This Article Abstract Full Text (PDF) An erratum has been published Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Hu, M.-C. Articles by Chung, B.-c. Search for Related Content PubMed PubMed Citation Articles by Hu, M.-C. Articles by Chung, B.-c. Pubmed/NCBI databases Substance via MeSH

Tissue-Specific, Hormonal, and Developmental Regulation of SCC-LacZ Expression in Transgenic Mice Leads to Adrenocortical Zone Characterization¹

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

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
We report here the study of the human CYP11A1 promoter in driving tissue-specific, developmentally and hormonally regulated reporter gene expression. A 4.4-kb fragment containing all known regulatory elements is more efficient than a short basal promoter fused to an upstream adrenal enhancer in driving reporter LacZ gene expression both in cell culture and in transgenic mice. The LacZ gene controlled by the 4.4- and 2.3-kb promoters was expressed in the adrenal cortex, testicular Leydig cells, ovarian corpora lutea, and granulosa cells. Transgene expression in the adrenals was stimulated by ACTH, indicating the presence of ACTH-responsive sequence. ß-Galactosidase activity was first detected in the adrenal primordia at 11.5 days postcoitum. Its expression continued throughout all stages of adrenal development in a pattern similar to that of the endogenous CYP11A1, which was expressed in all zones of the adrenal cortex, but was strongest in the X zone. The X zone grew before puberty but regressed afterward, as did the levels of CYP11A1 and LacZ gene expression in the X zone. Our study of the CYP11A1 promoter in transgenic mice led to characterization of the adrenocortical zones.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
STEROID HORMONES are synthesized primarily in the adrenal glands and gonads by a number of steroid hydroxylase enzymes. The first, and rate-limiting, step in the steroid synthetic pathway is catalyzed by cytochrome P450scc, the cholesterol side-chain cleavage enzyme, which converts the common precursor cholesterol into pregnenolone (1, 2, 3). P450scc is a member of the cytochrome P450 superfamily, which is characterized by the presence of a heme moiety for oxidation-reduction reactions. The gene encoding P450scc is termed CYP11A1 (2).

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).

View larger version (22K): [in this window] [in a new window]   Figure 1. Analysis of regulatory elements in the CYP11A1 gene. A, Schematic representation of the 4400-bp region of the 5'-flanking sequence of the human CYP11A1 gene. The locations of binding sites for Sp1-like proteins and SF-1 are shown. A cAMP-responsive region between -1500 and -1620 and adrenal-specific enhancers AdE1 and AdE2 are indicated. B, The LacZ reporter plasmid contains the basal promoter (-145 to +55) or the 4400-bp upstream sequence of the human CYP11A1 gene. Three copies of the SF-1-binding site and AdE1 were positioned upstream of the basal promoter. The recombinant plasmids were transfected into Y1 cells. The bars represent the ß-galactosidase activity relative to the basal promoter.

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.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Plasmids construction
The 5'-flanking region (-145 to +55) of the human CYP11A1 gene was cloned into vector pßgal-Basic (CLONTECH Laboratories, Inc., Palo Alto, CA) to generate plasmid pB145. A longer fragment (-4400 to +55) was cloned into pUC19/LacZ to generate plasmid pSCC4400. Both vectors carry the Escherichia coli LacZ gene fused to the SV40 polyadenylation site. Plasmids pSF1–145 and pAdE1–145 were generated by placing three copies of oligos that contained the SF-1-binding site or AdE1 (33), corresponding to the -1616/-1606 or -1975/-1845 regions of the human CYP11A1 gene, in front of the CYP11A1 promoter of pB145, respectively (Fig. 1).

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.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Analysis of CYP11A1 promoter in cell culture
The function of the 5'-flanking sequence of the human CYP11A1 gene in tissue-specific expression has been shown by transfection experiments (25, 33). Two SF-1-binding sites, an upstream cAMP-responsive sequence, and the adrenal enhancer sequences AdE1 and AdE2 have been identified (Fig. 1A). 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.

View larger version (26K): [in this window] [in a new window]   Figure 2. ß-Galactosidase activity in tissues of three transgenic mouse lines. Each of the three panels show ß-galactosidase activity in the tissues of 3- to 5-month-old transgenic (dark bars) and nontransgenic (open bars) littermates from lines 78, B47, and B64, respectively. The ovaries (O), testes (T), and male and female adrenals (A), brains (B), hearts (H), kidneys (K), livers (Li), lungs (Lu), and spleens (S) were tested.

