0013.7227/92/1311-01~1$03.00/0 Endocrinology Copyright (* 1992 by The Endocrine
Vol. 131, No. 1 I’rmted in USA.
Society
Inherited Congenital Adrenal Absent Cholesterol Side-Chain P450 Gene Expression* SONGYA PANG, XIMING YANG, MICHELL JOSE MANALIGOD, LESLIE P. BULLOCK,
Hyperplasia Cleavage
WANG, ROBERT TISSOT, J. IAN MASON
MARLIN
in the Rabbit: Cytochrome NINO,
AND
Departments of Pediatrics (S.P., X. Y., M. W., M.N.), Genetics (S.P., X. Y., R.T.), and Pathology (J.M.), University of Illinois College of Medicine, Chicago, Illinois 60612; the University of California (L.P.B.), Riuerside, California 92521; and the Cecil H. and Ida Green Center for Reproductive Biology Sciences, and the Departments of Biochemistry and Obstetrics and Gynecology, University of Texas Southwestern Medical Center (J.I.M.), Dallas, Texas 75235 ABSTRACT We investigated adrenal steroidogenic enzymes, their activity and mRNA expression, and in uitro biosynthesis of an enzyme in rabbits with congenital adrenal hyperplasia (CAH; weight: CAH, 19 f 5 mg/ adrenal; normal, 2.7 f 1.0 mg/adrenal). Serum pregnenolone (LV-P) levels in CAH newborn rabbits (12-36 h) were normal (mean/range, 438/51-2191 ng/dl), but corticosterone levels were low [0.05 ? 0.05 pg/ dl; P < 0.001 us. normal (0.66 f 0.57)]. Serum Na’ levels in CAH newborn rabbits were in the normal range (143 + 30 meq/liter), but K’ levels were elevated [7 f 1.1 meq/liter; P < 0.05 us. normal (5.9 * 0.6 meq/liter)]. Minced normal adrenal tissue incubated with [“HI cholesterol (30-100 pmol/flask) and ACTH (100 mu/flask) produced [:‘H]E-P (newborn, 21 and 45 fmol/lOO mg; adult, 3 and 5 fmol/lOO mg) and [“Hlcorticosterone (newborn, 23 fmol/lOO mg; adult, 11.3 fmol/lOO mg), but CAH adrenals produced no product (cl.3 fmol/lOO mg). Adrenal mitochondria from normal newborn rabbits produced L%P (4.4-7 nmol/mg protein), but CAH adrenals did not, while CAH adrenal mitochondria demonstrated over 4 times greater 11/j-hydroxylase activity. A Western blot of adrenal homogenate from normal
newborn rabbits revealed a cholesterol side-chain cleavage cytochrome P450 (P450scc)-immunoreactive species (mol wt, 53 x lo:‘), but this species was absent in CAH adrenals; CAH adrenals had a normal adrenodoxin and intensified 17n-hydroxylase cytochrome P450 (P450,,,,) band compared to normal adrenals. In vitro translation of RNA in a cell-free rabbit reticulocyte lysate system containing [““S] methionine yielded a precursor P45Oscc protein (mol wt, 58.5 x 10”) with normal adrenal RNA, but not with CAH adrenal RNA. P45Oscc mRNA was detected in all normal adrenals, but was not detected in all CAH adrenals. 21.Hydroxylase cytochrome P450 mRNA expression was detected at a similar level in both normal and CAH adrenals. We conclude that CAH in the rabbit is caused by inherited absent P45Oscc gene expression. The clinical, pathological, and biochemical manifestations of P45Oscc deficiency in the rabbit are nearly identical to the human disorder. Increased llfl-hydroxylase activity and increased P45017,, on Western blot of CAH adrenals indicate altered gene expression of other steroidogenic enzymes due to CAH. Further molecular analysis of the P45Oscc gene in this animal CAH model will facilitate understanding of P45Oscc deficiency CAH. (Endocrinology 131: 181186,1992)
C
cytochrome P450 (P45Oscc) in a mitochondrial enzyme complex that also involves adrenodoxin and NADPH adrenodoxin reductase (4, 6). Deficient P45Oscc activity in the conversion of cholesterol to A5-P results in the accumulation of cholesterol in the adrenal cortex, causing lipoid adrenal hyperplasia, a salt-wasting and feminizing disorder in humans (7-10). In animals, an inherited feminizing lethal form of CAH was first reported in rabbits by Fox and Crary (11). Markedly hypertrophied zona fasciculata containing large lipid droplets were described, but the biochemical defect causing CAH was not investigated. In this report we have further characterized CAH in this animal model; the clinical, pathological, and biochemical abnormalities appear identical to those in human P45Oscc deficiency CAH (4, 7-10). We have determined that CAH in affected animals is due to absent P45Oscc gene expression.
