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Institute of Molecular Biology, Academia Sinica (M.-C.H., S.-J.C., Y.-Y.H., N.-C.H., H.L., C.-c.C.); Institute of Life Sciences, National Defense Medical Center (M.-C.H.); and the Division of Endocrinology and Metabolism, Chang-Gung Memorial Hospital Medical Center (Y.-Y.H.), Taipei, Taiwan 11529, Republic of China
Address all correspondence and requests for reprints to: Dr. Bon-chu Chung, Institute of Molecular Biology, Academia Sinica, Nankang, Taipei, Taiwan 11529, Republic of China. E-mail: mbchung{at}sinica.edu.tw
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Introduction |
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Present in minute amounts, steroids are synthesized through intricately coordinated mechanisms. As a key step of steroid secretion, expression of the CYP11A1 gene is controlled in a tissue-specific, hormonally and developmentally regulated manner. P450scc exists mainly in the adrenal glands and gonads. It is also found in the placenta (4, 5), some parts of the brain (6), and other minor sites (7).
CYP11A1 expression is found in all three zones of the adrenal cortex, the zonae glomerulosa, fasciculata, and reticularis. The zona glomerulosa is engaged in the synthesis of mineralocorticoids, whereas the zonae fasciculata and reticularis mainly secrete glucocorticoids and androgens in the primates (8, 9). Other steroidogenic enzymes, including P450c21, P450c17, P450c11, and 3ß-hydroxysteroid dehydrogenase, are also expressed in the adrenal cortex. Rodents differ from primates in their adrenal physiology, because they do not express P450c17 in the adrenal (10). They use corticosterone as the major glucocorticoid, and their zona reticularis normally does not produce androgens (11).
Steroidogenesis in the adrenal is stimulated by ACTH, whose effect can be observed both acutely and chronically (11). The acute effect takes place within minutes due to the rapid transport of cholesterol from the lipid droplet to the inner mitochondrial membrane, site of P450scc function. Steroidogenic acute regulatory protein (StAR) plays a significant role during this process (12). This acute action is important for the rapid response of the adrenal to stress. The chronic action may take hours to occur. It involves activation of the steroidogenic genes (11). The expression of CYP11A1 in the zonae fasciculata and reticularis is stimulated by ACTH (13), using cAMP as an intracellular mediator (11, 14). In the zona glomerulosa, angiotensin II is the major stimulator of CYP11A1 expression, using calcium and protein kinase C as the signal transducers (15). This hormonal regulation maintains the ability of the adrenal cortex to secrete steroids.
In the ovary, CYP11A1 messenger RNA (mRNA) is found in both follicular granulosa and thecal cells, and in the corpus luteum (16). This expression pattern correlates well with the function of steroids in ovarian cycles. In the testis, CYP11A1 is expressed in the Leydig cells (17). In both male and female gonads, the expression of the CYP11A1 gene is under the control of gonadotropins, using cAMP as the second messenger inside the cell (18). Despite the extracellular peptide hormone and the intracellular signal transducer used, the regulation of the CYP11A1 gene in steroidogenic tissues is at the transcriptional level, increasing the CYP11A1 mRNA level upon stimulation (19). In the embryo, CYP11A1 is first expressed in mouse adrenal primordia at 11 days postcoitum and in the gonads from 12.5 days postcoitum when these organs start to form (20, 21). The expression of CYP11A1 correlates well with the requirement for steroid function during development.
Mechanisms controlling the expression of the CYP11A1 gene
have been under intensive investigation. The basal promoter is located
within 150 bp from the transcription start site of the human gene
(22, 23, 24). The binding sequence for steroidogenic factor 1 (SF-1),
TCAAGGTCA, is found in the promoter region of both the mouse and human
CYP11A1 gene (25, 26). SF-1 belongs to the nuclear receptor
family. In addition, an element (GGGGAGG) that binds weakly to Sp1-like
protein was found in the promoter region of the mouse, human, and
bovine CYP11A1 gene (25, 27, 28). The upstream sequence
displays cAMP-responsive activity (23, 24, 25, 29, 30, 31, 32). This region also
contains an SF-1-binding site at -1616/-1606 and an enhancer sequence
AdE1 -1932/-1822 of the human CYP11A1 gene; AdE1 can
increase reporter gene expression in steroidogenic cells (25, 33) (Fig. 1A
). In addition, a fragment located
between -2500 and -5000 of the mouse cyp11a gene can
enhance gene expression in MA-10 Leydig cells (34).