View larger version (49K): [in this window] [in a new window]   Figure 3. Detection of transgene expression in tissues of mouse line 78 by RT-PCR. RNAs were isolated from different tissues and amplified with either LacZ or glyceraldehyde-3-phosphate dehydrogenase primer as a control. -, Amplification with no RNA; +, amplification using LacZ plasmid as a template.

View larger version (119K): [in this window] [in a new window]   Figure 4. LacZ staining of adrenal and gonads of transgenic mouse lines. A, C, E, and G, LacZ staining of the entire tissue. Tissues on the right of the whole mount staining are from transgenic mice; those on the left are from nontransgenic littermates. B, D, F, and H, Sectioning of the transgenic tissues followed by ß-galactosidase staining. Adrenal (A and B) and ovary (E and F) from a strong expressing line (#78) and adrenal (C and D) from a weak expressing line (#76) are shown, both at 15 weeks. The arrowhead in F points to granulosa cells in the follicle that are also weakly stained. Gray lipid vacuoles in the X zone are degraded cell corpses. Testis obtained from B64 at 30 weeks are shown in G and H. F, Zona fasciculata; X, X zone; M, medulla; CL, corpus luteum; L, Leydig cell; SV, seminiferous vesicle.

View larger version (115K): [in this window] [in a new window]   Figure 5. In situ hybridization showing endogenous and transgenic gene expression. Adrenal (A and B) and ovary (C and D) sections from female transgenic mouse line 88 at 10 weeks were hybridized with LacZ (B and D) or CYP11A1 (SCC; A and C) riboprobe. F, Zona fasciculata; X, X zone; M, medulla; CL, corpus luteum; T, theca.

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.

View larger version (78K): [in this window] [in a new window]   Figure 6. Expression of LacZ during embryo development. Embryos from transgenic mouse line 78 at different gestational stages were stained for LacZ. Adp, Adrenal primordia; Ad, adrenal; Kd, kidney.

View larger version (110K): [in this window] [in a new window]   Figure 7. Expression of the transgene and the endogenous gene in male mice of different ages. Adrenal sections from line 78 mice at 3 and 5 weeks of age were stained for ß-galactosidase activity (A and C) or hybridized with the CYP11A1 (SCC; B and D) riboprobe. The adrenal X zone had disappeared by 5 weeks of age. The LacZ gene expression pattern was similar to SCC gene expression in the X zone and other adrenocortical zones. F, Zona fasciculata; X, X zone; M, medulla.

View larger version (145K): [in this window] [in a new window]   Figure 8. Expression of the transgene and the endogenous gene in female mice of different ages. Adrenal sections from female transgenic mice (line 78) at 3, 7, and 9 weeks of age were stained for ß-galactosidase activity (A, C, and E) or hybridized with the CYP11A1 (SCC; B and D) or LacZ (F) riboprobe. The adrenal X zone started to regress at 9 weeks. SCC and LacZ gene expression in the X zone follows the same sequence. F, Zona fasciculata; X, X zone; M, medulla.

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.

View larger version (40K): [in this window] [in a new window]   Figure 9. Stimulation of ß-galactosidase activity by ACTH injection in the adrenal glands of transgenic mice. Five female transgenic mice from line 78 in each group were injected with ACTH or NaCl once a day for 7 days. A, The ß-galactosidase activity from the adrenal homogenate. B, The total weight of the adrenal glands were determined.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

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.

	Acknowledgments

 
We thank the transgenic mouse facility at Academia Sinica for the generation of the transgenic mouse lines.

	Footnotes

 
¹ This work was supported by Grant DOH87-HR-609 from the National Health Research Institutes and by Academia Sinica, Republic of China.

