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-Reductase Type 1 Caused by Estrogen Excess
Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, Texas 75235-9046
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-reductase
type 1 have a decreased litter size. The average litter in homozygous
deficient females is 2.7 pups vs. 8.0 pups in wild type
controls. Oogenesis, fertilization, implantation, and placental
morphology appear normal in the mutant animals. Fetal loss occurs
between gestation days 10.75 and 11.0 commensurate with a midpregnancy
surge in placental androgen production and an induction of
5-reductase type 1 expression in the decidua of wild type mice.
Plasma levels of androstenedione and testosterone are 2- to 3-fold
higher on gestation day 9, and estradiol levels are chronically
elevated by 2- to 3-fold throughout early and midgestation in the
knockout mice. Administration of an estrogen receptor antagonist or
inhibitors of aromatase reverse the high rate of fetal death in the
mutant mice, and estradiol treatment of wild type pregnant mice causes
fetal wastage. The results suggest that in the deficient mice, a
failure to 5-reduce androgens leads to their conversion to
estrogens, which in turn causes fetal death in midgestation. These
findings indicate that the 5-reduction of androgens in female
animals plays a crucial role in guarding against estrogen toxicity
during pregnancy. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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). In one pathway, they are converted
into 5-reduced androgens by steroid 5-reductase isozymes (1). In
the other, they are converted into estrogens by the aromatase enzyme, a
cytochrome P450 of the endoplasmic reticulum (2). Conversion to these
end products is irreversible and mutually exclusive in that
5-reduced androgens are not aromatase substrates and estrogens are
not 5-reductase substrates (Fig. 1). The actions of 5-reduced
androgens and estrogens oppose one another: 5-reduced androgens
masculinize while estrogens feminize. Under physiological conditions in
each sex, a delicate balance is established in which appropriate
amounts of androstenedione and testosterone are converted into one or
the other class of hormones. The set point of this fulcrum is
differentially sustained in the two sexes by regulating the production
of testicular androgens in males and the expression of the aromatase
enzyme in females.
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-reduced
androgen-estrogen ratio due to genetic defects in the enzymes that
determine these metabolic fates (Fig. 1
). For example, mutations in the
aromatase gene cause an increase in the androgen-estrogen ratio leading
to virilization and polycystic ovarian disease in affected women (3, 4). Mutations in the 17ß-hydroxysteroid dehydrogenase type 3 gene
decrease the androgen-estrogen ratio and cause gynecomastia in affected
men (5). Two different 5-reductase genes encode the type 1 and type
2 isozymes in several mammalian species (2). Mutations in the human
5-reductase type 2 gene, which is normally expressed in the
urogenital tract and liver, cause male pseudohermaphroditism but do not
alter the 5-reduced androgen-estrogen ratio in men or women (6, 7).
This outcome is presumably due to the ability of the type 1 isozyme,
which is expressed in the liver and skin (8), to compensate for the
absence of the type 2 isozyme in affected individuals.
No mutations in the 5-reductase type 1 gene have yet been identified
in humans, perhaps because of the presence of the type 2 isozyme in
multiple tissues. However, the female mouse is an ideal animal model in
which to study the type 1 isozyme because very little 5-reductase
type 2 is expressed in the tissues of this mammal (9). To take
advantage of this expression pattern, and to explore the physiological
role of the type 1 isozyme, a line of mice with a mutation in the
5-reductase type 1 gene was developed (9). The absence of this
isozyme had no obvious effect in males but caused a parturition defect
and a reduction in litter size in females. The parturition defect was
traced to a failure of the uterus to synthesize 5-reduced androgens
in late gestation (9), a result that implicated the type 1 isozyme as
playing an anabolic role in steroid hormone metabolism.