ONGENITAL adrenal hyperplasia (CAH) is a family of well known human genetic disorders transmitted by an autosomal recessive trait that causes deficient steroidogenie enzyme activity in glucocorticoid and/or gonadal steroid biosynthesis (l-3). Inadequate glucocorticoid production, in turn, results in excess ACTH secretion, causing adrenocortical hyperplasia. The deficient steroidogenic enzyme activity may also cause under- or overproduction of mineralocorticoid, adrenal androgens, and gonadal sex steroids depending on the particular defect in steroidogenesis. Cholesterol is converted to pregnenolone (A5-P) by successive hydroxylation reactions at C-22 and C-20, followed by cleavage of the side-chain at C-20-22 (4-6). The conversion is mediated by a single cholesterol side-chain cleavage Received December 13, 1991. Address all correspondence and requests for reprints to: Songya Pang, M.D., Department of Pediatrics (M/C 856), 840 South Wood Street, Chicago, Illinois 60612. * This work was supported in part by grants from the USPHS (ROlHD-24360 and P50-HD-11149), the Washington Square Health Foundation at Chicago, and a Biomedical Research Grant to the University of Illinois College of Medicine. Presented in part at the 95th Annual Meeting of the Society for Pediatric Research, Anaheim, CA, May 7-11, 1990.
Materials Animal
and Methods
history and tissue collection
A strain of rabbits carrying the gene for CAH was derived by multiple out-crossings with carriers from the original strain (III vo/ah J) (11).
181
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182
INHERITED
CAH IN THE RABBIT
Newborn animals affected with CAH invariably died within the first 3 days of life. Blood samples were drawn from the ear median artery in live rabbits between 12-36 h of age. Adrenals were obtained at 1-3 days of age immediately after death by a method approved by the Animal Care Committee of the University of Illinois. The glands were freed of adhering fat, immediately frozen with liquid nitrogen, and stored at -70 C. The weight of adrenals was 2.7 & 1.0 mg/gland in phenotypically normal newborn litters (n = 50) and 195 mg/gland in CAH newborn litters (n = 20). Adrenal histological examination of CAH animals in our laboratory confirmed the hypertrophied zona fasciculata cells with multiple vacuolization as previously described (11). A pedigree study of our animals confirmed autosomal recessive transmission of the mutant trait for CAH (11).
Sexing of newborn
animals
The sex of phenotypically normal and CAH newborn rabbits was determined by a previously reported technique of examining both external and internal genitalia (12). Initially, sex was determined phenotypitally by gently exerting pressure on either side of the urogenital papillae of the external genital orifice. Male phenotype was assigned if a penis was everted from the genital orifice, and female if the slit-like vulva was present after protrusion of the genital orifice (Fig. 1). Ultimately, the sex of normal newborn rabbits and newborns affected with CAH was confirmed by examination of internal genitalia, including testes, ductus deferens, and gubernaculum in males, and ovaries, uterus, and chorda gubernaculi in females. The external genitalia of all male and female newborn rabbits with CAH were not distinguishable from normal female external genitalia (Fig. 1).