Received June 3, 1999.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Miller WL 1988 Molecular biology of steroid hormone synthesis. Endocr Rev 9:295–318[CrossRef][Medline]
2. Nelson DR, Koymans L, Kamataki T, Stegeman JJ, Feyereisen R, Waxman DJ, Waterman MR, Gotoh O, Coon MJ, Estabrook RW, Gunsalus IC, Nebert DW 1996 P450 superfamily: update on new sequences, gene mapping, accession numbers and nomenclature. Pharmacogenetics 6:1–42[Medline]
3. Guo IC, Wu LS, Lin JH, Chung BC 1994b Mechanism of trophic hormone action on the regulation of steroid biosynthesis. Cont Med Edu 4:414–423
4. Simpson ER, MacDonald PC 1981 Endocrine physiology of the placenta. Annu Rev Physiol 43:163–188[CrossRef][Medline]
5. Chung BC, Matteson KJ, Voutilainen R, Mohandas TK, Miller WL 1986 Human cholesterol side-chain cleavage enzyme, P450scc: cDNA cloning, assignment of the gene to chromosome 15, and expression in the placenta. Proc Natl Acad Sci USA 83:8962–8966[Abstract/Free Full Text]
6. Mellon SH, Deschepper CF 1993 Neurosteroid biosynthesis: genes for adrenal steroidogenic enzymes are expressed in the brain. Brain Res 629:283–292[CrossRef][Medline]
7. Keeney DS, Ikeda Y, Waterman MR, Parker KL 1995 Cholesterol side-chain cleavage cytochrome P450 gene expression in the primitive gut of the mouse embryo does not require steroidogenic factor 1. Mol Endocrinol 9:1091–1098[Abstract]
8. McNicol AM 1992 The human adrenal gland. In: James MHT (ed) The Adrenal Gland, ed 2. Raven Press, New York, pp 1–42
9. Orth DN, Kovacs WJ, Debold CR 1992 The adrenal cortex. In: Wilson JD, Foster DW (eds) Williams Textbook of Endocrinology, ed 8. Saunders, Philadelphia, pp 489–619
10. Nishihara M, Winters CA, Buzko E, Waterman MR, Dufau ML 1988 Hormonal regulation of rat Leydig cell cytochrome P-45017 mRNA levels and characterization of a partial length rat P-45017 cDNA. Biochem Biophys Res Commun 154:151–158[CrossRef][Medline]
11. Simpson ER, Waterman MR 1988 Regulation of the synthesis of steroidogenic enzymes in adrenal cortical cells by ACTH. Annu Rev Physiol 50:427–440[CrossRef][Medline]
12. Stocco DM, Clark BJ 1996 Regulation of the acute production of steroids in steroidogenic cells. Endocr Rev 17:221–244[CrossRef][Medline]
13. Alfano J, Brownie AC, Orme-Johnson WH, Beinert H 1973 Adrenal mitochondrial cytochrome P-450 and cholesterol side chain cleavage activity: difference in the response of the zona glomerulosa and zona fasciculata-reticularis to adrenocorticotropic hormone and its withdrawal. J Biol Chem 248:7860–7864[Abstract/Free Full Text]
14. Kramer RE, Rainey WE, Funkenstein B, Dee A, Simpson ER, Waterman MR 1984 Induction of synthesis of mitochondrial steroidogenic enzymes of bovine adrenocortical cells by analogs cyclic AMP. J Biol Chem 259:707–713[Abstract/Free Full Text]
15. Barrett PQ, Bollag WB, Isales CM, McCarthy RT, Rasmussen H 1989 Role of calcium in angiotensin II-mediated aldosterone secretion. Endocr Rev 10:496–518[CrossRef][Medline]
16. Goldring NB, Durica JM, Lifka J, Hedin L, Ratoosh SL, Miller WL, Orly J, Richards JS 1987 Cholesterol side-chain cleavage P450 messenger ribonucleic acid: evidence for hormonal regulation in rat ovarian follicles and constitutive expression in corpora lutea. Endocrinology 120:1942–1950[Abstract]
17. Payne AH 1990 Hormonal regulation of cytochrome P450 enzymes, cholesterol side-chain cleavage and 17-hydroxylase/C17–20 lyase in Leydig cells. Biol Reprod 42:399–404[Abstract]
18. Voutilainen R, Tapanainen J, Chung BC, Matteson KJ, Miller WL 1986 Hormonal regulation of P450scc (20,22-desmolase) and P450c17 (17-hydroxylase/17,20-lyase) in cultured human granulosa cells. J Clin Endocrinol Metab 63:202–207[Abstract]
