Developmental Roles of the Steroidogenic Acute Regulatory Protein (StAR) as Revealed by StAR Knockout Mice
Tomonobu Hasegawa,
Liping Zhao,
Kathleen M. Caron,
Gregor Majdic,
Takashi Suzuki,
Soichiro Shizawa,
Hironobu Sasano and
Keith L. Parker
Departments of Internal Medicine and Pharmacology (T.H., L.Z.,
G.M., K.L.P.) University of Texas Southwestern Medical Center
Dallas, Texas 75235
Department of Pathology (K.M.C.)
University of North Carolina-Chapel Hill Chapel Hill, North
Carolina 27599
Department of Pathology (T.S., S.S., H.S.)
Tohoku University School of Medicine Sendai, Miyagi, Japan,
980-8575
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ABSTRACT
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Steroidogenic acute regulatory protein (StAR) is
essential for adrenal and gonadal steroidogenesis, stimulating the
translocation of cholesterol to the inner mitochondrial membrane where
steroidogenesis commences. StAR mutations in humans cause congenital
lipoid adrenal hyperplasia (lipoid CAH), an autosomal recessive
condition with severe deficiencies of all classes of steroid hormones.
We previously described StAR knockout mice that mimic many features of
lipoid CAH patients. By keeping StAR knockout mice alive with
corticosteroid replacement, we now examine the temporal effects of StAR
deficiency on the structure and function of steroidogenic tissues. The
adrenal glands, affected most severely at birth, exhibited progressive
increases in lipid deposits with aging. The testes of newborn StAR
knockout mice contained scattered lipid deposits in the interstitial
region, presumably in remnants of fetal Leydig cells. By 8 weeks of
age, the interstitial lipid deposits worsened considerably and were
associated with Leydig cell hyperplasia. Despite these changes, germ
cells in the seminiferous tubules appeared intact histologically,
suggesting that the StAR knockout mice retained some capacity for
androgen biosynthesis. Sperm maturation was delayed, and the germ cells
exhibited histological features of apoptosis, consistent with
suboptimal androgen production. Immediately after birth, the ovaries of
StAR knockout mice appeared normal. After the time of normal puberty,
however, prominent lipid deposits accumulated in the interstitial
region, accompanied by marked luteinization of stromal cells and
incomplete follicular maturation that ultimately culminated in
premature ovarian failure. These studies provide the first systematic
evaluation of the developmental consequences of StAR deficiency in the
various steroidogenic organs.
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INTRODUCTION
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A critical component of regulated steroidogenesis is the induction
of steroid biosynthesis by pituitary trophic hormones. Temporally, this
hormonal induction is divided into two phases: acute effects that
reflect increased mobilization and delivery of cholesterol precursor to
the inner mitochondrial membrane, and chronic effects that result from
increased transcription of genes that encode key components of the
steroidogenic complex (reviewed in Ref. 1). A mitochondrial
phosphoprotein, designated the steroidogenic acute regulatory protein
(StAR), was identified and proposed to play key roles in the acute
induction of steroidogenesis (2). Compelling evidence for StARs
essential role in steroidogenesis came from analyses of patients with
congenital lipoid adrenal hyperplasia (lipoid CAH), an autosomal
recessive disorder characterized by severely impaired adrenal and
gonadal steroidogenesis coupled with characteristic lipid deposits in
the steroidogenic tissues (3, 4).
To further our understanding of the pathogenesis of lipoid CAH, we
generated StAR knockout mice (5). These mice established essential
roles of StAR in regulated steroidogenesis in mice and demonstrated a
spectrum of severity of lipid deposits in the primary steroidogenic
cells. In newborn StAR knockout mice, the adrenal glands lacked their
normal cellular architecture and had abundant lipid deposits,
presumably reflecting the fact that the mouse adrenal cortex normally
produces steroids in utero. In contrast, the testes
contained only scattered lipid deposits, while the ovaries appeared
completely normal. Based on a similar hierarchy in the impairment of
steroid production in patients with lipoid CAH, Bose et al.
(4) proposed a two-hit model of lipoid CAH. According to this model,
lipoid CAH patients initially retain some capacity for StAR-independent
steroidogenesis; thereafter, progressive lipid accumulation in
steroidogenic cells, driven at least partly by trophic hormone
stimulation, kills the cells and completely abrogates steroidogenic
capacity.