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Very few studies have been devoted to analysis of CYP11A1 promoter function in vivo. The 2.3 kb of the 5'-flanking region of the human CYP11A1 gene was used to drive simian virus 40 (SV40) T antigen expression for the generation of adrenal tumor and derivation of new steroidogenic cell lines (38). The presence of only two transgenic mice in this study preludes detailed analysis of promoter function in vivo. To investigate further the mechanism controlling CYP11A1 gene expression in vivo, we generated transgenic mice harboring the human CYP11A1 promoter fused to the LacZ reporter gene. We show that either the 2300-bp or the 4400-bp fragment can drive the transgene expression specifically in the adrenals and gonads. This adrenal LacZ gene expression is responsive to ACTH stimulation. Also, transgene expression in the embryonic and postnatal adrenals is controlled by developmental programming, similar to CYP11A1 gene regulation. We have analyzed the gene expression pattern of the adrenocortical zones during development and showed that the previously poorly characterized X zone is probably engaged in steroid production.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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).
Transfection and reporter gene assay
Twenty micrograms of LacZ reporter constructs and 6
µg pGL2-control-containing luciferase gene were electroporated into
Y1 cells at 0.4 kV and 975 µF. The cell extract was prepared 72
h after transfection. ß-Galactosidase activity was measured using a
chemiluminescent detection assay (Galacto-Light, Tropix, Bedford, MA)
and was normalized against luciferase activity as an internal
control.
Generation and analysis of transgenic mice
Three DNA fragments were used to generate transgenic mice as
described by Hogan (39). One was excised from the plasmid pAdE1–145
(Fig. 1B) with SalI and SmaI to release a 5-kb
fragment containing three AdE1 in front of 145 bp of the
CYP11A1 promoter/LacZ/SV40 poly(A). The second
was excised from the plasmid pSCC4400 (Fig. 1B) with SpeI
and EcoRI to release a 6.7-kb fragment containing 2300 bp of
CYP11A1 promoter/LacZ/SV40 poly(A). The third was
excised from the plasmid pSCC4400 (Fig. 1B) with SalI and
EcoRI to release a 8.8-kb fragment containing 4400 bp of
CYP11A1 promoter/LacZ/SV40 poly(A). FVB mice were
used as embryo donors, and ICR mice were used as recipient foster
mothers. Subsequently, the transgene was maintained in the inbred FVB
background. Injection fragments were purified by agarose gel
electrophoresis and microinjected at a concentration of 1–5 ng/µl
(in 10 mM Tris-HCl, pH 7.4, and 0.1 mM
EDTA).
Twelve, 15, and 16 transgenic founders were obtained from the AdE1–145 (5-kb), 2300-bp (6.7-kb), and 4400-bp (8.8-kb) fragment, respectively. Transgenic founders were mated with FVB nontransgenic mice to generate progeny. Genomic DNA was prepared from tail biopsies using the QIAamp Tissue Kit (QIAGEN, Chatsworth, CA) and screened for the presence of the transgene by PCR. PCR amplification for LacZ was performed using primers 5'ßGal (5'-CGTTTTACAACGTCGTGACTGGGAAAACCC-3') and 3'ßGal (5'-ATGTGAGCGAGTAACAACCCGTCGGATTCT-3'), producing a 352-bp fragment. The genomic DNA of embryos was prepared from the tail or placenta, and the transgene was identified by the same method. The sexes of the embryos were determined by PCR with Sry primers 5'-AAGCGCCCCATGAATGCATT-3' and 5'CGATGAGGCTGATATTTATA-3' (40).
RNA was isolated from mouse tissues by TRIzol reagent from Life Technologies, Inc. (Grand Island, NY), and then digested with deoxyribonuclease I before RT-PCR reactions to make sure that the DNA was completely removed from the RNA preparation. The glyceraldehyde-3-phosphate dehydrogenase was amplified with primers 5'-GCTGTAGCCAAATTCGTTGTC-3' and 5'-GATGACATCAAGAAGGTGGTG-3' to generate a 198-bp fragment (41).