In the current study, we show that an imbalance in the ratio of
5-reduced androgens to estrogens underlies the reduction in litter
size in the 5-reductase type 1-deficient mice. Approximately half of
embryos die between gestation days 10.75 and 11 in animals homozygous
for a null mutation in the type 1 gene
(Srd5a1-/- mice). Fetal loss correlates with a
transient increase in the plasma levels of androstenedione and
testosterone and with a chronic elevation of plasma estradiol. The
increase in the latter hormone is shown to cause fetal death. The
results indicate that the type 1 isozyme plays an important catabolic
role in sex steroid homeostasis by controlling the availability of
substrate for estrogen synthesis.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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). Similarly, the litter size of
Srd5a1+/- heterozygotes was 8.5. In contrast,
the average number of pups in the litters of
Srd5a1-/- homozygous mice was only
2.7. Essentially equal percentages of male and female pups were born to
homozygous mothers (51% male, 49% female, n = 78). When
homozygous mutant males were mated with wild type females, the litters
were of normal size (
= 8.0), whereas crosses
between wild type males and homozygous mutant females produced small
litters ( = 3.0). Thus, the 5-reductase type
1 genotype of the mother, and not that of her mate or pups, determined
litter size.
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. In both
wild type and Srd5a1-/- females,
about 80% of embryos survived through gestation day 10.75. However,
approximately half of the surviving embryos in the mutant mice died
between day 10.75 and 11.0, whereas no subsequent embryo loss occurred
in the wild type mice (Fig. 2).
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-Reductase Type 1-reductase type 1 in wild
type mice, RNA was isolated from the placentas and surrounding decidua,
ovaries, livers, and brains of pregnant and nonpregnant animals and
subjected to blot hybridization. The steady state levels of type 1 mRNA
did not change during pregnancy in the ovaries (data not shown);
however, the levels of type 1 mRNA transiently increased in the
placenta/decidua between days 6 and 10 (Fig. 3A). In the
brain, the level of type 1 mRNA was low in nonpregnant animals but
substantially increased upon pregnancy (Fig. 3B). In the liver, a
transient and modest increase in type 1 mRNA was detected on days 9–14
of gestation (Fig. 3C). The levels of control mRNAs (ß-actin in
the placenta/decidua, CRH in the brain, and cyclophilin in the liver)
remained constant during these times (Fig. 3).
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-reductase type 1 mRNA in
the placenta/decidua led to an increase in enzyme mass, immunoblotting
assays were performed (Fig. 4). A protein of
approximately 22 kDa that comigrated with a recombinant 5-reductase
type 1 standard was induced in the tissue on gestation days 6 through
11 coincident with the observed increase in mRNA. The induction of
5-reductase type 1 protein in the uterus was monitored as a control.
In agreement with previous results (9), the content of uterine
5-reductase type 1 increased in late but not midgestation (Fig. 4).
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-Reductase Type 1 Expression in the Decidua-reductase type 1 on
gestation day 8. An antisense probe revealed type 1 transcripts in
decidual cells of the tissue (Fig. 5, A and B), whereas
a sense probe produced no specific hybridization pattern (Fig. 5, C and
D). Cells containing the type 1 mRNA were concentrated in the decidua
basalis, which is the region of the decidua opposed to the mesometrial
aspect of the uterus. No specific hybridization was detected in the
decidua capsularis, which is located on the antimesometrial side of the
uterus, or in any of the extraembryonic membranes of the
fetal-placental unit (Fig. 5, A and B). Cells of the decidua basalis
differentiate from endometrial connective tissue cells of the uterus
(10). Thus, the finding that maternal decidual cells express
5-reductase type 1 is consistent with results from the breeding
experiments (Table 1), which showed that the fecundity defect was
maternal in origin.
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-reductase type 1-deficient mice was associated with
misexpression of one or more of the placental lactogens, the expression
of placental lactogen 1 and 2 and proliferin were compared in wild type
and knockout placentas in RNA blotting experiments. No differences in
the induction patterns of these peptide hormone mRNAs were detected
(data not shown). In agreement with these findings, no obvious
morphological differences were apparent at gross or microscopic levels
in the day 11 placentas associated with living or dead embryos in the
mutant animals, nor were the patterns of desmin expression, a product
of maternal decidual cells (12), different between these two tissue
groups (data not shown).