In this report, we used corticosteroid replacement to keep StAR
knockout mice alive for differing periods of time after birth, thereby
allowing us to assess the temporal effects of StAR deficiency. Our
results, which demonstrate progressive pathological changes in the
gonads after the time of normal sexual maturation, strongly support the
two-hit model of lipoid CAH.
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RESULTS
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Steroid Hormone Levels in StAR Knockout Mice
Previous samples for analyses of hormone levels in newborn
StAR knockout mice potentially contained maternally derived steroids.
By rescuing StAR knockout mice with corticosteroid replacement as
described in Materials and Methods, we were able to examine
the effect of StAR deficiency on circulating steroid hormones later in
life. As shown in Table 1
, plasma
corticosterone, the major circulating glucocorticoid in mice, was
markedly decreased in StAR knockout mice at 8 weeks of age, although
levels still exceeded the lower limit of assay sensitivity. This
finding suggests that the adrenal cortex retained some residual
capacity for corticosteroid production, even at 8 weeks. Although
markedly decreased, plasma testosterone in males also was detectable.
In females, progesterone was decreased and ovarian morphology was
markedly abnormal (see below), Surprisingly, estradiol levels in StAR
knockout females did not differ significantly from wild-type values.
Given the low levels of circulating estradiol, it is unclear whether
this finding reflects unimpeded production of ovarian steroids or
limitations in the assay that mask defects in ovarian steroidogenesis.
As discussed below, the uterus and oviducts of StAR knockout females
were markedly hypoplastic, suggesting that estrogen action on target
tissues is impaired despite the "normal" circulating levels.
Developmental Effects of StAR Deficiency in the Adrenal Glands
The adrenal cortex is the best studied steroidogenic organ
in human patients with lipoid CAH. In StAR knockout mice, the adrenal
cortex, which exhibited the most marked abnormalities in newborn pups
(5), underwent progressive increases in lipid deposition, as revealed
by vacuolated regions in hematoxylin-eosin-stained sections (Fig. 1
) and marked lipid deposits demonstrated
by oil red O staining (Fig. 2
). At 8
weeks, these changes were especially marked in the zona glomerulosa and
zona fasciculata, while the zona reticularis was relatively preserved
and the adrenal medulla lacked discernable pathological findings.
Unlike human patients with lipoid CAH (6), cholesterol crystals were
not present in the mouse adrenal sections, and there was no evidence of
adrenocortical hyperplasia at 8 weeks (i.e. the
cortex-medulla ratio was not increased in StAR knockout adrenals). The
postnatal administration of corticosteroids to StAR knockout mice
before weaning at 3 weeks may have partially suppressed ACTH release
and ameliorated the progression of the adrenal lesions. By 16 weeks,
adrenocortical hyperplasia was apparent, accompanied by markedly
increased lipid deposits (data not shown).

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Figure 1. Histology of Wild-Type and StAR Knockout Adrenal
Glands from Mice at Different Ages
As described in Materials and Methods, adrenal glands
were isolated from wild-type (WT) and StAR knockout (KO) mice either
immediately after birth (PO) or at 8 weeks of age (8w), and sections
were prepared and stained with hematoxylin-eosin. Panel A, WT, P0.
Panel B, WT, 8w. Panel C, KO, P0. Panel D, KO, 8w. c, Cortex; m,
medulla.
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Figure 2. Oil Red O Staining of Lipid Deposits in
Steroidogenic Organs of Wild-Type and StAR Knockout Mice
Steroidogenic organs were harvested shortly after birth (P0, adrenal
and testis; P7, ovary) or at 8 weeks of age from wild-type or StAR
knockout mice. Panel A, WT adrenal, P0. Panel B, WT adrenal, 8w. Panel
C, WT testis, P0. Panel D, WT testis, 8w. Panel E, WT ovary, P7. Panel
F, WT ovary, 8w. Panel G, KO adrenal, P0. Panel H, KO adrenal, 8w.
Panel I, KO testis, P0. Panel J, KO testis, 8w. Panel K, KO ovary, P7.
Panel L, KO ovary, 8w.