The transgene copy number was determined by Southern blot analysis. Genomic DNA (20 µg) was separated by 0.8% agarose gel electrophoresis after PstI digestion, transferred to the membrane, and hybridized with 32P-labeled LacZ gene fragment or the CYP11A1 5'-flanking sequence. The plasmid DNA pAdE1–145 (0.039, 0.195, and 0.39 ng) or pSCC4400 (0.08, 0.4, and 0.8 ng) was used as the standard for 1, 5, and 10 copies of transgene. About 1–12 copies were found in the AdE1–145, and about 1–100 copies of transgenes were found in the 2300- and 4400-bp transgenic animals. Transgene rearrangement was found in one of the founders containing the 4400-bp transgene.
ß-Galactosidase assays
Tissue lysate was assayed for ß-galactosidase activity using
the chemiluminescent detection assay. Tissues were homogenized in the
smallest volume of lysis buffer [100 mM potassium
phosphate (pH 7.8), 0.2% Triton X-100, 0.5 mM
dithiothreitol, 0.2 mM phenylmethylsulfonylfluoride, and 5
µg/ml leupeptin] and centrifuged, and the supernatant was incubated
at 48 C for 1 h to inactivate the endogenous ß-galactosidase
activity. Tissue lysate (10 µl) was mixed with 50 µl reaction
buffer (Galacton-Plus substrate diluted 100-fold with Galacton-Light
Reaction Buffer Diluent) and incubated at room temperature for 1
h. Light Emission Accelerator was injected, and the sample was counted
for 5 sec in a luminometer.
Tissue sectioning and ß-galactosidase histochemistry
Tissues were fixed with 2% paraformaldehyde and 0.2%
glutaraldehyde in PBS for 30 min at 4 C, washed with PBS, and then
incubated in 1 mg/ml Bluo-gal
(5-bromo-indolyl-ß-O-galactopyranoside, a substrate for
LacZ) (42) reaction buffer (3 mM potassium
ferricyanide, 3 mM potassium ferrocyanide, 1.5
mM magnesium sulfate, 0.2% sodium deoxycholate, 0.1%
Nonidet P-40, and 0.15 mg/ml chloroquine in PBS) at room temperature
overnight. For sectioning, tissues were immersed in OCT compound,
followed by freezing at -20 C and storing at -70 C. Frozen sections
(10 µm) were fixed in 0.2% glutaraldehyde, 100 mM sodium
phosphate (pH 7.4), 5 mM EGTA, and 2 mM
MgCl2 for 5 min; washed with PBS; and then incubated in 1
mg/ml Bluo-gal reaction buffer at room temperature overnight.
In situ hybridization
Frozen sections (10 µm) were fixed with 4% paraformaldehyde
in PBS for 2 h. Tissue sections were treated with proteinase K (1
µg/ml) in PBST (0.1% Tween-20 in PBS) for 2 min and then fixed with
4% paraformaldehyde in PBS for 20 min. After PBST washes, slides were
treated with anhydride acetate (2.5 µl/ml) in 0.1 M
triethanolamine (pH 8.0) for 10 min and then washed with PBST. Slides
were incubated with prehybridization solution [50% formamide, 5
x SCC (standard saline citrate), and 0.1% Tween-20] at 68 C for
4 h and then hybridized with digoxigenin (DIG)-labeled riboprobes
in 50% formamide, 5 x SSC, 0.1% Tween-20, 50 µg/ml heparin,
and 10 µg/ml yeast transfer RNA overnight at 68 C. DIG-labeled
riboprobes were synthesized using RNA polymerase with labeling mix (1
mM ATP, 1 mM CTP, 1 mM GTP, 0.65
mM UTP, and 0.35 mM DIG-11-UTP). The
CYP11A1 probe was made with the T3 RNA polymerase and
plasmid p424 that contains about 600 bp of the 5' CYP11A1
complementary DNA cloned in pBluescript KS. The LacZ probe
was generated with the Sp6 RNA polymerase from plasmid
pLacZGem4 that contains 1250 bp of the 5'
LacZ-coding region cloned in pGEM-4. After hybridization,
tissues were washed with 50% formamide in 2 x SSC at 65 C for
1 h and then in 2 x SSC at 37 C for 10 min three times.