Serum Steroid Hormone Levels
The lack of a gross placentation defect in the mutant mice
suggested that fetal death might be due to the absence of an essential
steroid produced by 5-reductase type 1 or to the presence of a toxic
metabolite that was normally catabolized by the enzyme. To test these
hypotheses, plasma levels of androstenedione, testosterone,
dihydrotestosterone, and estradiol were measured by RIA in pregnant
wild type and knockout mice. The data of Fig. 6 show
that in wild type females serum levels of the three androgens were low
on days 6 and 7 of gestation, transiently increased between days 8 and
10, with a peak on day 9, and then returned to baseline levels. In
knockout females, similar temporal increases in androstenedione and
testosterone, but not dihydrotestosterone, were observed. However, the
mean serum concentrations of androstenedione and testosterone were as
much as 5-fold higher in the mutant animals during this period (Fig. 6). A statistical analysis indicated that the androstenedione levels on
day 9 in knockout mice were not significantly different from those in
wild type animals (P = 0.1, Wilcoxon rank sum test),
whereas the testosterone and dihydrotestosterone levels were
significantly different (P = 0.03 and 0.01,
respectively).
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), which led to a statistically significant difference on day 18
(P = 0.04) and a nonsignificant difference on day 19
(P = 0.8). Together these data suggested that plasma levels of both estrogen precursors (androstenedione and testosterone) and estradiol itself were higher before and during the time of fetal death in the mutant mice.
A Bioassay for Fetal Death
To determine whether excess androgens or estradiol caused fetal
death, we developed a bioassay in which pellets containing different
amounts of individual steroids were subcutaneously implanted on day 5
of gestation. The pregnancies were allowed to proceed until gestation
day 11 or 12, at which time the animals were killed and the living
embryos were counted. The results obtained in wild type and
Srd5a1-/- mice are summarized in
Table 2. Pellets containing 0.5 mg of androstenedione
killed approximately half of the embryos in wild type mice and appeared
to increase the frequency of embryo death in the knockout animals.
Testosterone had no statistically significant effects in two wild type
mice in this assay, and in the mutant mice this hormone may have
conferred a mild protective effect (Table 2). Estradiol at doses
between 0.5 and 0.005 mg killed essentially all embryos in mice of both
genotypes. When the estradiol dose was decreased to 0.0025 mg, the
frequency of embryo survival in the wild type animals (
50%) was
approximately the same as that seen in the untreated knockout mice
(Table 1). This dose did not decrease embryo survival in the mutant
mice. Ten additional 5-reduced steroids or combinations of steroids,
which are listed in Materials and Methods, were tested in
the assay and did not affect embryo survival in animals of either
genotype (data not shown).
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). Bleeding
did not occur in the abdominal space, but rather was limited to the
uterine lumen and the encircled fetal-placental units.
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), estrogen was
thought to be the more likely hormone responsible for fetal death. If
this interpretation is correct, then compounds that inhibit aromatase
or block estrogen binding to the estrogen receptor should restore
normal embryo survival to
Srd5a1-/- mice. The data of Table 3 show that injection of aromatase inhibitors
(4-hydroxyandrostenedione or Arimidex) on gestation days 6 through 10
prevented excess fetal death in the mutant mice. Approximately 79% of
the knockout embryos survived in these experiments, which was a
frequency identical to that observed in untreated wild type mice (Fig. 2). Similar results were obtained when pellets containing either 5.0 or
7.5 mg of the estrogen receptor antagonist tamoxifen were implanted on
day 5 of gestation: administration of this drug led to more than 70%
embryo survival frequencies (Table 3).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-reductase type 1 in mice causes a fecundity
defect of maternal origin. The pregnancies of these animals are normal
until day 10.75, but between gestation day 10.75 and 11, approximately
half of the embryos die. In wild type mice, expression of
5-reductase type 1 increases to high levels in the decidua between
gestation days 6 and 11. This increase correlates with a burst of
androgen synthesis by the placenta that leads to elevated plasma levels
of androstenedione, testosterone, and dihydrotestosterone. In the
knockout mice, serum levels of androstenedione and testosterone
increase during this same time period, whereas plasma
dihydrotestosterone levels remain unchanged. The serum estradiol levels
are elevated throughout early and midgestation in the
Srd5a1-/- mice. Antagonists of
estrogen synthesis and action correct the fecundity defect in knockout
animals, a finding consistent with the concept that fetal death in
midpregnancy is caused by estradiol toxicity. This scenario is further
supported by the observation that exogenous estradiol causes fetal
death in the offspring of wild type mothers. Given the known pathways
of androgen and estrogen biosynthesis (Fig. 1), the simplest
interpretation of these results is that 5-reductase type 1 normally
plays an important catabolic role in pregnant females by converting
androgens to their nonaromatizable forms and that prevention of this
action causes estradiol levels to increase and cause fetal demise.