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Developmental Effects of StAR Deficiency in the Testes
The testes of lipoid CAH patients have been studied in less
detail (7, 8). Because concurrent treatment with sex steroids was not
required for survival, we were able to study the full developmental
effects of StAR deficiency in the gonads of StAR knockout mice.
Anatomically, the testes in StAR knockout mice are found in the
inguinal canal. A similar position is observed in testicular
feminization mice, which have deficient androgen action due to a
mutation in the androgen receptor (9). As shown in Figs. 2
and 3
, the
testes of newborn StAR knockout mice appeared relatively intact, with
only scattered foci of lipid deposits in the interstitial region. These
deposits presumably are in remnants of fetal Leydig cells, a distinct
population of cells that produce androgens in utero and then
regress postnatally (10, 11). As StAR knockout mice aged, lipid
accumulated throughout the interstitial region, consistent with
recruitment of the adult population of Leydig cells. At 8 weeks of age
(Fig. 3
), the cytoplasm of the Leydig
cells appeared foamy, and oil red O staining (Fig. 2
) revealed
progression in the severity of their lipid deposits. Moreover, Leydig
cell hyperplasia was noted in the StAR knockout testes (Fig. 3G
); at 8
weeks of age, oil red O-positive areas accounted for 10.5% of the area
of StAR knockout testes (vs. 3.18% of wild-type testes).
These studies document a progressive worsening in the integrity of the
steroidogenic cells, precisely as predicted by the two-stage model.

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Figure 3. Histology of the Testes from Wild-Type and StAR
Knockout Mice at Different Ages
Testes were isolated from wild-type and StAR knockout mice at P0, 4w,
or 8w. Higher power views of sections from the KO mice at 8w are shown
in the right panels. Panel A, WT, P0. Panel B, WT, 4w.
Panel C, WT, 8w. Panel D, KO, P0. Panel E, KO, 4w. Panel F, KO, 8w.
Panel G, KO 8w, high power. Note Leydig cell hyperplasia in the
interstitial region. Panel H, KO, 8w, high power. The solid
arrow points to multinucleated germ cells within the
seminiferous tubules, and the dashed arrow points to a
germ cell with chromatin condensation.
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Previous studies have suggested that spermatogenesis absolutely
requires androgens (reviewed in Ref. 12). Despite marked lipid deposits
in the Leydig cells of StAR knockout testes at 8 weeks, microscopic
examination revealed residual capacity for spermatogenesis. The basal
spermatogonia in the seminiferous tubules were intact (Fig. 3
) and
mature spermatids were detected in the epididymis (data not shown). In
addition, as shown in Fig. 4
, StAR
knockout testes at 8 weeks had comparable immunohistochemical staining
for CREM, a marker for postmeiotic germ cells (13), and cyclin
A1, which is expressed during the first meiotic division (14). These
results, in conjunction with the low-butdetectable plasma
testosterone levels (Table 1
), indicate that StAR knockout testes
retain sufficient androgen biosynthesis to support germ cell
maturation, a finding that persists even at 24 weeks of age (data not
shown). Examination at earlier stages of development, however, showed
that the initial wave of spermatogenesis in StAR knockout testes is
delayed. Whereas wild-type seminiferous tubules at 4 weeks contained
mature elongated spermatids (Fig. 3B
), germ cell maturation in StAR
knockout mice had not progressed beyond step 14 [stage II/III
elongating spermatids (Fig. 3E
)]. Moreover, the tubules contained
multinucleated giant cells (Fig. 3H
, solid arrow), and
individual germ cells exhibited chromatin condensation (dashed
arrow), features consistent with programmed cell death. TUNEL
assays (Fig. 4
) revealed considerably increased labeling in
germ cells of some tubules, consistent with DNA fragmentation.
Collectively, these findings suggest that androgen production in
StAR knockout mice is inadequate to maintain optimal
spermatogenesis.

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Figure 4. Expression of Markers of Germ Cell Maturation and
TUNEL Assay of Programmed Cell Death in Testis Sections
Sections from WT or KO testes at 8w were analyzed as described in
Materials and Methods by immunohistochemistry with
antisera specific for CREM or cyclin A or TUNEL assay. Panel A, WT,
anti-CREM antibody. Panel B, WT, anti-cyclin A antibody. Panel C, WT,
TUNEL assay. Panel D, KO, anti-CREM antibody. Panel E, KO, anti-cyclin
A antibody. Panel F, KO, TUNEL assay. Panel G, KO, TUNEL assay,
high-power.