19. John ME, John MC, Boggaram V, Simpson ER, Waterman MR 1986 Transcriptional regulation of steroid hydroxylase genes by corticotropin. Proc Natl Acad Sci USA 83:4715–4719[Abstract/Free Full Text]
20. Ikeda Y, Shen WH, Ingraham HA, Parker KL 1994 Developmental expression of mouse steroidogenic factor-1, an essential regulator of the steroid hydroxylases. Mol Endocrinol 8:654–662[Abstract]
21. Hatano O, Takayama K, Imai T, Waterman MR, Takakusu A, Omura T, Morohashi K-i 1994 Sex-dependent expression of a transcription factor, Ad4BP, regulating steroidogenic P-450 genes in the gonads during prenatal and postnatal rat development. Development 120:2787–2797[Abstract]
22. Chung BC, Hu MC, Lai CC, Lin CH 1989 The 5'-region of the P450XIA1 (P450scc) gene contains a basal promoter and an adrenal-specific activating domain. Biochem Biophys Res Commun 160:276–281[CrossRef][Medline]
23. Hum DW, Miller WL 1993 Transcriptional regulation of human genes for steroidogenic enzymes. Clin Chem 39:333–340[Abstract]
24. Moore CC, Hum DW, Miller WL 1992 Identification of positive and negative placenta-specific basal elements and a cyclic adenosine 3',5'-monophosphate response element in the human gene for P450scc. Mol Endocrinol 6:2045–2058[Abstract]
25. Guo IC, Tsai HM, Chung BC 1994a Actions of two different cAMP-responsive sequences and an enhancer of the human CYP11A1 (P450scc) gene in adrenal Y1 and placental JEG-3 cells. J Biol Chem 269:6362–6369
26. Rice DA, Kirkman MS, Aitken LD, Mouw AR, Schimmer BP, Parker KL 1990 Analysis of the promoter region of the gene encoding mouse cholesterol side-chain cleavage enzyme. J Biol Chem 265:11713–11720[Abstract/Free Full Text]
27. Momoi K, Waterman MR, Simpson ER, Zanger UM 1992 3',5'-cyclic adenosine monophosphate-dependent transcription of CYP11A (cholesterol side chain cleavage cytochrome P450) gene involves a DNA response element containing a putative binding site for transcription factor Sp1. Mol Endocrinol 6:1682–1690[Abstract]
28. Venepally P, Waterman MR 1995 Two Sp1-binding sites mediate cAMP-induced transcription of the bovine CYP11A gene through the protein kinase A signaling pathway. J Biol Chem 270:25402–25410[Abstract/Free Full Text]
29. Inoue H, Watanabe N, Higashi Y, Fujii Kuriyama Y 1991 Structures of regulatory regions in the human cytochrome P-450scc (desmolase) gene. Eur J Biochem 195:563–569[Medline]
30. Watanabe N, Inoue H, Fujii-Kuriyama Y 1994 Regulatory mechanisms of cAMP-dependent and cell-specific expression of human steroidogenic cytochrome P450scc (CYP11A1) gene. Eur J Biochem 222:825–834[Medline]
31. Rodriguez H, Hum DW, Staels B, Miller WL 1997 Transcription of the human genes for cytochrome P450scc and P450c17 is regulated differently in human adrenal NCI-H295 cells than in mouse adrenal Y1 cells. J Clin Endocrinol Metab 82:365–371[Abstract/Free Full Text]
32. Moore CCD, Brentano ST, Miller WL 1990 Human P450scc gene transcription is induced by cyclic AMP and repressed by 12-O-tetradecanoylphorbol-13-acetate and A23187 through independent cis elements. Mol Cell Biol 10:6013–6023[Abstract/Free Full Text]
33. Chou SJ, Lai KN, Chung B 1996 Characterization of the upstream sequence of the human CYP11A1 gene for cell type-specific expression. J Biol Chem 271:22125–22129[Abstract/Free Full Text]
34. Payne AH, Youngblood GL 1995 Regulation of expression of steroidogenic enzymes in Leydig cells. Biol Reprod 52:217–225[Abstract]
35. Ikeda Y, Lala DS, Luo X, Kim E, Moisan MP, Parker KL 1993 Characterization of the mouse FTZ-F1 gene, which encodes a key regulator of steroid hydroxylase gene expression. Mol Endocrinol 7:852–860[Abstract]