Laboratory
1992 No 1
tane-ethyl acetate, 99:l for A5-I’ and 60:40 for B), TLC (CHC13-methanol. 92:8). HPLC-1 (methanol-H1O, 60:40 for A5-P and 50:50 for B), and HPLC-2 (acetonitrile-H20, 60:40 for A5-I’ and 50:50 for B). The radioactivity of 3H and 14C was assessed using dual channels of a Packard scintillation counter (Downers Grove, IL). The constant ratio of [‘H]A5P to [“CIA5-P and of [3H]B to 5i4C ] B from the fractions of consecutive HPLC indicated the purity of H-labeled steroids produced from [‘HI cholesterol. [3H]A5-P and [3H]B were estimated by correcting for the loss of added “C-labeled steroids.
Adrenal
mitochondrial
steroid formation
Mitochondria were prepared as previously described (16). The protein concentration was determined by the method of Lowry et al. (17). Mitochondrial A5-I’ formation was determined using a RIA previously described (15). Medium (1 ml) containing approximately 1 mg mitochondrial protein was preincubated at 37 C with and without exogenous cholesterol (100 PM) for 10 min. o,L-Isocitrate (10 mM) was added to initiate the sterol side-chain cleavage reaction. Aliquots of the reaction mixture (0.2 ml at 0, 5, and 10 min) were assayed for A5-P by RIA. Mitochondrial 1 l/3-hydroxylase (P4501ip) activity was determined by preincubating mitochondrial protein (0.5 mg protein) in buffers, as previously described (15), supplemented with 1 mM CaC12, 10 PM [“Cl deoxycorticosterone (DOC) at 37 C for 10 min. The assay was initiated by the addition of 10 ~1 NADPH (7.5 mg/ml). Aliquots from the reaction mixture at 5 and 30 min were extracted with methanol, 30 pg DOC, and B, and the extracts were subjected to TLC (CHQmethanol, 95:5). P450,,p activity (picomoles per min/mg protein) was estimated from the fractional (percent) conversion of [r4C]DOC to [“C]B.
Western immunoblot
method
Na+ and K+ were determined by flame photometry. A5-I’ and corticosterone (B) concentrations were determined by specific RIA after extraction with an organic solvent and purification using Celite column chromatography (13). The inter- and intraassay variations in the RIA for A5-P and B were 12-15% and 8-lo%, respectively.
In vitro minced adrenal
Endo. Voll31.
incubation
and steroidogenesis
study
Adrenal glands from each animal group were pooled and minced with fine scissors. Incubation of the minced adrenals (48-100 mg/flask) was carried out according to a previously described method (14), with an initial oreincubation in an atmosphere of 95% 02-S% CO, at 37 C for 1 h. The preincubation medium was replaced in all flasks with fresh medium (5 ml). ACTH (100 mu/50 ~1 H20) was added to a flask; ACTH (100 mu) and [3H]cholesterol (30-100 pmol/20 PL ethanol; [1,2,6,7-N‘Hlcholesterol; 93.8 Ci/mmol) were added to a second flask; while in the third flask, ACTH, [3H]cholesterol, and cycloheximide (50 pg/20 ~1 H,O) were added. All flasks were incubated for 2 h. The medium was collected from the flask containing only ACTH. The media and rinses from the second and third flasks were extracted twice with equal volumes (10 ml) of methvlene chloride (15). A5-I’ and B compounds (100 fig/liter ethanol) were first added to the extracts as carriers, and additionally, 7000 cpm (100 ~1 ethanol) [4-‘4C]A5-P (NEC-375 A5-I’; 60 mCi/mmol) and [4-Y]B (NEC-421; 60 mCi/mmol) were added to each tube as internal markers. The dried residues were subjected to four consecutive chromatographic purifications using Celite columns (isooc-
The adrenal homogenate (50 pg protein/lane) was resolved by onedimensional electrophoresis on a 7.5% polyacrylamide gel containing 0.1% sodium dodecyl sulfate (SDS) at 4 C and 12 mamp for 18 h; followed bv transblottine. to a nitrocellulose filter at 4 C and 100 V for 1 h (18, 19). The nitrovcellulose filter was incubated in a sequential manner with a solution of BLOTTO buffer containing antibovine adrenal P45Oscc immunoglobulin G (IgG), antibovine adrenal adrenodoxin, or antiporcine testicular 17cu-hvdroxvlase cytochrome I’450 (I’45017a) IgG (20 ig/ml) for 1 h at room temperature (18, 19). The filter wasthen washed five times with BLOTTO buffer. For immunoblots of P45Oscc and adrenodoxin, the filter was incubated with the buffer containing antirabbit IgG alkaline phosphatase for 2 h at room temperature. After washing with BLOTTO buffer, the filter was treated with an alkaline phosphatase color reagent kit (Bio-Rad, Richmond, CA). For the immunoblot of P450 rTn, the filter was incubated with [rz51]protein-A (lo6 dpm/ ml) for 2 h at room temperature. After washing with buffer, the filter was analyzed using autoradiography.