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Developmental Effects of StAR Deficiency in the Ovaries
We next examined the ovaries of StAR knockout mice. At 1 week of
age (P7), both wild-type and StAR knockout ovaries contained immature
follicles at the one- and two-cell stages (Fig. 5
), and no lipid deposits were seen in
oil red O staining (Fig. 2
). By 8 weeks of age, when sexual maturation
normally has occurred, the StAR knockout ovaries exhibited impaired
follicular maturation and contained abundant lipid deposits, primarily
in stromal cells that exhibited a luteinized appearance (Fig. 2
) and
expressed high levels of the cholesterol side-chain cleavage enzyme
(Fig. 5
, N and P). Although some follicles contained luteinized theca
cells, there were no corpora lutea (i.e. structures composed
of well developed luteinized granulosa and theca interna cells with
dissolution of the basement membrane) in any section examined (Fig. 5
and data not shown). The markedly decreased level of circulating
progesterone further suggests that the decreased steroidogenesis
impaired ovulation. In contrast, both granulosa cells and theca interna
cells appeared relatively normal in the remaining follicles. Although
their circulating estrogen levels were not clearly decreased, StAR
knockout females had sexually immature uterus and oviducts (data not
shown), suggesting impaired estrogen action.

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Figure 5. Histology of Ovaries from Wild-Type and StAR
Knockout Mice at Different Ages
Ovaries were isolated from WT and StAR KO mice at the indicated ages
and were processed as described in Materials and
Methods. Top panels show hematoxylin-eosin
staining of sections. Middle panels show
photomicrographs of ovarian sections analyzed by in situ
hybridization with an antisense probe for cholesterol side-chain
cleavage enzyme. The bottom panels show oil red O
staining. Panel A, WT, P7, low power. Panel B, WT 8w, low power. Panel
C, WT, 24 w, low power. Panel D, WT, P7, high power. Panel E, WT,
8w, high power. Panel F, WT, 24w, high power. Panel G, KO, P7, low
power. Panel H, KO, 8w, low power. Panel I, KO, 24 w, low power.
Panel J, KO, P7, high power. Panel K, KO, 8w, high power. Panel L, KO,
24w, high power. Panel M, WT, 8w, brightfield, P450scc. Panel N, WT,
8w, darkfield, P450scc. Panel O, KO, 8w, brightfield, P450scc. Panel P,
KO, 8w, darkfield, P450scc. Panel Q, WT, 24w, oil red O. Panel R, KO,
24w, oil red O. CL, Corpus luteum.
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By 24 weeks, the StAR knockout ovaries were larger than the
wild-type ovaries. They retained only a few scattered follicles and
were composed largely of vacuolated stromal cells, consistent with the
onset of premature ovarian failure (Fig. 5
, I and L). These studies,
which provide the first detailed analyses of ovarian histopathology in
the absence of StAR, again are consistent with the two-hit model of
lipoid CAH.
Developmental Effects of StAR Deficiency on Accessory Sex
Organs
Limited analyses of 46, XY patients with lipoid CAH suggested that
structures derived from the wolffian ducts were normal, while the
external genitalia were completely feminized (15). In contrast, there
were well documented examples of 46, XX patients who underwent menarche
and breast development (4). The ability to maintain StAR knockout mice
until the time of normal puberty allowed us to examine the effect of
StAR deficiency on development of accessory sex organs. We noted
previously that the epididymal structures of wild-type and StAR
knockout mice were indistinguishable at birth (5); comparable histology
of the epididymis and vas deferens also was seen in older StAR knockout
mice (data not shown). In contrast, while the seminal vesicles appeared
relatively normal at birth, they were clearly hypoplastic at 4 and 8
weeks of age (Fig. 6
). Microscopically,
the seminal vesicles contained only simple tubular structures with few
convolutions (data not shown). Finally, the prostate was hypoplastic,
both immediately after birth and at 8 weeks of age (Fig. 6
), and on
microscopic examination contained only primary branching tubules (data
not shown). These findings suggest that residual capacity for androgen
synthesis in the absence of StAR, although sufficient to support
survival of wolffian structures in utero, is insufficient to
virilize more distal accessory sex glands at the time of normal
puberty.