36. Morohashi K, Zanger UM, Honda S, Hara M, Waterman MR, Omura T 1993 Activation of CYP11A and CYP11B gene promoters by the steroidogenic cell-specific transcription factor, Ad4BP. Mol Endocrinol 7:1196–1204[Abstract]
37. Sadovsky Y, Crawford PA, Woodson KG, Polish JA, Clements MA, Tourtellotte LM, Simburger K, Milbrandt J 1995 Mice deficient in the orphan receptor steroidogenic factor 1 lack adrenal glands and gonads but express P450 side-chain-cleavage enzyme in the placenta and have normal embryonic serum levels of corticosteroids. Proc Natl Acad Sci USA 92:10939–10943[Abstract/Free Full Text]
38. Mellon SH, Miller WL, Bair SR, Moore CC, Vigne JL, Weiner RI 1994 Steroidogenic adrenocortical cell lines produced by genetically targeted tumorigenesis in transgenic mice. Mol Endocrinol 8:97–108[Abstract]
39. Hogan B, Beddington R, Costantini F, Lacy E 1994 Manipulating the Mouse Embryo: A Laboratory Manual, ed 2. Cold Spring Harbor Laboratory, Cold Spring Harbor
40. Luo X, Ikeda Y, Parker KL 1994 A cell-specific nuclear receptor is essential for adrenal and gonadal development and sexual differentiation. Cell 77:481–490[CrossRef][Medline]
41. Nurnberg W, Artuc M, Nawrath M, Lovric J, Stuting S, Moelling K, Czarnetzki BM, Schadendorf D 1995 Human c-myb is expressed in cervical carcinomas and transactivates the HPV-16 promoter. Cancer Res 55:4432–4437[Abstract/Free Full Text]
42. Aguzzi A, Theuring F 1994 Improved in situ ß-galactosidase staining for histological analysis of transgenic mice. Histochemistry 102:477–481[CrossRef][Medline]
43. Morley SD, Viard I, Chung BC, Ikeda Y, Parker KL, Mullins JJ 1996 Variegated expression of a mouse steroid 21-hydroxylase/ß-galactosidase transgene suggests centripetal migration of adrenocortical cells. Mol Endocrinol 10:585–598[Abstract]
44. Brinkmeier ML, Gordon DF, Dowding JM, Saunders TL, Kendall SK, Sarapura VD, Wood WM, Ridgway EC, Camper SA 1998 Cell-specific expression of the mouse glycoprotein hormone -subunit gene requires multiple interacting DNA elements in transgenic mice and cultured cells. Mol Endocrinol 12:622–633[Abstract/Free Full Text]
45. Keri RA, Nilson JH 1996 A steroidogenic factor-1 binding site is required for activity of the luteinizing hormone ß subunit promoter in gonadotropes of transgenic mice. J Biol Chem 271:10782–10785[Abstract/Free Full Text]
46. Wolffe AP 1997 Transcription control: repressed repeats express themselves. Curr Biol 7:R796–R798
47. Dorer DR 1997 Do transgene arrays form heterochromatin in vertebrates? Transgenic Res 6:3–10[CrossRef][Medline]
48. Garrick D, Fiering S, Martin DIK, Whitelaw E 1998 Repeat-induce gene silencing in mammals. Nat Genet 18:56–59[CrossRef][Medline]
49. Compagnone NA, Bulfone A, Rubenstein JL, Mellon SH 1995 Expression of the steroidogenic enzyme P450scc in the central and peripheral nervous systems during rodent embryogenesis. Endocrinology 136:2689–2696[Abstract]
50. Masui K, Tamura Y 1926 The effect of gonadectomy on the structure of the suprarenal gland of mice, with reference to the functional relation between this gland and the sex gland of the female. J Coll Tokyo Imp Univ 7:353–376
51. Howard E 1927 A transitory zone in the adrenal cortex which shows age and sex relationships. Am J Anat 40:251–293[CrossRef]
52. Chester Jones I 1952 The disappearance of the X-zone of the mouse adrenal cortex during first pregnancy. Proc R Soc Lond B 139:398–410
53. Deacon CF, Mosley W, Jones IC 1986 The X zone of the mouse adrenal cortex of the Swiss albino strain. Gen Comp Endocrinol 61:87–99[CrossRef][Medline]
54. Mitani F, Mukai K, Ogawa T, Miyamoto H, Ishimura Y 1997 Expression of cytochromes P450aldo and P45011 ß in rat adrenal gland during late gestational and neonatal stages. Steroids 62:57–61[CrossRef][Medline]