Tissues were incubated with blocking solution (0.2% Tween-20, 0.2%
Triton X-100, and 2% sheep serum preheated 30 min at 55 C in PBS) for
4 h at room temperature and then incubated with anti-DIG-AP
(1:5000 dilution) in blocking solution at 4 C overnight. Slides were
washed three times with PBST for 30 min each, then in 1 mM
levamisol in PBST for 30 min. Samples were equilibrated with detection
solution [0.1 M Tris (pH 9.5), 50 mM
MgCl2, 0.1 M NaCl, 0.1% Tween, and 1
mM levamisol] for 10 min three times before incubation in
1 ml detection solution containing 0.33 mg/ml nitro blue tetrazolium
and 0.175 mg/ml 5-bromo-4-chloro-3-indolyl-phosphate. The coloring
reaction was stopped by washing with 0.1% Tween-20.
Physiological manipulation
Mice were housed under standard laboratory conditions (12 h of
light and 12 h of dark; lights on at 0700 h) and were fed
laboratory chow and water ad libitum. Five female transgenic
mice (line 78) were injected with 1 IU ACTH (Cortrosyn,
Organon, Oss, Holland), sc, once a day for 7 days. Mice
were anesthetized with ether on the last day 1.5 h after
injection. Blood was promptly collected from the heart via needle
aspiration, spun down to isolate serum, and stored at -20 C.
Corticosterone was measured using a 125I RIA kit (ICN Biomedicals, Inc., Costa Mesa, CA). Adrenal lysate was assayed
for ß-galactosidase activity as described above.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). To determine whether the upstream
SF-1-binding site also contributes to basal transcription, three copies
of the SF-1-binding sequence from -1616 to -1606 were placed in front
of the 145-bp basal promoter, which already has an SF-1-binding site.
Additional copies of the SF-1-binding sites did not further enhance the
basal promoter activity (Fig. 1B). However, three copies of the AdE1
enhancer increased transcription by 5-fold, indicating that AdE1 can
further activate the basal promoter. The longest promoter, covering
4400 bp of the 5'-flanking sequence, activated transcription by about
8-fold.
Detection of gene expression in transgenic mice
Both AdE1–145 and SCC4400 function as promoters in cultured cells
(Fig. 1). To investigate their function in vivo, we
generated transgenic mouse lines by injecting AdE1–145, 2300-bp
promoter, or 4400-bp promoter linked to LacZ/poly(A) into
fertilized mouse eggs. Twelve, 15, and 16 transgenic lines were
obtained from the AdE1–145, 2300-bp, and 4400-bp constructs,
respectively. Transgene copy numbers, calculated from band intensities
of Southern blot hybridization, ranged from 1–12 for AdE1–145 and
1–100 for the 2300-bp and 4400-bp lines. The transgenic mouse lines
were screened for transgene expression by inspection of
ß-galactosidase staining in the adrenal glands and gonads. Eight
mouse lines (B15, B47, B49, B53, B64, 3, 5, and 8) containing the
2300-bp construct and three mouse lines (76, 78, and 88) containing the
4400-bp construct showed significant transgene expression in adrenals
and gonads. Transgene rearrangement was found in one mouse line (13)
containing the 4400-bp construct. This mouse line had weak transgene
expression in many tissues.
None of the 12 AdE1–145 transgenic mouse lines showed detectable
levels of transgene expression. These data suggested that AdE1–145 did
not activate reporter gene sufficiently in vivo, although
its function could be detected in cell culture (Fig. 1). The 4400-bp
5'-flanking sequence had higher activity than AdE1–145 in cultured
cells and directed significant tissue-specific transgene expression in
transgenic mice. The shorter 2300-bp sequence also showed similar
activity in transgenic mice. These mouse lines were analyzed
further.
Tissue-specific expression of LacZ gene
ß-Galactosidase activity assay. To examine the location of
transgene expression, ß-galactosidase activity was measured from
homogenates of the adrenal, ovary, testis, brain, heart, kidney, lung,
and spleen of male and female of 3- to 4-month-old transgenic progeny
using a luminescence-based assay. All mouse lines containing the 2300-
or 4400-bp constructs showed strong ß-galactosidase activity in the
adrenals and gonads, but not in other tissues (Fig. 2). The levels of ß-galactosidase
expression varied in different lines. Different expression levels in
different mouse lines generated by the same construct are commonly
observed (43, 44, 45), because transgene expression is affected by the
integration site and copy number (46, 47, 48). Despite variation in the
extent, expression of the reporter gene driven by the 2300- or 4400-bp
human CYP11A1 5'-flanking sequence was tissue specific,
appearing mainly in the adrenals and gonads. Therefore, the 2300-bp
promoter is sufficient to direct heterologous gene expression
specifically in steroidogenic tissues.