Steroid 5-reductase occurs in two isozymic forms called type 1 and
type 2 that were initially postulated to fulfill distinct physiological
roles. The type 1 isozyme was predicted to play a catabolic role in
steroid hormone metabolism based on a high level of expression in
tissues that break down steroids such as the liver and kidney and a
micromolar affinity for steroid substrates (13). Conversely, a
preferential expression of the type 2 isozyme in androgen target
tissues of the male reproductive tract, coupled with a nanomolar
affinity for steroid substrates, implied an anabolic role for the type
2 isozyme. Men that lack the type 2 isozyme exhibit symptoms of
androgen deficiency, which confirms the anabolic role of this isozyme
in humans (6).
The physiological role of the type 1 isozyme has been assessed in mice
containing a null allele at the Srd5a1 locus. The analysis
of these animals reveals that the function of the type 1 isozyme is
more complex than originally proposed in that it plays both anabolic
and catabolic roles in pregnant female mice. In an anabolic capacity,
the type 1 isozyme synthesizes a 5-reduced androgen, most likely
5-androstane-3,17ß-diol, which is required for the delivery of
young at term (9). In a catabolic capacity, the current studies show
that the type 1 isozyme breaks down androgens and, in so doing,
prevents their conversion to estrogens.
Male and female mice do not appear to require 5-reductase type 1 for
phenotypic sexual differentiation or for the maintenance of steroid
hormone homeostasis under normal conditions (9). The adverse
consequences of enzyme loss are not realized until pregnancy, when the
endocrine system of the female changes drastically in ways that reveal
essential catabolic and anabolic functions of the type 1 isozyme. The
first requirement is one of catabolism and is manifest in midpregnancy
at a time when circulating levels of several androgens are transiently
increased to very high levels. These increases have been noted before
(14, 15, 16, 17) and appear to be peculiar to the mouse in that the rat does
not exhibit midpregnancy androgen surges (18). The source of the murine
androgens is the placenta (17), and we show here that 5-reductase
type 1 increases in the associated decidua with the same time course as
androgen biosynthesis (
Figs. 3–5). The physiological role of androgens
at midpregnancy is not known.
The serum levels of estradiol that accumulate in the knockout mice are
consistently at least 2-fold higher throughout days 6–14 of gestation,
which span the time of fetal death (Fig. 6). Estrogen is among the most
powerful of steroid hormones in mammals (19), and even slight changes
in the estrogen response system can affect reproduction (20). This
potency is further demonstrated by the ability of 21-day release
pellets containing as little as 5 µg of hormone to kill all fetuses
in a wild type animal (Table 2). The identification of estradiol as the
toxic steroid in the type 1-deficient mice is further supported by the
ability of an estrogen antagonist and aromatase inhibitors to reverse
the fecundity defect in the mutant animals (Table 3). We conclude that
the most likely culprit causing fetal death in the knockout animals is
estrogen excess.
