| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
ARTICLES |
-Reductase Enzymes
Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, Texas 75390; and Division of Reproductive Sciences, Oregon Regional Primate Research Center (D.L.H.), Beaverton, Oregon 97006
Address all correspondence and requests for reprints to: Dr. David W. Russell, Department of Molecular Genetics, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75390-9046. E-mail: russell{at}utsw.swmed.edu
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-reductase 1 and 2 were produced by gene
targeting and breeding. Male mice without 5-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-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-reductases
(GI 208335X) decreased prostate and coagulating gland weights of
control mice, but had no effect in those lacking 5-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-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-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. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The conversion of T to DHT is catalyzed by steroid 5-reductase, a
membrane-bound enzyme that uses NADPH as a cofactor to reduce the
4,5 bonds of various substrates
(8). There are two 5-reductase genes, and their encoded
proteins, designated type 1 and type 2, share approximately 50%
sequence identity (9). The enzymes have divergent
biochemical and pharmacological properties, and they exhibit different
tissue-specific and cell type-specific expression patterns that reflect
their biological roles. For example, the type 2 enzyme is expressed in
the mesenchyme of the urogenital sinus and genital tubercle of the rat
(10, 11), a cell type that plays an instructive role in
the formation of the prostate and external genitalia (12).
Many species, including humans, monkeys, dogs, rats, and mice, have two
5-reductase genes, indicating that the duplication event that gave
rise to the type 1 and 2 enzymes occurred early in evolution. The
pan-specific distribution and duplication of the enzyme in mammals
also underscores the need for DHT in androgen action.
The contributions of individual 5-reductase enzymes to sexual
differentiation and reproductive biology are being elucidated in part
through analysis of mutations in the encoding genes. Naturally
occurring mutations in the human 5-reductase 2 gene cause male
pseudohermaphroditism in which the Wolffian ducts virilize normally due
to the actions of T, but the prostate and external genitalia fail to
form in the absence of DHT (13). An induced mutation in
the mouse 5-reductase 1 gene constructed by homologous recombination
in embryonic stem (ES) cells produced male mice that were
indistinguishable from wild-type counterparts, presumably due to the
presence of an active type 2 enzyme. In contrast, female mice that
lacked the type 1 enzyme exhibited partially penetrant defects in
parturition and fecundity (14, 15, 16). These results
confirmed the idea that the two 5-reductase enzymes have different
endocrine roles and suggested that the type 1 enzyme may be of greater
importance in the female and the type 2 enzyme of greater importance in
the male (17).
In the current study we report the construction and analysis of mice
that lack 5-reductase 2. The absence of this enzyme has no effect in
the female, but causes a mild virilization defect in the male, marked
by a reduction in the size of the secondary sexual glands. This
phenotype is far less severe than that observed in 5-reductase
2-deficient human males, in which the enzyme’s absence leads to a
failure of these tissues to differentiate from their respective
anlagen. Crossing the type 2-deficient mice with the previously
constructed type 1-deficient mice produced animals that lack all known
5-reductase enzyme activity. The phenotype of female mice missing
both enzymes is no more severe than that of mice without the type 1
enzyme. The modest virilization defect observed in type 2-deficient
male mice is more pronounced in animals lacking both enzymes; however,
the prostate, penis, and scrotum are male in character in these mice,
and the animals are fertile. These findings indicate that T is
sufficient for formation of the male phenotype in mice and suggest that
the conversion of T to DHT represents a signal amplification
mechanism.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-Reductase type
2-deficient mice were propagated by matings between animals homozygous
for the introduced null allele. Crossing type 2-deficient mice with
type 1-deficient mice (14) produced double knockout
animals, which were propagated by crossing females heterozygous for the
type 1 mutation and homozygous for the type 2 mutation with males
homozygous for both type 1 and 2 mutations. Breeding animals of these
genotypes avoided the parturition and fecundity defects in females
associated with loss of the 5-reductase 1 gene (14) and
produced 50% of offspring with the desired double knockout genotype.
All studies were conducted in accordance with the standards of humane
animal care described in the NIH Guide for the Care and Use of
Laboratory Animals using protocols approved by an institutional animal
care and research advisory committee.
ES cell work
A bacteriophage P1 clone encompassing the mouse 5-reductase
type 2 gene from the 129SvJ strain was isolated from a genomic DNA
library (Genome Systems, St. Louis, MO). A targeting
vector derived from the pPolIIshort-neobPA-HSVTK plasmid
(18) was assembled using standard methods of genetic
engineering and contained a 6.8-kb BglII fragment from the
type 2 gene as a long arm and a 2.3-kb XhoI fragment from
the gene as a short arm (Fig. 1A
). The
6.8-kb DNA fragment spanned exons 3–5, and the corresponding
intervening sequences and was generated by long PCR from the
bacteriophage P1 clone (19). The 2.3-kb XhoI
DNA fragment spanned a portion of intervening sequence 1 and was
isolated directly from the recombinant bacteriophage P1. The 2.5 kb of
DNA that normally separate these two fragments in the type 2 gene were
replaced by a neomycin resistance cassette (Fig. 1A).
|
-reductase 2 allele. The presence of
the mutation in the offspring of chimeric males was detected by a
PCR-based assay.