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Figure 6. Effect of StAR Knockout on Male Accessory Sex
Organs
The male accessory sex glands were harvested from WT and StAR KO mice
at the indicated ages. For P0, sagittal sections were stained with
hematoxylin-eosin and analyzed by photomicroscopy. For 4w and 8w, the
structures were processed as described in Materials and
Methods. Panel A, WT, P0. Panel B, KO, P0. Panel C, WT and KO
seminal vesicles, 4w. Panel D, WT and KO seminal vesicles, 8w. Panel E,
WT and KO prostate, 4w. Panel F, WT and KO prostate, 8w.
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DISCUSSION
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In this report, we extend our analyses of the StAR knockout
mice, examining the temporal effects of StAR deficiency on
steroidogenic organs in vivo and defining the effects of the
StAR knockout on development of male accessory sex organs and sperm
maturation. Collectively, these studies provide new insights into the
pathogenesis of StAR deficiency, particularly with respect to
developmental events in gonads that support the two-hit model for the
pathogenesis of lipoid CAH (5). As predicted by this model, we observed
a hierarchy of involvement of the different steroidogenic organs.
Adrenal glands were affected severely at birth and subsequently showed
progressive increases in lipid deposition. Although the Leydig cells
contained scattered foci of lipid deposits shortly after birth, more
generalized lipid deposits were not seen until the time of normal
sexual maturation. Hyperplastic Leydig cells containing florid lipid
deposits ultimately filled the interstitial region. As noted previously
(5), the ovaries appeared normal at birth and lacked any lipid
deposits. When kept alive until after the time of normal sexual
maturation, the ovaries exhibited histological abnormalities, with
decreased numbers of follicles, absent corpora lutea, and proliferation
of vacuolated stromal cells that strongly expressed P450scc (Fig. 5
).
These findings strongly support the concept that persistent trophic
hormone stimulation of StAR-deficient steroidogenic organs leads to
progressively worsening lipid deposits and diminished function of
steroidogenic cells.
Steroidogenic cells obtain cholesterol from multiple sources, including
de novo biosynthesis, hydrolysis of cholesterol esters, and
uptake from circulating lipoproteins [in mice, predominantly high
density lipoprotein (HDL) taken up by the scavenger receptor-B1
pathway]. The StAR knockout mice provide a model system to address the
relative contributions of these different pathways to the lipid
deposits that accumulate in steroidogenic cells. In situ
hybridization analyses did not reveal increased expression of SR-B1 in
StAR knockout adrenals or testes (T. Hasegawa, unpublished
observation). However, preliminary studies indicate that double
knockout mice lacking both ApoA1 and StAR, although severely deficient
in steroidogenesis, accumulate considerably less lipid in their
steroidogenic cells (T. Hasegawa, unpublished observation). These
findings implicate circulating HDL as the major source of the
cholesterol that accumulates in StAR knockout mice.
Few studies have examined testicular structure, spermatogenesis, or
development of male secondary sexual organs in 46,XY subjects with
lipoid CAH. Although differing in age and location of testes
(i.e. abdominal vs. inguinal), most lipoid CAH
patients have had normal appearing Sertoli cells, Leydig cell
hyperplasia, and decreased numbers of germ cell precursors; internal
genitalia have included normal epididymis and vas deferens, with no
description of the prostate. Similarly, StAR knockout mice had normal
appearing epididymis and vas deferens and mature spermatids within the
epididymis (data not shown), supporting some residual capacity for
androgen biosynthesis. They did exhibit some signs of impaired
spermatogenesis, with delayed germ cell maturation at 4 weeks of age
and increased apoptosis in developing spermatocytes. Moreover, the
seminal vesicles and prostate were markedly hypoplastic, and there was
no virilization of external genital structures.
