNAs detected with each probe are indicated on the left of the autoradiograms. The fold changes in mRNA levels between the various animal groups are indicated below the autoradiograms and were determined by scanning densitometry using signals from a cyclophilin cDNA probe as a loading control.