The aromatase mRNA (and presumably protein) are not expressed in the
murine placenta (our unpublished observations); thus the placenta is
not the source of excess serum estradiol in the mutant mice. Rather,
the androgenic precursors produced by this tissue and others must be
converted into estrogens in another organ, such as the ovary, in which
aromatase is actively expressed throughout gestation (21, 22), or in
some unidentified extraglandular location. The levels of aromatase mRNA
in the ovary during gestation were similar between wild type and
knockout females (data not shown), suggesting that a supraphysiological
level of this enzyme in the mutant mice was not the cause of increased
plasma estradiol. The absence of 5-reductase in the liver may
contribute to higher estradiol levels since circulating androgens would
be expected to have a longer half-life and thus a greater chance of
conversion into estrogens. In addition, the induction of 5-reductase
type 1 mRNA in the brain with pregnancy (Fig. 2B) may serve to regulate
estrogen levels in wild type mice through classical endocrine feedback
mechanisms involving the hypothalamic-pituitary-gonadal axes.
There are two apparent inconsistencies in the data reported here.
First, we did not observe a direct correlation between serum estradiol
and androgen levels in the knockout mice. Thus, on gestation day 9,
when serum levels of androstenedione and testosterone doubled or
tripled in the knockout mice, there was no corresponding spike of
estradiol (Fig. 6). A brief elevation of estradiol was observed on days
12–15, but this rise was several days after the day 9 peak of
androgens. Second, the time of fetal death (day 10.75–11) did not
correlate with the largest observed increase in plasma androgens (day
9), as would be expected if these precursors were converted into
circulating estrogens.
Several possible explanations exist for these inconsistencies. First,
chronic exposure of the fetuses to excess estradiol before day 10.75
may lead to death shortly thereafter. Second, the embryos may become
hypersensitive to estrogen around day 10.75 as a consequence of a
developmental change in the fetal-placental unit. Third, death may be
caused by an acute accumulation of estradiol in the placenta/decidua
before the time of death that is not reflected in plasma hormone
levels. This latter explanation is supported by the observation that
5-reductase type 1 is induced in the decidua on gestation days 6–11
(Fig. 3). In normal mice, this induction would protect the fetus from
perilous estrogen accumulation. Little is known concerning the turnover
of estrogens and androgens in wild type mice, much less in mutant
animals; thus we can not interpret these results from the standpoint of
kinetics. Despite these overall uncertainties, the ability of excess
estradiol to recapitulate the knockout phenotype in wild type mice and
of estrogen antagonists to restore fecundity in the mutant mice argues
in favor of an estrogen excess theory of fetal death. Finally, a mild
protective effect was observed upon testosterone administration to
knockout mice (Table 2). This result may be a consequence of the
ability of activated androgen receptor to oppose the deleterious
effects of excess estradiol.
The mechanism by which estradiol brings about loss of fetal life in
midgestation has not been explored. Inspection of the uteri of
estrogen-treated animals revealed evidence of hemorrhage, which was
limited to the fetal-placental units within the organ (Fig. 7). Similar
observations were made in some, but not all, untreated knockout mice.
Estradiol may thus alter the permeability or development of the
maternal and fetal blood vessels, causing the fetuses to bleed to
death. Estradiol affects the production of nitric oxide by the
endothelium (reviewed in 23 , which suggests the testable
hypothesis that aberrant production of this potent vasodilator may lead
to excess bleeding and fetal death in the
Srd5a1-/- mice. The observation that tamoxifen
blocks estrogen action in the knockout mice (Table 3) suggests that the
hormone is acting through a receptor-based mechanism; however, the
target tissue and the mechanism by which tamoxifen exerts this
protective effect remain to be determined. No differences in the level
of estrogen receptor mRNA were detected in the uteri and ovaries of
mutant mice vs. wild type controls (data not shown). Thus,
it is unlikely that enhanced estrogen sensitivity brings about fetal
death in the mutant animals.
The administration of estrogens during early, mid, and late gestation
has previously been shown to disrupt implantation (24), cause fetal
death and resorbtion (25), or delay parturition (26). In the knockout
mice studied here, implantation appears normal; however, fetal death
occurs in midgestation as reported (25). The delay in parturition seen
when ovarian extracts were administered late in gestation (26) is
similar to the parturition defect described in the type 1-deficient
mice (9). However, tamoxifen did not reverse the parturition defect in
these animals (data not shown), whereas 5-reduced androgens did
(9).