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). It is not clear whether the RT-PCR
product in the heart could reflect an artifact in the highly sensitive
RT-PCR method, because no ß-galactosidase activity was detected in
any of the mouse lines. However, expression in the brain could be real,
because the brain of line 76 also had a small amount of
ß-galactosidase activity (data not shown). A low level of
CYP11A1 mRNA is present in some parts of the brain (6, 49).
Despite these minor variations, RT-PCR results showed LacZ
mRNA expression in the adrenals and gonads.
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). This result is consistent with
expression of the endogenous CYP11A1 gene. In those lines in
which the expression level was high, such as 78 and B64, staining of
ß-galactosidase was detected in almost all cortical cells (Fig. 4, A
and B). In lines in which expression level was low, such as 76 and 88,
a variegated staining pattern in the adrenal gland was observed (Fig. 4, C and D). Line 88 exhibited homogeneous expression in the X zone and
variegated expression in the zona fasciculata of the adrenal cortex
(Fig. 5B). Similar observations were
reported in LacZ transgenic mice driven by the
21-hydroxylase promoter (43).
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and 5D), and sections of ovary have ß-galactosidase staining in
the corpora lutea and follicles (arrowhead in Fig. 4F). In
the testis, most lines showed ß-galactosidase staining in the Leydig
cells, but not in the seminiferous vesicles (Fig. 4, G and H). This
correlates with the endogenous CYP11A1 gene expression in
the Leydig cells. The presence of the transgene only in the adrenal
cortex, ovarian follicle, corpus luteum, and testicular Leydig cells
indicates that transgene expression is specific to steroidogenic
cells.
In situ hybridization. To determine the distribution of
transgene mRNA, we performed in situ hybridization of frozen
tissue sections using DIG-labeled CYP11A1 and
LacZ antisense RNA probe. The endogenous CYP11A1
mRNA was expressed throughout the adult adrenal cortex (Fig. 5A).
LacZ mRNA was also detected in the adrenal cortex in line 88
(Fig. 5B), an expression pattern consistent with ß-galactosidase
activity staining data (not shown). In the ovary, the highest levels of
CYP11A1 mRNA were found in the corpora lutea (Fig. 5C), and
weaker expression was detected in the follicular thecal and granulosa
cells. Similar results were obtained based on LacZ mRNA
expression (Fig. 5D). Both the in situ hybridization and
activity staining analyses demonstrated that LacZ gene
expression in the corpora lutea correlated well with the endogenous
CYP11A1 expression pattern.
Developmental regulation of transgene expression
To determine whether the transgene expression was regulated
by the CYP11A1 promoter during development, we generated
transgenic embryos by mating transgenic males of line 78 with
nontransgenic females. ß-Galactosidase activity was detected in the
adrenal primordia of transgenic embryos on day 11.5 (E11.5) of
gestation (Fig. 6). The nontransgenic
littermates did not show any blue staining. On E14.5 and E18.5, when
the adrenal glands are fully formed, higher ß-galactosidase activity
was observed in the transgenic adrenal glands. We further examined the
sex of transgenic embryos by PCR analysis with Sry primers.
Both female and male transgenic embryos had high levels of
ß-galactosidase activity. These results demonstrated that the
transgene could be expressed in the adrenals of both sexes during
embryo development, like the endogenous CYP11A1 gene.
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). A similar expression pattern was
detected for the endogenous CYP11A1 gene by in
situ hybridization (Fig. 7B). This region, called the X zone, was
first described in mice by Masui and Tamura (50). The X zone is a
transient layer that disappears concomitantly with puberty in the male.
It persists for a longer time in the female and gradually degenerates
with age. However, the first pregnancy causes the rapid disappearance
of the X zone in the female mice (51, 52, 53). Consistent with earlier
reports, we found that the X zone had almost completely disappeared by
5 weeks in the male (Fig. 7, C and D). Both ß-galactosidase activity
and the CYP11A1 gene were expressed in the remaining zones
of the male adrenal cortex (Fig. 7, C and D).