55. Chung BC, Guo IC, Chou SJ 1997 Transcriptional regulation of the CYP11A1 and ferredoxin genes. Steroids 62:37–42[CrossRef][Medline]
56. Guo IC, Chung BC 1999 Cell-type specificity of human CYP11A1 TATA box. J Steroid Biochem Mol Biol 69:329–334[CrossRef][Medline]
57. Hum DW, Aza Blanc P, Miller WL 1995 Characterization of placental transcriptional activation of the human gene for P450scc. DNA Cell Biol 14:451–463[Medline]
58. Yu RN, Ito M, Saunders TL, Camper SA, Jameson JL 1998 Role of Ahch in gonadal development and gametogenesis. Nat Genet 20:353–357[CrossRef][Medline]
59. Nishida S, Mochizuki K 1963 Preliminary report on the function of the X-zone of the mouse adrenal cortex. Arch Histol Jap 23:213–227[Medline]
60. Mullins JJ, Sigmund CD, Kane Haas C, Gross KW, McGowan RA 1989 Expression of the DBA/2J Ren-2 gene in the adrenal gland of transgenic mice. EMBO J 8:4065–4072[Medline]
61. Kananen K, Markkula M, Mikola M, Rainio EM, McNeilly A, Huhtaniemi I 1996 Gonadectomy permits adrenocortical tumorigenesis in mice transgenic for the mouse inhibin -subunit promoter/simian virus 40 T-antigen fusion gene: evidence for negative autoregulation of the inhibin -subunit gene. Mol Endocrinol 10:1667–1677[Abstract]
62. Tanaka S, Matsuzawa A 1995 Comparison of adrenocortical zonation in C57BL/6J and DDD mice. Exp Anim 44:285–291[CrossRef][Medline]
63. Tanaka S, Nishimura M, Kitoh J, Matsuzawa A 1995 Strain difference of the adrenal cortex between A/J and SM/J mice, progenitors of SMXA recombinant inbred group. Exp Anim 44:127–130[CrossRef][Medline]
64. Mitani F, Suzuki H, Hata J, Ogishima T, Shimada H, Ishimura Y 1994 A novel cell layer without corticosteroid-synthesizing enzymes in rat adrenal cortex: histochemical detection and possible physiological role. Endocrinology 135:431–438[Abstract]
65. Linn F, Heidmann I, Saedler H, Meyer P 1990 Epigenetic changes in the expression of the maize A1 gene in Petunia hybrida: role of numbers of integrated gene copies and state of methylation. Mol Gen Genet 222:329–336[CrossRef][Medline]
66. Scheid OM, Paszkowski J, Potrykus I 1991 Reversible inactivation of a transgene in Arabidopsis thaliana. Mol Gen Genet 228:104–112[CrossRef][Medline]
67. Assaad FF, Tucker KL, Signer ER 1993 Epigenetic repeat-induced gene silencing (RIGS) in Arabidopsis. Plant Mol Biol 22:1067–1085[CrossRef][Medline]
68. Dorer DR, Henikoff S 1994 Expansions of transgene repeats cause heterochromatin formation and gene silencing in Drosophila. Cell 77:993–1002[CrossRef][Medline]
69. Sabl JF, Henikoff S 1996 Copy number and orientation determine the susceptibility of a gene to silencing by nearby heterochromatin in Drosophila. Genetics 142:447–458[Abstract]
70. Davis BP, MacDonald RJ 1988 Limited transcription of rat elastase I transgene repeats in transgenic mice. Genes Dev 2:13–22[Abstract/Free Full Text]
71. Sharpe JA, Wells DJ, Whitelaw E, Vyas P, Higgs DR, Wood WG 1993 Analysis of the human -globin gene cluster in transgenic mice. Proc Natl Acad Sci USA 90:11262–11266[Abstract/Free Full Text]
72. Chen CT, Guo IC, Chung BC 1995 Regulation of cholesterol side-chain cleavage cytochrome P450 in mouse testis Leydig cell line I-10. DNA Cell Biol 14:803–810[Medline]
73. Youngblood GL, Payne AH 1992 Isolation and characterization of the mouse P450 17-hydroxylase/C17–20-lyase gene (Cyp17): transcriptional regulation of the gene by cyclic adenosine 3',5'-monophosphate in MA-10 Leydig cells. Mol Endocrinol 6:927–934[Abstract]

This Article Abstract Full Text (PDF) An erratum has been published Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Hu, M.-C. Articles by Chung, B.-c.