Finally, we can ask whether the current results have any bearing on
reproduction in women. There are well-documented cases of recurrent
miscarriage occurring in midgestation (27), which ostensibly could be
due to estrogen excess caused by the absence of the 5-reductase type
1 isozyme. However, unlike the rodent placenta, the human placenta is
laden with aromatase, which produces very high levels of estrogen in
the amniotic fluid (28). Thus, the human fetal-placental unit must have
developed a mechanism that protects the fetus from the toxic effects of
estrogen that we observe in mice.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Embryo survival in wild type and homozygous knockout mice was determined as follows. Pregnant females arising from timed matings were killed on gestation days 9–19 and their uteri were removed. The number of fetuses was determined by counting implantation sites. Fetal-placental units were dissected from the uteri and scored for the presence or absence of a beating heart in the embryo with the aid of a low-power microscope.
Organs were dissected from timed pregnant females on the indicated days of gestation. Placental tissues removed on gestation days 6–8 refer to fetal-placental units and the immediately opposed decidua. After gestation day 8, embryos were removed from this tissue before RNA or protein isolation.
All animal experiments were carried out using protocols approved by the University of Texas Southwestern Medical Center Institutional Animal Care and Research Advisory Committee.
RNA Blotting
RNA isolation and blotting were performed as previously
described (9). Radiolabeled cDNA probes were prepared by random
hexanucleotide priming or by the PCR (30). Complementary DNA probes
included mouse 5-reductase type 1 (9), human ß-actin (31), mouse
CRH (32), mouse placental lactogen 1 (33), mouse placental lactogen 2
(34), mouse proliferin (35), mouse estrogen receptor (36), rat
aromatase (37), and rat cyclophilin (38). These were obtained from
individual investigators (ß-actin, CRH, proliferin, aromatase) or by
the PCR using published cDNA sequences. Each RNA blotting experiment
was repeated two to three times with samples isolated from different
sets of animals.
Immunoblotting
Immunoblotting of 5-reductase type 1 protein was carried out
as previously described (8). The primary antibody used in these
experiments was raised against a multiantigen peptide (Bio-Synthesis,
Lewisville, TX) composed of the amino acid sequence
RAKEHHEWYLRKFEEYPKSRKILI, which corresponds to residues 233–256 of the
human type 1 isozyme (2). Before use, the antiserum was
affinity-purified on a Sepharose 4B column to which a peptide
(LRKFEEYPKFRKIIIP) was coupled (39). This sequence is a hybrid derived
from the carboxy termini of the rat and human type 1 isozymes (2). The
purified antiserum was used at a concentration of 2 µg/ml in the
blotting reactions. Antigen-antibody complexes were detected by
enhanced chemiluminescence. The placental expression of the
intermediate filament protein desmin was followed by immunoblotting
with an antibody from Sigma (D-8281, St. Louis, MO). Each
immunoblotting experiment was repeated two or more times using tissues
isolated from different animals.
In Situ mRNA Hybridization
Transcripts of the 5-reductase type 1 gene were detected by
in situ hybridization in 5 µm sections of day 8
placenta/decidua as described previously (40).
[33P]-Radiolabeled RNA probes in sense and antisense
orientation were transcribed in vitro from a cDNA encoding
amino acids 1–94 of the murine type 1 enzyme. Exposure times were 21
days. After development, tissue sections were photographed under
lightfield and darkfield illumination on a Leitz Labrolux S
Photomicroscope (Rockleigh, NJ) outfitted with a Bunton low
magnification darkfield illuminating condenser.
Hormone Measurements
Blood was drawn from the inferior vena cava of pregnant animals
between days 6 and 19 of gestation and 1 day postpartum (labeled as
d20). Blood samples were collected from three to seven wild type or
Srd5a1-/- females for each time point with the
exception of day 20, wild type, for which steroids in only one animal
were measured. Serum was collected and stored at -20 C until steroid
analyses were performed.