RNA blotting
Total RNA was prepared from tissues using RNA Stat 60
(Tel-Test B, Inc., Friendswood, TX). Polyadenylated
[poly(A)+] RNA was purified by
oligo(deoxythymidine)-cellulose chromatography using a kit (Pharmacia
LKB, Piscataway, NJ). The purified RNA was
size-fractionated by electrophoresis through 1.4% (wt/vol) agarose
gels, transferred to nylon filters (Biotrans, ICN Biomedicals, Inc., Cleveland, OH) by capillary blotting, and subjected to
blot hybridization using standard methods (19). A probe
corresponding to exon 1 of the encoding gene was used to detect the
5-reductase type 2 mRNA. cDNAs purchased from Incyte Genomics (St.
Louis, MO) were used as hybridization probes in the experiments
presented in Fig. 7.
|
-androstan-3,17ß-diol
glucuronide, and LH were measured in the sera of 3-month-old male mice
by RIA (22). Blood was drawn from the inferior vena cava,
cells were removed by centrifugation, and the resulting sera were
stored at -20 C until analyzed. T, androstenedione, and E2 were
extracted into ether, separated by chromatography on Sepharose LH-20,
and subjected to RIA at the Oregon Regional Primate Research Center
(Beaverton, OR). 5-Androstan-3,17ß-diol glucuronide and DHT
levels were measured using kits from Diagnostics Systems Laboratories, Inc. (Webster, TX). Dr. Terry Nett (Animal
Reproduction and Biotechnology Laboratory, Colorado State University,
Ft. Collins, CO) measured LH concentrations.
Tissue hormone measurements
Androstenedione and T levels were measured in the prostates and
seminal vesicles of 3-month-old male mice. Prostates (5–15 mg) and
seminal vesicles (50–150 mg) were dissected and stored at -80 C until
analyzed. For extraction, the tissues were thawed, weighed on a
microbalance, and homogenized in 1 ml PBS at 4 C using a Polytron
(Brinkmann Instruments, Inc., Westbury, NY). Steroids were
extracted into ether from the homogenates and subsequently measured as
described above and previously (22).
Steroid 5-reductase enzyme activity
Tissues were dissected from 3-month-old wild-type and knockout
animals and homogenized in 10 mM potassium phosphate (pH
7.0), 150 mM potassium chloride, 0.3 M sucrose,
and 1 mM EDTA. Protein concentrations were determined using
a kit (Bio-Rad Laboratories, Inc., Hercules, CA).
5-Reductase type 1 enzyme activity was assayed by incubating tissue
homogenates (150 µg protein) in 0.1 M Tris-citrate buffer
(pH 7.0) containing 5 µM [14C]T
(NEN Life Science Products, Boston, MA) and 5
mM NADPH (Sigma, St. Louis, MO) in a total
volume of 0.5 ml for 1 h at 37 C. 5-Reductase type 2 enzyme
activity was measured similarly, except that the pH of the 0.1
M Tris-citrate buffer was 5.0. In both assays, steroids
were extracted into 5 ml methylene chloride at the end of the
incubation period and taken to dryness under a stream of nitrogen. The
resulting pellets were dissolved in 20 µl chloroform-methanol (2:1,
vol/vol), spotted onto Silica Gel 150 TLC plates (catalogue no.
4855-82, Whatman, Clifton, NJ), and resolved by
development in chloroform-ethylacetate (3:1, vol/vol). Radioactive
steroids were detected by autoradiography using Kodak
XAR-5 film (Eastman Kodak Co., Rochester, NY). Exposure
times were 12–16 h. The sensitivity of the assay under these
conditions was approximately 1 pmol/min·mg protein.
Animal studies
Castration was performed at 10–12 wk of age. Animals were
anesthetized, the abdomen/scrotal area was cleaned, and a single
incision was made. The testis, epididymes, and epididymal fat pad were
removed after ligation of the spermatic vein. The incision was closed
with sutures and staples. The animals were killed 9–10 d later, and
tissue weights were determined after dissection.
Steroid hormones were administered by sc implantation of pellets containing T or DHT (5 mg, 21-d release; Innovative Research of America, Sarasota, FL). Pellets were inserted on the backs of 10-wk-old animals as previously described (14). Twenty days after pellet insertion, the animals were killed, and blood and tissues were harvested.
Inhibitor studies were performed using GI 208335X
(N-[1-(4-trifluoromethylphenyl)-cycopentenyl]3-oxo-4-aza-5-androst-1-ene-17ß-carboxamide,
empirical formula
C31H39F3N2O2,
Mr = 528), a compound that inhibits both
5-reductase type 1 and 2 (obtained from Dr. Stephen Frye,
GlaxoSmithKline, Research Triangle Park, NC). Adult male mice, 10–12
wk old, were injected sc with 10 mg/kg BW GI 208335X for 9 d. On d
10 of the experiment, the animals were killed, and blood and tissues
were collected.
Morphometry
Semiquantitative morphometric analyses to determine the
numbers of epithelial and stromal cells in androgen-dependent tissues
were carried out as previously described (23). Briefly,
tissues were dissected from wild-type, 5-reductase 1 and 2 double
knockout, and castrated wild-type mice; fixed in Bouin’s
solution; sectioned at 5 µm; stained with hematoxylin and eosin; and
examined by light microscopy (Eclipse 1000, Nikon,
Melville, NY). The numbers of stromal cells, epithelial cells, and
glandular lumens present in 20 grids from each tissue section were
counted with the aid of the NIH Image version I.58 software program
(http://rsb.info.nih.gov/nih-image/). These data were expressed as
ratios of cell types to determine the effects of gene and organ loss on
tissue architecture.