Developmentally, the seminal vesicles, epididymis, and ductus deferens
all arise from the Wolffian ducts, whereas the prostate develops from
the urogenital sinus. The apparent discrepancy in the development of
organs derived from the wolffian ducts probably reflects differing
thresholds for paracrine actions of androgens in the immediate vicinity
of the testes (i.e. epididymis and ductus deferens)
vs. endocrine actions at more distal sites (i.e.
seminal vesicles). The necessity to convert testosterone to
dihydrotestosterone for full virilization may further exacerbate the
effects of the testosterone deficiency, as supported by the prostate
hypoplasia in patients with genetic defects in the type 2 isozyme of
steroid 5
-reductase (16). However, knockout mice lacking both type 1
and 2 isozymes of 5
-reductase undergo normal male sexual
differentiation (M. Mahendroo and D. Russell, personal communication),
suggesting that there may be species-dependent differences in these
processes.
In part because their gonads are not removed to prevent malignant
transformation, even less is known about the structure and function of
ovaries in 46,XX patients with lipoid CAH. The spontaneous onset of
breast development and menarche in 46,XX patients with lipoid CAH was
one factor that prompted the two-hit model. The worsening ovarian
histopathology in StAR knockout mice, beginning at the time of normal
puberty, is entirely consistent with this model. Under persistent
gonadotropin stimulation, the ovaries of StAR knockout mice developed
progressive lipid deposition, particularly within the stromal cells.
Although their serum estradiol levels did not differ from those in
wild-type mice, the uterus and oviducts of StAR knockout mice were
markedly hypoplastic, indicating that estrogen production was
inadequate to stimulate normal development of these secondary sex
organs. No corpora lutea were detected, and progesterone levels were
markedly impaired, indicating that the ovaries of StAR knockout mice
were anovulatory. In this regard, the relative roles of defects in
estrogen vs. progesterone biosynthesis in the ovaries of
StAR knockout mice warrant further study. Ovulation also is impaired in
knockout mice lacking progesterone receptor (17), suggesting that
abnormal production of progesterone at least partly explains the
ovarian phenotype.
A recent study documented the expression of StAR transcripts in
the rat brain, colocalizing with transcripts for cholesterol side chain
cleavage enzyme and 3ß-hydroxysteroid dehydrogenase in the
hippocampus, dentate gyrus, and granular and Purkinje cells of the
cerebellum (18). Given the proposed roles of endogenous steroids,
designated neurosteroids, within the central nervous system (19), it is
of interest to analyze neuronal function in StAR knockout mice. To
date, the histology of different brain regions where StAR is expressed
does not appear distinguishable from the same regions in wild-type mice
(L. Zhao, unpublished observation). However, it is important to note
that neurophysiological analyses of StAR knockout mice have not been
performed, and this area needs further investigation. Ultimately, we
may need to make tissue-specific knockouts that ablate StAR in the
brain but retain its expression in the primary steroidogenic tissues to
delineate specific roles of StAR in the central nervous system.
We noted previously that some StAR knockout mice exhibited signs
of respiratory distress. However, analyses of the lungs of newborn StAR
knockout mice did not reveal any consistent histological abnormalities,
and in situ hybridization analyses showed comparable
expression of genes encoding surfactant proteins A and C in wild-type
and knockout mice (T. Hasegawa, unpublished observation). Although it
is tempting to ascribe the apparent respiratory distress to impaired
lung maturation secondary to glucocorticoid deficiency, results to date
have not elucidated the molecular basis for this aspect of the StAR
knockout phenotype.
In summary, our studies, which demonstrate progressive
histopathological changes in the gonads after the time of normal
puberty, strongly support the two-hit model of lipoid CAH. The StAR
knockout mice, and immortalized cell lines derived from their
steroidogenic organs, hold considerable promise as a system to expand
our understanding of the mechanisms by which StAR facilitates
cholesterol translocation to the inner mitochondrial membrane. Through
studies such as these, we hope to increase our understanding of how
StAR makes its essential contributions to adrenocortical and gonadal
steroidogenesis and endocrine function.
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MATERIALS AND METHODS
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Reagents
Oligonucleotides and reagents for PCR were purchased from
Life Technologies, Inc. (Gaithersburg, MD) and PE Applied Biosystems (Norwalk, CT), respectively. Radionuclides
were purchased from Amersham Pharmacia Biotech (Arlington
Heights, IL), and routine laboratory reagents were purchased from
Sigma (St. Louis, MO). Anti-CREM antiserum was purchased
from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), and
anti-cyclin A antiserum was a generous gift from Dr. Debra Wolgemuth
(Columbia University, New York, NY).