Estradiol, androstenedione, testosterone, and dihydrotestosterone levels were quantified in serum by RIA after chromatographic separation of steroids on Sephadex LH-20 columns (Pharmacia, Inc., Piscataway, NJ). Steroid measurements were performed at the Oregon Regional Primate Research Center (Beaverton, OR). All samples were analyzed in a single, large experiment. The intraassay coefficients were: estradiol, 5.8%; androstenedione, 12%; testosterone, 1.0%; dihydrotestosterone, 9.8%. The average blank values were 1.2 pg for estradiol, 3.8 pg for androstenedione, 2.8 pg for testosterone, and 7.0 pg for dihydrotestosterone.
Steroid Pellet Studies
On day 5 of pregnancy (plug day = 0) animals were
anesthetized by intraperitoneal injection of 1.6 mg Nembutal (Abbott
Laboratories, North Chicago, IL) dissolved in approximately 100 µl
saline. A 1-cm incision was made through the back skin, one or more
steroid pellets (Innovative Research of America, Sarasota, FL) were
inserted, and the incision was closed with wound clips. Animals were
thereafter killed on day 11 or 12 of gestation, and embryo survival was
determined by the presence of a beating heart. Twenty-one-day time
release pellets were employed that contained the steroids or drugs
indicated in Tables 2 and 3.
Several additional 5-reduced steroids were tested for their ability
to reverse the fecundity defect in Srd5a1-/-
mice. Pellets were inserted as described above on gestation day 5.
Pregnant females were then killed on gestation day 17 or 18, and the
number of live and resorbed fetuses was determined. None of the
following 5-reduced steroids were effective in reversing the
fecundity defect: dihydroprogesterone, 1.5 or 25 mg, 30-day release;
dihydrotestosterone, 0.5 or 1.5 mg, 21-day release;
5-androstan-3,17ß-diol, 0.13 or 0.91 mg, 14-day release;
5-androstan-3ß,17ß-diol, 0.13 mg, 14-day release;
androstanedione, 0.13 mg, 14-day release; androsterone, 0.22 mg, 21-day
release; 5-androstan-3,17ß-diol together with
5-androstan-3ß,17ß-diol, 0.13 mg each, 14-day release;
epiandrosterone, 1 mg, 14-day release; allodihydrocortisone, 7.5 mg,
14-day release.
Aromatase Inhibitor Studies
The aromatase inhibitors 4-hydroxyandrostenedione (Sigma, St.
Louis, MO) and Arimidex [ZD1033,
2,2'-(5-(IH-1,2,4-triazol-1-ylmethyl)-1,3-phenylene)-bis(2-methylpropiononitrile),
Zeneca Pharmaceuticals, Maccelsfield, England], were administered on
days 6–10 of gestation by daily subcutaneous injections.
4-Hydroxy-androstenedione (1.5 mg) was injected daily in 75 µl
propylene glycol. Arimidex (0.15 mg) was injected daily in 50 µl
triolene (Sigma, St. Louis, MO). On gestation day 11 or 12, the treated
female was killed, and the number of live and dead embryos was
determined by observation of a heartbeat.
Statistical Analysis
The Students t test and Wilcoxon rank sum test were
used to determine significant differences in litter sizes reported in
Table 1. The significance of changes in embryo survival with
drug/hormone treatments (Tables 2 and 3) was determined using Fisher’s
exact test. The Mantel-Haenszel 
2 test with continuity
correction was used to compare significance of embryo survival data in
wild type vs. Srd5a1-/- animals from day
9 to day 10.75 and from days 11 through 19 (Fig. 2). Steroid hormone
profiles of wild type and Srd5a1-/- animals
were compared using the Wilcoxon rank sum test. For all statistical
tests employed, P < 0.05 indicates significance.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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This research was supported by grants from the NIH (GM-43753), the Perot Family Foundation, and a postdoctoral fellowship awarded to M.S.M. from the Lalor Foundation.
Received for publication December 5, 1996. Revision received January 31, 1997. Accepted for publication February 25, 1997.
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