Assessment of reproductive function
Reproduction in knockout animals was assessed by harem mating
experiments in which individual males of different 5-reductase
genotypes were housed with three wild-type C57BL/6J females from
0800–1300 h daily. At 1300 h females were examined for the
presence of a copulatory plug in the vagina (Exp 1) or for the presence
of sperm in the vagina by lavage (Exp 2). In Exp 1, wild-type (n =
3), 5-reductase type 2 knockout (n = 3), and 5-reductase
type 1 and 2 double knockout (n = 3) males were used, and the
experiment was continued over a 40-d period. The efficiencies of
plugging were 47.5%, 19.2%, and 17.5% for wild-type, type
2-deficient, and type 1- and 2-deficient male animals, respectively.
Females with plugs were removed from the cages on the day of
insemination and replaced with fresh females over the course of the
experiment. The frequency of pregnancies carried to term in the plugged
females was 60%, 64%, and 51% in matings with wild-type, type
2-deficient or type 1- and 2-deficient males, respectively. The average
litter sizes resulting from these pregnancies were 6.4, 7.5, and 6.6
pups, respectively.
In the second experiment wild-type (n = 3) and 5-reductase type
1- and 2-deficient males (n = 3) were mated with three C57BL/6J
females over a 23-d period. Daily lavages were performed to determine
the presence of sperm in the vagina as an indicator of mating. In
matings involving wild-type and double knockout males, the mating
frequencies were 32% and 44%, the pregnancy rates were 96% and 60%,
and the average litter sizes were eight and eight pups,
respectively.
Sperm counts were measured by dissection of the vas and cauda epididymes into 1 ml PBS. Three small incisions were made in each fragment, and the tissues were gently massaged to release the sperm. The tissue and sperm were placed in a 5% CO2 incubator for 1 h to facilitate dispersal. Sperm counts were determined by light microscopy using a hemocytometer.
Microarray analysis
Total RNA was prepared from seminal vesicles/coagulating glands
of wild-type and mutant mice by extraction with TRIzol (Life Technologies, Inc./BRL, Gaithersburg, MD) and checked for
integrity by agarose gel electrophoresis. The RNA was shipped to Incyte
Genomics (St. Louis, MO) and used to prepare
poly(A)+ RNA, which was converted into
fluorescent dye-labeled cDNA for use as hybridization probes with the
mouse GEM1 microarray containing approximately 8000 cDNA clones.
Differentially expressed cDNAs in the tissues of the wild-type and
double knockout mice were identified, purchased as plasmid DNAs, and
used in the RNA blotting experiments of Fig. 7
.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-reductase 2 were produced by homologous
recombination in ES cells. Schematics of the normal allele, the
targeting vector used for recombination, and the expected structure of
the mutant allele are shown in Fig. 1A. The targeting vector was
designed to inactivate the gene by replacing a DNA segment encompassing
a portion of intron 1 through a portion of exon 3 with a bacterial gene
encoding neomycin resistance. ES cells with the desired recombination
event were detected at a frequency of 1% after electroporation and
plus-minus selection, and of these, four were injected into blastocysts
to produce three lines of chimeric male mice. Two lines transmitted the
mutation to their offspring, as judged by PCR- and Southern
blotting-based genotyping assays. Mice from these lines exhibited the
same phenotype and were used interchangeably in the studies described
below.
Blotting experiments were performed to assess the effects of the
introduced mutation on 5-reductase 2 mRNA levels. As indicated by
the data in Fig. 1B, three mRNAs of 4.2, 2.0, and 1.2 kb were detected
in the epididymis of wild-type mice. In animals heterozygous for the
deletion allele, these same three mRNAs were present together with two
additional species of 4.0 and 1.4 kb, whereas animals homozygous for
the mutation had only the latter two mRNAs. RT-PCR and DNA-sequencing
experiments showed that the aberrant mRNAs associated with the
deletion allele contained exon 1 spliced to exon 5. The size variation
in mRNAs containing this arrangement of exons arose from the use of
different polyadenylation sequences located within the 3'-flanking
region of the gene (see schematics of Fig. 1B). The translational
reading frame of exon 1 is not in phase with that of exon 5; thus, the
mRNAs transcribed from the deletion allele were predicted to encode a
truncated and inactive 5-reductase 2 enzyme of 116 amino acids. To
ensure that the induced mutation eliminated enzyme activity, a
truncated cDNA encoding the first 116 amino acids of the protein was
generated and introduced into cultured human embryonic kidney 293 cells
by transfection. Assay of the transfected cells using
[14C]T substrate revealed that the truncated
protein had no measurable 5-reductase enzyme activity (data not
shown).
To confirm that the introduced mutation led to loss of enzyme activity
in vivo, extracts were prepared from the epididymes of adult
male mice with different 5-reductase genotypes and assayed for
activity (Fig. 2). This tissue is a
particularly rich source of 5-reductase 2 in the mouse
(14), and the contributions of each enzyme to total
activity can be partially distinguished by carrying out assays in
buffers of different pH (24, 25). Extracts from wild-type
mice converted T into DHT at both an acidic pH (type 2 enzyme activity;
Fig. 2A, lane 2) and a neutral pH (type 1 enzyme activity; Fig. 2A, lane 8). In contrast, extracts from mice with a mutation in the type 1
gene exhibited normal or slightly elevated levels of 5-reductase
activity at pH 5.0 (lane 3) and reduced activity at pH 7.0 (lane 9).