Generation of StAR Knockout Mice
All animal studies were approved by the Institutional
Review Committee at University of Texas Southwestern Medical Center.
The StAR knockout mice were generated as previously described (5) and
maintained as +/- heterozygotes, which were crossed to produce StAR
knockout pups. Mice were maintained in the University of Texas
Southwestern Animal Resources Center on a 12-h light, 12-h dark cycle
and were given food and water ad libitum. StAR genotypes
were determined by PCR analyses of tail DNA using forward
(5'-AAGAGCTCAACTGGAGAGCAC-3') and reverse (5'-TACTTAGCACTTCGTCCCCGT-3')
primers. Genetic sex was determined by PCR analyses for Sry as
previously described (20).
Corticosteroid Rescue of StAR Knockout Mice
StAR knockout mice were rescued with a modification of a
previously described steroid replacement regimen that includes both
glucocorticoids and mineralocorticoids (21). Stock solutions of
dexamethasone 21-phosphate (4 mg/ml in H2O),
fludrocortisone acetate (5 mg/ml in 95% ethanol), and hydrocortisone
(4 mg/ml in 95% ethanol) were prepared and stored at 4 C. A
corticosteroid cocktail was made by diluting the stock solutions in
olive oil (1:10,000 for dexamethasone and fludrocortisone, 1:10 for
hydrocortisone). All newborn pups were injected with 0.05 ml sc once
daily until StAR knockout pups were identified by PCR analysis. Steroid
injections then were continued in StAR knockout pups until weaning,
when the mice were provided ad libitum with 0.9% sodium
chloride as drinking solution and all steroid injections were
stopped.
Histological Analyses
Histological analyses were carried out with 4%
paraformaldehyde or Bouins fixed tissue specimens with hematoxylin
and eosin staining, or frozen tissue sections for oil red O staining
with hematoxylin counterstaining. Areas positive for oil red O staining
were measured in 20 high-power fields (40x: 3.2 x 18 mm) in
representative specimens using CAS 200 morphometrical analysis as
described (22). To examine prostate morphology, glandular and stromal
compartments were separated by a modification of a published method
(23). Immunohistochemical analyses were carried out with primary
antibodies including anti-CREM (1:100 dilution) and anti-cyclin A1
(1:500 dilution). Control reactions were performed with normal rabbit
serum (DAKO Corp., Carpinteria, CA). The TUNEL assay for
DNA fragmentation was performed with a kit purchased from
Intergen (Purchase, NY) according to the manufacturers
protocol. In situ hybridization was performed with an
antisense probe specific for the cholesterol side-chain cleavage enzyme
as previously described (24).
Hormone Assays
RIAs were performed by Dr. David Hess, Oregon
Regional Primate Center using serum collected from +/+ and -/- mice
at 8 weeks of age. Steroids analyzed included corticosterone [limit of
detection, 2 ng/ml; intraassay % coefficient of variation (CV), 8.5%;
% recovery, 88.3], progesterone (limit of detection, 30 pg/ml;
intraassay % CV, 10.7%; % recovery, 87.8), testosterone (limit of
detection, 0.1 ng/ml; intraassay % CV, 6.2%; % recovery, 69.1), and
estradiol (limit of detection, 3.0 pg/ml; intraassay % CV, 9.5%; %
recovery, 79.1). The means and SEs of each group were
calculated, and the statistical significance of differences was
determined by the Mann-Whitney U test.
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ACKNOWLEDGMENTS
|
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We thank Dr. Beverly Koller and Ann Latour for
invaluable assistance in producing the StAR knockout mice, Drs.
Martin Matzuk and William Rainey for helpful discussions, Dr. Deborah
Wolgemuth for the anticyclin A1 antiserum, Dr. David Hess for
measurements of circulating steroid hormones, and Dr. Yelena Krimkevich
for excellent technical assistance.
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FOOTNOTES
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Address requests for reprints to: Dr. Keith L. Parker, Division of Endocrinology and Metabolism, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75235-8857. E-mail: kparke{at}mednet.swmed.edu
This work was supported by grant support from the NIH (DK-54028 and
DK-54480 to K.L.P.).
Received for publication April 11, 2000.
Revision received May 22, 2000.
Accepted for publication May 30, 2000.
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