In vitro RNA translation
in a cell-free system
Adrenal RNA was isolated as previously described (20). A lo-pg sample of RNA was translated using a commercially available rabbit reticulocyte lysate kit containing [35S]methionine (DuPont-New England Nuclear, Boston, MA), as previously described (21). [35S]Methionine translation products (6 X lo6 cpm) were immunoisolated after incubation with antibovine P45Oscc IgG (20 rg) overnight at 4 C and then immu-
FIG. 1. External genitalia of newborn rabbits: phenotypically normal male, CAH male, and phenotypically normal female. Note that the external genitalia of the CAH male is not distinguishable from that of the normal female. The arrows indicate a penile structure in a normal newborn male and the slit-like vulva in a CAH newborn male (center) and a normal female.
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INHERITED
CAH
IN THE
RABBIT
183
noprecipitated with Pansorbin-A (20 ~1) at room temperature for 10 min. The washed Pansorbin pellet was suspended in an electrophoresis sample buffer, incubated at 100 C for 3 h, and centrifuged. The supernatant was subjected to 7.5-12% gradient SDS-polyacrylamide gel electrophoresis with subsequent flourography.
exogenous cholesterol substrate. CAH adrenal mitochondria did not produce detectable A5-I’. CAH adrenal mitochondrial P450,,,, activity was 3.5-fold greater than that in normal adrenals.
Northern
Immunoblots of P45Oscc, adrenodoxin, of newborn rabbits (Fig. 2)
RNA analysis
Adrenal RNA was loaded (15 pg/lane) on a 1.2% denaturing agarose gel for electrophoresis and stained with acridine for RNA quantification. RNA was extracted from the adrenals (n = l/lane) of individual proven homozygous normal adult rabbits (wt/wt alleles), pooled adrenals (n = 6&8/lane) of phenotypically normal newborn rabbits born from proven heteroLygous parents, and adrenals (n = I-2/lane) of individual CAIi newborn rabbits. RNA was transferred to a Nytran membrane (Schleicher and Schuell, Keene, NH) and hybridized to human 1’450s~~ (a gift from Dr. W. L. Miller, Umversity of Cahforma, San Francisco, CA) (22) or human 1’450~21 cDNA vrobes (American Tvue Culture Coliection, Rockvrlle, MD) (23) in the $esence~ of 50% formamide. The final washing was performed using IX SCC buffer-0.1% SDS at 55 C and autoradiographed.