Mice with a mutation in the type 2 gene had a marked reduction in
activity at pH 5.0 (lane 4) and a decrease at pH 7.0 (lane 10). The
latter result indicated that approximately 50% of the activity
detected at pH 7.0 was due to the type 2 enzyme. Extracts from mice
lacking both 5-reductase 1 and 2 exhibited no 5-reductase enzyme
activity at either pH (lanes 5 and 11). In a similar series of
experiments, no 5-reductase enzyme activity was detected at acidic
(Fig. 2B) or neutral (data not shown) pH in extracts from double
knockout mice prepared from adult testis, prostate, seminal vesicle, or
coagulating gland or male and female embryonic d 18 urogenital
tracts.
|
-reductase genotypes were grossly normal. The anogenital
distances of newborn animals harboring the introduced mutations were
not different from those of controls, they did not exhibit nipple
formation, and the animals appeared to undergo puberty within the same
time period as normal males based on the onset of mating at 6–8 wk of
age.
Genetic deficiencies in 5-reductase did alter steroid hormone levels
in the sera of 3-month-old male mice (Table 1). T levels increased in both the
type 2-deficient and double knockout animals; however, the observed
differences did not reach statistical significance. DHT levels
decreased in double knockout mice to below the detectable range of the
RIA (Table 1). Androstenedione levels were increased in the type
2-deficient mice, but not to statistical significance, and were
significantly increased in double knockout mice (P =
0.03; Table 1). E2 and LH levels were unchanged between wild-type and
knockout mice, and in agreement with the latter data, no changes were
detected in pituitary levels of LH - or ß-subunit mRNAs or
proteins (data not shown). Finally, the levels of
5-androstan-3,17ß-diol glucuronide, a DHT metabolite, were
significantly reduced in both type 2-deficient and double knockout mice
(P = 0.001; Table 1).
|
-reductase type 2 gene or of both the type 1 and
type 2 genes caused a reduction in the weights of androgen-dependent
tissues. The prostates of wild-type mice increased in weight from about
0.2 to 0.6 mg/g BW between postnatal wk 6 and 24 (Fig. 3A). This trend was also evident in mice
lacking the type 2 gene and in the double knockout animals; however, in
animals of these genotypes the tissues were less than 50% of the
weights of age-matched wild-type controls (Fig. 3A). Similar decreases
in the weights of the seminal vesicles and coagulating glands were
observed in the knockout vs. wild-type animals during
postnatal wk 3–24 (Fig. 3B). The weights of the testes were not
different between wild-type and type 2-deficient mice, whereas those of
the double knockout mice were slightly smaller (Fig. 3C).
Semiquantitative morphometric analyses (23) of the
prostates of the type 2-deficient and double knockout mice revealed
that the decrease in weight was due to cellular hypotrophy rather than
hypoplasia. The stromal to epithelial cell ratio in wild-type mice
(n = 3) was 1.1, and those in the type 2-deficient (n = 4)
and double knockout mice (n = 4) were 0.73 and 0.75, respectively.
In a control experiment the ratio of stromal to epithelial cells
increased to 2.3 in the prostates of wild-type mice castrated 14 d
before tissue harvest.
|
). In
contrast, these steroids accumulated to markedly high levels in the
prostates of type 2-deficient and double knockout mice. Large increases
in androstenedione and T also were detected in the seminal vesicles and
coagulating glands of the mutant mice (Table 2). These data and those
presented in Fig. 3 indicated that mice deficient in 5-reductase
type 2 or both type 1 and type 2 had smaller prostates, seminal
vesicles, and coagulating glands despite prominent elevations in the
tissue content of T.
|
-reductase type 2 or of both the type 1 and 2 enzymes had
effects on male fertility and fecundity (26, 27). For
example, in a harem mating experiment in which formation of a vaginal
plug was monitored as an end point, double knockout males plugged 18%
of wild-type females compared with a 47.5% efficiency measured for
wild-type males. When the presence of sperm in the vagina was monitored
in a second experiment as an indication of mating, double knockout
males impregnated 44% of wild-type females. The increase in apparent
mating frequency detected in the latter assay was probably a
consequence of the smaller secondary sexual organs in the knockout
mice. These would be expected to secrete less seminal fluid upon
ejaculation, which, in turn, would decrease the formation of copulatory
plugs.
In agreement with the mating frequency data, sperm counts in double
knockout mice (17.9 ± 8.8 x 106;
n = 11) were not statistically different from those in wild-type
controls (25 ± 8.5 x 106; n =
8). The data in Table 3 indicated that
the litter sizes fathered by type 2-deficient or type 1- and
2-deficient male mice were no different from those of wild-type males.
Loss of the type 1 enzyme in females caused a marked decrease in litter
size, as expected from previous studies (15); however,
loss of the type 2 enzyme did not affect this parameter, and the
fecundity of double knockout females was not lower than that of type
1-deficient animals (Table 3).
|
-reductase
genotypes were castrated, and the weights of the prostate and seminal
vesicle/coagulating gland were measured. In wild-type mice,
orchidectomy caused statistically significant declines in the weights
of the prostate (Fig. 4A) and seminal
vesicle/coagulating gland (Fig. 4B). In mice without the type 2 enzyme
or those lacking the type 1 and 2 enzymes, removal of the testes
produced a further decline in the weights of both tissues (Fig. 4, A
and B). These data suggested that the internal genitalia of
5-reductase knockout mice remained responsive to T.
|
indicated that these
tissues also retained their sensitivity to DHT in intact animals.