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and 8).
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, A and B). The female X zone
persisted until 7 weeks of age (Fig. 8, C and D), but by 9 weeks, the X
zone had involuted (Fig. 8E). Cells showing fatty degeneration
appeared, and the involution continued with age until the lipid
vacuoles were clearly visible at 15 weeks of age (brown
pigments in Fig. 4, B and D). Detection of LacZ mRNA by
in situ hybridization showed LacZ transcripts in
the same cell types as those exhibiting ß-galactosidase activity and
CYP11A1 mRNA (Fig. 8F). These results indicated that the
4.4-kb region of the 5'-flanking sequence was sufficient to regulate
transgene expression in the adrenals during different stages of
development in both sexes.
Hormonal regulation of transgene expression
To test whether this 4.4-kb region carries information for
hormonal regulation, we injected ACTH into transgenic mice from line 78
for 7 days. The plasma corticosterone levels in ACTH-injected mice
(1688 ± 186 ng/ml) were much higher than those in saline-injected
mice (108 ± 127 ng/ml). Similarly, ß-galactosidase activity in
the stimulated group was also increased by about 6-fold after ACTH
injection (Fig. 9A) compared with saline
injection. The total weight of the adrenal did not increase
significantly after ACTH injection (Fig. 9B). The results indicate that
the transgene contains cues for ACTH stimulation.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Three methods were used to analyze tissue-specific transgene expression. ß-Galactosidase activity was detected from homogenates of the adrenal and gonad, but was absent in the other tissues examined. Second, RT-PCR analysis demonstrated the expression of LacZ mRNA in the adrenals and ovaries. Finally, tissue sections with ß-galactosidase activity staining and in situ hybridization showed that the LacZ gene was expressed in the adrenal cortex, ovarian follicle, corpus luteum, and testicular Leydig cells, which correlates well with the endogenous CYP11A1 gene expression in steroidogenic tissues.
We found that ß-galactosidase activity was also present in the
adrenal primordia on E11.5 in line 78 transgenic embryos. Thus, the
4.4-kb fragment of the CYP11A1 gene promoter was able to
drive transgene expression in embryonic adrenal glands. The parallel
expression of the LacZ and CYP11A1 genes
continued after birth. We found that in addition to the zonae
glomerulosa and fasciculata, the X zone of the adrenal cortex was
especially high in LacZ and CYP11A1 gene
expression. Mice have a distinct X zone in the adrenal cortex only in
the early stages of development (51, 52, 53, 58), but its function is
unclear. An earlier report indicated that the X zone may secrete an
androgenic substance (59), but P450c17 is not expressed in rat (10) or
mouse (Hu, M.-C., and B.-c. Chung, data not shown) adrenal. The X zone
expresses Ren-2 and the inhibin 
-subunit gene (60, 61), but previous
studies provided no information about steroidogenic gene expression. In
our study, we found the CYP11A1 gene had higher expression
in the X zone than in the zona fasciculata. In addition,
3ß-hydroxysteroid dehydrogenase is expressed in the X zone (data not
shown). Therefore, the X zone may have steroidogenic activity.
Very little of the zona reticularis was observed in our adrenal cryosections, although a thin zona reticularis layer between the zona fasciculata and the X zone could be recognized in paraffin sections of some mouse strains (62, 63). It could be that the morphology of the cryosections was not good enough to observe the thin cell layers in adrenal cortex. The X zone appears to be a dynamic and major zone of the mouse adrenal cortex before puberty. Because it abundantly expresses steroidogenic genes, such as CYP11A1 and 3ß-hydroxysteroid dehydrogenase, we suspect that the X zone has a similar function as the zona reticularis and may play a role in steroid synthesis during development. It will be interesting to find out whether other genes related to the regulation of steroidogenesis, such as StAR or ACTH receptor, are also expressed in the X zone.