Administration of this androgen increased prostatic weight by 22% in
the type 2-deficient mice and by 41% in the double knockout animals
and increased seminal vesicle/coagulating gland weights by 50% and
27%, respectively. Exogenous DHT did not increase tissue weights in
wild-type mice (Fig. 5), presumably due to the presence of maximum
hormone levels in these tissues.
|
-reductase inhibitors have been identified, and in several
instances these have been used to produce a pharmacological knockout of
the enzyme (28). To establish the effects of one of these
inhibitors in adult wild-type and 5-reductase deficient mice,
animals (n = 7–9/genotype) were treated with GI 208335X. This
compound inhibits both 5-reductase type 1 and type 2 when delivered
sc to mice (Frye, S., personal communication). As shown by the data in
Fig. 6A, GI 208335X treatment of
2.5-month-old wild-type males for 9 d caused a 25% decrease in
the weight of the prostate. In contrast, the drug did not affect
prostatic weights in animals lacking both 5-reductase type 1 and 2.
Similar findings were obtained when the coagulating glands were
examined; the drug caused a 45% decrease in the weight of this gland
in wild-type mice, but had no consequences in the double knockout mice
(Fig. 6A). The seminal vesicles of animals of both genotypes decreased
in weight with inhibitor treatment; however, the decline observed in
the wild-type animals (60%) was larger than that observed in the
double knockouts (28%; Fig. 6B). Penis weights did not change in
animals of either genotype (Fig. 6B).
|
-reductase were assessed in the seminal vesicle/coagulating gland
by cDNA microarray technology. RNA was prepared from pooled glands
dissected from wild-type (n = 2) or double knockout (n = 2)
animals and converted into cDNA in the presence of a deoxynucleoside
triphosphate derivatized with a fluorescent dye. Different dyes were
used to label the wild-type and knockout cDNA probes, which were
subsequently hybridized to a commercially available microarray
containing about 8000 mouse cDNAs. Changes in gene expression were
measured by fluorescence spectroscopy, and the data were organized
according to rank order differences in the levels of a given mRNA. In
this sample of genes, over 20 mRNAs were found to differ by more than
2-fold between mice of different genotypes. Among the differentially
expressed mRNAs were those encoding metallothionein, transglutaminase,
and EST AA124355, which, as indicated by the RNA blotting data shown in
Fig. 7, were expressed at 5-fold higher
levels in wild-type mice than in the double knockout mice. The
metallothionein and transglutaminase genes are known to be regulated by
androgen (29, 30). These androgen-dependent mRNAs were
also present at lower levels in mice lacking just the type 2 enzyme
(Fig. 7).
To determine whether androgens would restore the expression levels of
these genes in the mutant mice to those observed in wild-type mice,
double knockout animals (n = 7–9/genotype) were treated for
21 d with T or DHT, and changes in each mRNA were measured by RNA
blotting. T administration increased the level of metallothionein mRNA
to approximately 60% of that in wild-type mice, whereas the levels of
the transglutaminase and EST AA124355 mRNAs were increased to 130% and
100%, respectively, of those found in the controls (Fig. 7). DHT also
increased the amount of each mRNA, but only to levels that were
40–60% of those in wild-type mice (Fig. 7). These data confirmed the
androgen responsiveness of the three genes and suggested that they were
sensitive to T and DHT. This conclusion was supported by the finding
that treatment of wild-type mice with the GI 208335X inhibitor
decreased levels of the metallothionein and EST AA 124355 mRNAs, but
did not alter transglutaminase mRNA levels (Fig. 7).
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-reductase
gene expression in mice. The most surprising findings are that males
lacking 5-reductase type 2 or both 5-reductase type 1 and type 2
have properly formed internal and external genitalia and are fertile.
These outcomes are in marked contrast to the situation in the human
male, in whom mutations in the 5-reductase type 2 gene severely
impede the formation of the prostate and external genitalia, and they
suggest that the urogenital tract of the mouse requires only T for the
differentiation of these tissues. The requirement for DHT cannot be
completely dispensed with in the mouse, in that animals without
5-reductase have smaller prostates and seminal vesicles, and the
expression of androgen-responsive genes is decreased in these
tissues. The hypotrophy of the secondary sexual glands and the
alterations in gene expression occur despite the accumulation of T in
the tissues, which further underscores the potency of DHT and the need
for signal amplification.
Loss of the 5-reductase type 2 gene in female mice did not produce
adverse consequences, suggesting that this enzyme is not required for
normal endocrine function in this sex. The fecundity and parturition
defects observed in type 1-deficient mice (14, 15) were
not worsened in the double knockout females, nor were new phenotypes
evident. These findings support the hypothesis that the type 1 enzyme
has evolved to play an essential role in the female, whereas the type 2
enzyme subserves in the male (17).
A simple explanation for the unexpected virilization observed in the
knockout male mice would be the existence of a third 5-reductase. A
putative type 3 enzyme might be unique to the mouse, thus explaining
the phenotypic differences observed between this and other species upon
loss of 5-reductase. We were, however, unable to detect
5-reductase enzyme activity in multiple tissues from adult, fetal,
and newborn double knockout mice under different assay conditions using
a high specific activity T substrate. Expression cloning experiments in
which 600,000 cDNA clones were screened from a liver library
constructed from a 3-month-old male double knockout mouse also failed
to yield a third form of the enzyme. Finally, an inhibitor of the type
1 and 2 enzymes (GI 208335X) reduced the sizes of androgen-dependent
tissues in wild-type mice, but had little or no effect in double
knockout animals. We thus can offer no experimental evidence in support
of a third 5-reductase enzyme.
DHT levels assessed by RIA in the sera of 5-reductase knockout
animals were reduced to below the reliable level of detection of the
assay. Any residual amount of DHT may be due to cross-reaction between
the antibody employed and one or more steroid metabolites that are
elevated in the mutant mice. The amounts of C19
and C21 steroids with 5ß stereochemistry are
increased in patients with 5-reductase 2 deficiency (4, 31, 32, 33), and it seems likely that the same is true in the
knockout mice. RIA measurements of 5-androstan-3,17ß-diol
glucuronide levels revealed that this metabolite was also present at
background levels in the sera of type 2-deficient and type 1- and
2-deficient mice. This steroid is produced from DHT by reductive
3-hydroxysteroid dehydrogenases, and its presence in the plasma of
humans is directly proportional to the level of DHT
(34, 35, 36). Inasmuch as these findings translate to the
mouse, the absence of 5-androstan-3,17ß-diol glucuronide from
the knockout animals suggests that circulating levels of DHT are, in
fact, very low or absent. The steroid metabolites that accumulate in
the mutant mice remain to be determined by chemical analysis.
There are two papers in the literature that report the consequences of
treating mice with 5-reductase inhibitors (37, 38). In
one, the compound 6-methylene-4-pregnene-3,20-dione was
administered to pregnant female mice of the ICR/JCL strain at 400 mg/kg
BW from d 12–19 of gestation (37). This regimen decreased
the anogenital distance in postnatal d 1 male offspring, caused
hypospadiac urethras, decreased the number of prostatic buds formed
along the urogenital sinus, promoted nipple formation, and decreased
the epithelial volumes of the prostatic buds and coagulating glands.
These abnormalities were not present in 3-month-old animals delivered
from dams treated with the drug in utero, and these adults
were fertile. In contrast, 129Sv/Ev/C57BL/6J mixed strain newborn male
mice lacking 5-reductase type 1 and 2 and born to double knockout
mothers do not have reductions in their anogenital distances, do not
have nipples, and have properly formed penile urethras. In a second
published study, administration of finasteride at doses
ranging from 25–250 mg/kg·d to adult CD-1 male mice caused Leydig
cell hyperplasia and adenomas and increased plasma LH levels
(38). Again, in contrast to these results, the double
knockout mice lacking 5-reductase studied here do not show these
symptoms. We do not know whether the differences between the
inhibitor-treated and knockout mice are due to the use of different
mouse strains in the experiments, to possible androgenic or
antiandrogenic effects of the inhibitors, or to other explanations.
In contrast to these results, extensive data are available from studies
with humans and rats treated with 5-reductase inhibitors
(28), and there is a good correlation between many of
these findings and those reported here for 5-reductase knockout
mice. For example, inhibition of 5-reductase in man causes a
decrease in prostatic DHT and a corresponding increase in T
(39), as seen in 5-reductase knockout mice. Treatment
of male rats with 5-reductase inhibitors causes a decrease in
copulatory plug formation (26, 27), and the same
phenomenon is observed in the knockout mice together with a decrease in
the mRNA encoding transglutaminase, an enzyme associated with plug
formation (40). These similarities suggest that the
phenotypes of the knockout mice may accurately reflect the endocrine
roles of 5-reductase and DHT in this species.
With these considerations in mind, T alone appears sufficient for
formation of the urogenital tract in the mouse. The involvement of a
single androgen for phenotypic sexual differentiation has not been
observed previously in several species, including humans, in whom
mutation of the 5-reductase type 2 gene causes male
pseudohermaphroditism (13), or rats and tammar wallabies,
which when given inhibitors of 5-reductase develop a phenotype
similar to that of 5-reductase 2-deficient men (5, 7, 41, 42). Although there are differences between these species in the
biosynthetic origins of DHT, the absolute requirement for the hormone
is inviolate. For example, in rats, rabbits, and humans, T is
synthesized by the fetal testes, secreted into the circulation, and
then converted to DHT by 5-reductase in target tissues (43, 44). In contrast, 5-androstan-3,17ß-diol is the secreted
testicular androgen in the wallaby, which is then converted back to DHT
by oxidative 3-hydroxysteroid dehydrogenases in the prostate and
other target tissues (45).
Several hypotheses have been put forth to explain why two androgens are
needed for formation of the male phenotype. First, different classes of
androgen-responsive genes may exist, including those that respond
solely to T or DHT or equally well to either androgen. Unique
transcriptional responses could be mediated at the DNA level by the
existence of androgen response elements of different sequences or
contexts that are recognized by particular AR-ligand complexes.