The thickness of the X zone is related to age. The X zone probably
degenerates in response to androgen (52). In males, the X zone is
evident at 3 weeks of age, but starts to degenerate at puberty; the X
zone is completely gone in the adrenals of 5-week-old males (Fig. 7, C
and D). In females, the X zone persists for a longer period, but
eventually starts to degenerate with age (Fig. 8E). The expression of
CYP11A1 in the X zone parallels the developmental sequences
of this zone. The expression of LacZ in the X zone also
parallels that of the CYP11A1 gene, indicating a strict
correlation during these developmental events. There is an additional
region, between the zona fasciculata and the X zone, that was low in
CYP11A1 and Ren-2 gene expression (60). This region may have
properties similar to those of the cell layer between the rat zona
glomerulosa and fasciculata, which does not express steroidogenic genes
but shows active DNA replication (64). It has been suggested that this
layer contains the progenitor cell of the rat adrenal cortex. As this
region was weak in CYP11A1 and Ren-2 gene expression, and
its size appeared to be reduced when the X zone had degenerated, we
suspect that the region between the zona fasciculata and the X zone
might contain the progenitor cells for the X zone. However, the
characteristics and functions of this region need further study.
Transgene expression in transgenic animals is usually affected by promoter strength, integration site, and copy number (46, 47, 48). Many copies of the transgenes in tandem repeats frequently result in poor expression in plants (65, 66, 67), Drosophila (68, 69), and mice (48, 70, 71). A similar situation occurred in our study. Two mouse cell lines with low copy numbers, lines 78 (3 copies) and B64 (2 copies), had the best expression levels. By contrast, line 76 had 35 copies but poor transgene expression. This poor gene expression may explain the variegated pattern of LacZ gene expression. On the other hand, the insertion site effect or insufficient promoter strength may also contribute to the variegated expression that we observed.
The 2.3-kb 5'-flanking region of the human CYP11A1 gene fused to SV40 T antigen has previously been used to generate transgenic mice (38). Tumors developed in their adrenals, consistent with our data, but no ovarian or testis tumors were detected. However, only two female transgenic mice were reported in that study. In our study, we showed that the 2.3-kb fragment can indeed direct ß-galactosidase expression in the gonads.
Both the 2.3- and 4.4-kb constructs directed LacZ gene expression in the adrenal and gonad. Although varying from line to line, overall the expression levels directed by the 2.3- and 4.4-kb constructs do not appear to differ. They both drive reporter gene expression in the adrenal, testis, and ovary to a comparable level. We have previously shown the presence of an enhancer element, AdE, located at about -1.9 kb of the human CYP11A1 gene, which could drive gene expression in the adrenal and testis Leydig cells (33, 72). The localization of the enhancer is consistent with the current transgenic mouse data. The Leydig enhancer has also been localized to between -2500 and -5000 bp of the mouse cyp11a1 gene (73). As this mouse enhancer has not been well characterized, we do not know whether it functions similarly to the AdE of the human enhancer.
In summary, we have characterized important regulatory elements involved in the expression of the human CYP11A1 gene. The 2.3-kb promoter fragment was able to drive tissue-specific and hormonally regulated expression of the reporter gene. In addition, reporter gene expression was developmentally regulated from the embryonic stage throughout adulthood. This led to characterization of the transient nature of the adrenocortical X zone. We found that the X zone abundantly expressed steroidogenic genes.