Alternatively, a ligand-dependent response could be mediated at the
protein level by receptor-associated chaperones (46) or by
the recruitment of different coactivators to a target gene by receptors
activated by one or the other androgen. Despite the attractiveness of
these hypotheses, there is presently little convincing evidence to
support distinct gene networks or cofactors, and differential
hybridization experiments in the 5-reductase double knockout mice
have to date failed to reveal genes with an absolute dependence on
DHT.
A second hypothesis to explain the requirement for two androgens, and one that is endorsed by the data presented here, invokes a need for signal amplification in some target tissues. The requirement for a stronger signal may arise in a tissue due to poor vascularization and hence diminished access to hormone, to the presence of high levels of catabolic enzymes that specifically degrade one or the other androgen (47), or to the expression of receptor-binding proteins that destabilize a particular liganded complex (46). The signal amplification hypothesis is supported by biochemical studies that document a longer half-life and a higher DNA binding affinity for the human DHT-AR complex vs. the T complex (48, 49, 50, 51, 52). As a consequence of these properties, the effective dose of DHT required to activate an androgen-responsive marker gene by 50% is about 10-fold lower than that required to achieve the same level of induction with T. At higher hormone concentrations, however, the extents of activation achieved with both androgens are the same (49).
With respect to the virilized phenotypes of 5-reductase knockout
mice, T levels in the androgen target tissues of adult animals were
20–240 times higher than those measured in wild-type controls. If T
also accumulates to this extent in fetal tissues, then this buildup
would diminish or abolish the need for DHT and, hence, signal
amplification. The accumulation of T to such high levels distinguishes
the mouse from other species, in that dogs treated with a
5-reductase inhibitor accumulate androstenedione, but not T, in
androgen target tissues (53), and T accumulates in the
prostates of inhibitor-treated humans to levels that are only 2–11
times higher than those in untreated tissues (39). T may
accumulate in mouse tissues due to low levels of oxidative
17ß-hydroxysteroid dehydrogenase enzyme activity
(54, 55, 56), which would otherwise convert this active
androgen into the far less potent steroid androstenedione, as happens
in the dog (53). This interpretation supports the proposed
important role of 17ß-hydroxysteroid dehydrogenases in regulating
tissue levels of active and inactive steroid hormones (57, 58) and, we believe, may also explain the unexpected
virilization of double knockout mice.
|
Acknowledgments |
|---|
-reductase
inhibitors, and an anonymous reviewer for suggestions concerning the
measurement of steroid hormones by RIA. |
Footnotes |
|---|
Abbreviations: ES, Embryonic stem; poly(A)+, polyadenylated.
Received May 8, 2001.
Accepted for publication August 2, 2001.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-reductase deficiency in man: an inherited
form of male pseudohermaphroditism. Science 186:1213–1215-reductase. Endocrinology 16:807–812
-reductase inhibitor on androgen physiology in the
immature male rat. Endocrinology 125:2434–2438-reductase
inhibitor-induced abnormalities. Teratology 42:483–496[CrossRef][Medline]
-androstan-17ß-ol-3-one by rat prostate in
vivo and in vitro. J Biol Chem 243:2012–2021-reductase:
two genes/two enzymes. Annu Rev Biochem 63:25–61[Medline]
-reductase in the urogenital tract of the
fetal rat. Mol Endocrinol 9:1561–1570-reductase in the genital tubercle of the fetal rat. Dev
Dynamics 209:117–126[CrossRef][Medline]
-reductase 2 deficiency. Endocr Rev 14:577–593-Reduced
androgens play a key role in murine parturition. Mol Endocrinol 10:380–392-reductase type 1 caused by estrogen
excess. Mol Endocrinol 11:917–927-reductase type 1 deficient mice
is due to impaired cervical ripening. Mol Endocrinol 13:981–992-reductase. Rev Reprod 4:179–183[Abstract]
-reductase in
cultured human fibroblasts. J Biol Chem 251:5895–5900-reductase isozymes. J Biol Chem 268:17404–17412-reductase inhibitor, finasteride, is due to
deficits in copulatory plug formation. Reprod Toxicol 5:353–362[CrossRef][Medline]
-reductase inhibitor. Reprod Toxicol 5:337–346[CrossRef][Medline]
-reductase
inhibitors in androgen-dependent disorders. Curr Opin Chem Biol 1:254–259[CrossRef][Medline]
-reductase
deficiency. Am J Med 62:170–191[CrossRef][Medline]
-reductase deficiency. Clin Endocrinol
(Oxf) 23:43–53[Medline]
-metabolites in parents of male
pseudohermaphrodites with 5-reductase deficiency: detection of
carriers. J Clin Endocrinol Metab 60:553–558-Androstanediol
kinetics in man. J Clin Invest 54:646–653
-,17ß-Androstanediol glucoseronide in plasma: a marker of
androgen action in idiopathic hirsutism. J Clin Invest 69:1203–1206
-androstanediol glucuronide
levels in plasma and random urines in male pseudohermaphroditism caused
by 5-reductase deficiency. Metabolism 38:817–821[CrossRef][Medline]
-reductase inhibitor. J Endocrinol 128:395–401-reductase inhibitor: a possible mechanism. Fund Applied Toxicol 22:211–219[CrossRef][Medline]