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Acknowledgments |
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Footnotes |
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Received June 3, 1999.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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  E. Missaghian, P. Kempna, B. Dick, A. Hirsch, R. Alikhani-Koupaei, B. Jegou, P. E Mullis, B. M Frey, and C. E Fluck Role of DNA methylation in the tissue-specific expression of the CYP17A1 gene for steroidogenesis in rodents J. Endocrinol., July 1, 2009; 202(1): 99 - 109. [Abstract] [Full Text] [PDF]
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 A. C. Kim, F. M. Barlaskar, J. H. Heaton, T. Else, V. R. Kelly, K. T. Krill, J. O. Scheys, D. P. Simon, A. Trovato, W.-H. Yang, et al. In Search of Adrenocortical Stem and Progenitor Cells Endocr. Rev., May 1, 2009; 30(3): 241 - 263. [Abstract] [Full Text] [PDF]
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 M. Bielinska, H. Parviainen, S. Kiiveri, M. Heikinheimo, and D. B. Wilson REVIEW PAPER: Origin and Molecular Pathology of Adrenocortical Neoplasms Vet. Pathol., March 1, 2009; 46(2): 194 - 210. [Abstract] [Full Text] [PDF]
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 I.-C. Guo, C.-Y. Huang, C.-K. L. Wang, and B.-c. Chung Activating Protein-1 Cooperates with Steroidogenic Factor-1 to Regulate 3',5'-Cyclic Adenosine 5'-Monophosphate-Dependent Human CYP11A1 Transcription in Vitro and in Vivo Endocrinology, April 1, 2007; 148(4): 1804 - 1812. [Abstract] [Full Text] [PDF]
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 H.-C. Lan, H.-J. Li, G. Lin, P.-Y. Lai, and B.-c. Chung Cyclic AMP Stimulates SF-1-Dependent CYP11A1 Expression through Homeodomain-Interacting Protein Kinase 3-Mediated Jun N-Terminal Kinase and c-Jun Phosphorylation Mol. Cell. Biol., March 15, 2007; 27(6): 2027 - 2036. [Abstract] [Full Text] [PDF]
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T. Yazawa, T. Mizutani, K. Yamada, H. Kawata, T. Sekiguchi, M. Yoshino, T. Kajitani, Z. Shou, A. Umezawa, and K. Miyamoto Differentiation of Adult Stem Cells Derived from Bone Marrow Stroma into Leydig or Adrenocortical Cells Endocrinology, September 1, 2006; 147(9): 4104 - 4111. [Abstract] [Full Text] [PDF]
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W.-Y. Chen, L.-J. Juan, and B.-c. Chung SF-1 (Nuclear Receptor 5A1) Activity Is Activated by Cyclic AMP via p300-Mediated Recruitment to Active Foci, Acetylation, and Increased DNA Binding Mol. Cell. Biol., December 1, 2005; 25(23): 10442 - 10453. [Abstract] [Full Text] [PDF]
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 J. C.Y. Wong, N. Alon, C. Mckerlie, J. R. Huang, M. S. Meyn, and M. Buchwald Targeted disruption of exons 1 to 6 of the Fanconi Anemia group A gene leads to growth retardation, strain-specific microphthalmia, meiotic defects and primordial germ cell hypoplasia Hum. Mol. Genet., August 15, 2003; 12(16): 2063 - 2076. [Abstract] [Full Text] [PDF]
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 R. J. Mann, R. A. Keri, and J. H. Nilson Consequences of Elevated Luteinizing Hormone on Diverse Physiological Systems: Use of the LH{beta}CTP Transgenic Mouse as a Model of Ovarian Hyperstimulation-induced Pathophysiology Recent Prog. Horm. Res., January 1, 2003; 58(1): 343 - 375. [Abstract] [Full Text] [PDF]
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 P. J. Spencer, J. W. Crissman, W. T. Stott, R. A. Corley, F. S. Cieszlak, A. M. Schumann, and J. F. Hardisty Propylene Glycol Monomethyl Ether (PGME): Inhalation Toxicity and Carcinogenicity in Fischer 344 Rats and B6C3F1 Mice Toxicol Pathol, August 1, 2002; 30(5): 570 - 579. [Abstract] [PDF]
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S. Kiiveri, J. Liu, M. Westerholm-Ormio, N. Narita, D. B. Wilson, R. Voutilainen, and M. Heikinheimo Differential Expression of GATA-4 and GATA-6 in Fetal and Adult Mouse and Human Adrenal Tissue Endocrinology, August 1, 2002; 143(8): 3136 - 3143. [Abstract] [Full Text] [PDF]
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 M.-C. Hu, N.-C. Hsu, N. B. El Hadj, C.-I Pai, H.-P. Chu, C.-K. L. Wang, and B.-c. Chung Steroid Deficiency Syndromes in Mice with Targeted Disruption of Cyp11a1 Mol. Endocrinol., August 1, 2002; 16(8): 1943 - 1950. [Abstract] [Full Text] [PDF]
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M.-C. Hu, N.-C. Hsu, C.-I Pai, C.-K. L. Wang, and B.-c. Chung Functions of the Upstream and Proximal Steroidogenic Factor 1 (SF-1)-Binding Sites in the CYP11A1 Promoter in Basal Transcription and Hormonal Response Mol. Endocrinol., May 1, 2001; 15(5): 812 - 818. [Abstract] [Full Text]
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