-reductase,
suppresses prostatic dihydrotestosterone in men with benign prostatic
hyperplasia. J Clin Endocrinol Metab 74:505–508[Abstract]
-Dihydrotestosterone formation is necessary for embryogenesis of
the rat prostate. Endocrinology 122:1159–1164-reductase inhibitor, finasteride, on the developing
prostate and testis of a marsupial. J Androl 18:123–130-androstane-3,17ß-diol. Proc Natl Acad
Sci USA 97:12256–12259-dihydrotestosterone interact differently with the
androgen receptor to enhance transcription of the MMTV-CAT reporter
gene. Mol Cell Endocrinol 88:15–22[CrossRef][Medline]
-dihydrotestosterone-receptor complexes formed
within cultured human genital skin fibroblasts. J Steroid Biochem 18:121–125[CrossRef][Medline]
-reductase sufficient genital
skin fibroblasts and the effects of 5-reductase deficiency on its
formation. J Steroid Biochem 45:467–476[CrossRef]
-reductase inhibitor on androgen-mediated growth of the dog
prostate. Endocrinology 113:569–573-hydroxysteroid dehydrogenase activity. J Biol
Chem 268:12964–12969This article has been cited by other articles:
 |
|
 |
|
  D. E W Livingstone, S. L Grassick, G. L Currie, B. R Walker, and R. Andrew Dysregulation of glucocorticoid metabolism in murine obesity: comparable effects of leptin resistance and deficiency J. Endocrinol., May 1, 2009; 201(2): 211 - 218. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. B. Askew, R. T. Gampe Jr., T. B. Stanley, J. L. Faggart, and E. M. Wilson Modulation of Androgen Receptor Activation Function 2 by Testosterone and Dihydrotestosterone J. Biol. Chem., August 31, 2007; 282(35): 25801 - 25816. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 U. Simanainen, C. M. Allan, P. Lim, S. McPherson, M. Jimenez, J. D. Zajac, R. A. Davey, and D. J. Handelsman Disruption of Prostate Epithelial Androgen Receptor Impedes Prostate Lobe-Specific Growth and Function Endocrinology, May 1, 2007; 148(5): 2264 - 2272. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Wistuba, J. Mittag, C M. Luetjens, T. G Cooper, C.-H. Yeung, E. Nieschlag, and K. Bauer Male congenital hypothyroid Pax8-/- mice are infertile despite adequate treatment with thyroid hormone J. Endocrinol., January 1, 2007; 192(1): 99 - 109. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. J. Ovando, C. M. Vezina, B. P. McGarrigle, and J. R. Olson Hepatic Gene Downregulation following Acute and Subchronic Exposure to 2,3,7,8-Tetrachlorodibenzo-p-dioxin Toxicol. Sci., December 1, 2006; 94(2): 428 - 438. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Heikkila, R. Prunskaite, F. Naillat, P. Itaranta, J. Vuoristo, J. Leppaluoto, H. Peltoketo, and S. Vainio The Partial Female to Male Sex Reversal in Wnt-4-Deficient Females Involves Induced Expression of Testosterone Biosynthetic Genes and Testosterone Production, and Depends on Androgen Action Endocrinology, September 1, 2005; 146(9): 4016 - 4023. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Iranmanesh and J. D. Veldhuis Combined Inhibition of Types I and II 5 {alpha}-Reductase Selectively Augments the Basal (Nonpulsatile) Mode of Testosterone Secretion in Young Men J. Clin. Endocrinol. Metab., July 1, 2005; 90(7): 4232 - 4237. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Cheung-Flynn, V. Prapapanich, M. B. Cox, D. L. Riggs, C. Suarez-Quian, and D. F. Smith Physiological Role for the Cochaperone FKBP52 in Androgen Receptor Signaling Mol. Endocrinol., June 1, 2005; 19(6): 1654 - 1666. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. A. Henderson and B. Robaire Effects of PNU157706, a Dual 5{alpha}-Reductase Inhibitor, on Rat Epididymal Sperm Maturation and Fertility Biol Reprod, February 1, 2005; 72(2): 436 - 443. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Umar, M. P. Ooms, T. M. Luider, J. A. Grootegoed, and A. O. Brinkmann Proteomic Profiling of Epididymis and Vas Deferens: Identification of Proteins Regulated during Rat Genital Tract Development Endocrinology, October 1, 2003; 144(10): 4637 - 4647. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Killian, K. Pratis, R. J. Clifton, P. G. Stanton, D. M. Robertson, and L. O'Donnell 5{alpha}-Reductase Isoenzymes 1 and 2 in the Rat Testis During Postnatal Development Biol Reprod, May 1, 2003; 68(5): 1711 - 1718. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Ivell and S. Hartung The molecular basis of cryptorchidism Mol. Hum. Reprod., April 1, 2003; 9(4): 175 - 181. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Rosati, G. Danza, A. Guarna, N. Cini, M. L. Racchi, and M. Serio New Evidence of Similarity between Human and Plant Steroid Metabolism: 5{alpha}-Reductase Activity in Solanum malacoxylon Endocrinology, January 1, 2003; 144(1): 220 - 229. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Matsui, M. Sakari, T. Sato, A. Murayama, I. Takada, M. Kim, K.-i. Takeyama, and S. Kato Transcriptional regulation of the mouse steroid 5{alpha}-reductase type II gene by progesterone in brain Nucleic Acids Res., March 15, 2002; 30(6): 1387 - 1393. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
0.05) from those of wild-type mice.
A, Prostate; B, seminal vesicles plus coagulating glands; C, testes.