Results Serum electrolyte
levels in newborn
rabbits
Serum Na’ levels in seven CAH rabbits were in the normal newborn range (n = 20; 143 * 30 meq/liter); K’ levels (7 + 1.1 meq/liter) were significantly higher (P < 0.05) than those in normal newborn rabbits (5.9 f 0.6 meq/liter). Serum steroid levels in newborn
rabbits
Serum A5-I’ levels in six CAH newborns (258 f 200 ng/ dl) were lower than those in normal newborns (438 f 655 ng/dl), but were in the normal range; serum B levels (0.05 f 0.05) were unequivocally lower (P < 0.001) than those in the newborn animals (0.66 ? 0.57 +g/dl). Radiolabeled and total steroid levels in medium from incubation with minced adrenals of normal newborn and adult and CAH newborn and term fetal animals (Table 1) [“H]A5-P and [“H]B produced from [3H]cholesterol in the minced adrenal tissue of phenotypically normal newborn and adult animals were detected in the medium. Cycloheximide treatment inhibited the production of [3H]A5-P and [3H]B in normal adrenals, as evidenced by the decreased or undetectable [3H]A5-P or [3H]B levels in the medium. The CAH adrenals of newborn rabbits and fetuses did not produce detectable levels of [3H]A5-P or [3H]B. Total A5-P and B concentrations in the medium of normal newborn and adult adrenals increased 2.4- to 2.8-fold and 43 to 4.7-fold, respectively, after incubation with ACTH. In the medium of term CAH fetal adrenals, the A5-I’ concentration in the basal state was higher, while the B concentration was lower than those in normal adrenals, but the concentrations decreased or were unchanged after incubation with ACTH. Adrenal
mitochondrial
steroidogenic
enzyme activities
(Table 2)
Adrenal mitochondria of normal newborn rabbits produced A5-P in the absence and presence (3-fold increase) of
and P45017,, in adrenals
A P450scc-immunoreactive band (mol wt, 53 x 103) was detected in adrenals of phenotypically normal newborn rabbits. This band was absent in adrenals from CAH newborn rabbits, indicating the absence of P45Oscc (Fig. 2A). A control sample, mouse Leydig tumor cells, revealed the expected immunoreactive P45Oscc band at mol wt 51 X lo1 (Fig. 2A). lmmunoblots of normal and CAH adrenals revealed an adrenodoxin-immunoreactive species band at mol wt 12 x 10’. Immunoblots of normal adrenal demonstrated a P450,,,, band (mol wt, 55 x 10”; Fig. 2B) on autoradiography, which comigrated with bovine P450j7,, (not shown in the figure). This immunoreactive band was markedly intensified in the CAH adrenal, indicating an increased level of P450,,,, in hyperplastic adrenals (Fig. 28). In vitro translation
of
newborn
rabbit adrenal
RNA (Fig. 3)
The adrenal RNA translation products of normal newborn rabbits showed a radiolabeled protein band at mol wt 58.5 X lo1 on autoradiography. This indicated the biosynthesis of a higher mol wt mitochondrial P45Oscc precursor protein from normal newborn rabbit adrenal RNA. The adrenal RNA translation products of CAH newborn rabbits did not yield the labeled protein band at mol wt 58.5 X lo3 on autoradiography, indicating the absence of P45Oscc biosynthesis from CAH adrenal RNA. P45Oscc mRNA
and P45Oc21 mRNA
expression
(Fig. 4)
P45Oscc mRNA [-2.0 kilobases (kb)] was detected from the adrenals of individual proven homozygous normal adult rabbits (lanes 1, 3, and 10) and from the pooled adrenals of phenotypicaily normal newborn rabbits (lanes 4-6, Fig. 4A). P45Oscc mRNA was not detected in all CAH newborn adrenals (lanes 2 and 7-9, Fig. 4A). P45Oscc mRNA of phenotypically normal newborn adrenals showed two levels of expression, similar to (lane 5) and lower than (lanes 4 and 6) those of adult adrenals, These findings were consistently noted in subsequent Northern blot analysis. The integrity of RNA in newborn rabbit adrenals was confirmed by the presence of a control P4502i, mRNA in pooled normal newborn adrenals (lane 11) and a CAH adrenal (lane 12, Fig. 4A). In addition, 28s and 18s ribosomal markers in the corresponding lanes depict the integrity and equal quantities of 28s and 18s RNA markers used for the Northern blot hybridization.
Discussion The presentation of CAH animals, including pedigree data, sexual differentiation, invariable death, and markedly hy-
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INHERITED
184 TABLE
1. In vitro
Endo. Voll31.
1992 No 1
steroidogenic capacity of minced normal newborn, adult adrenals, CAH newborn, and CAH term fetal adrenals Medium
Adrenals pooled, tissue/flask (mg)
Normal adult (n = 4), 96-100 mg Normal newborn (n = 56), 18,” 50-52
CAH IN THE RABBIT
cow. after incubation
5~.P (ng/dl. 100 mg) Base
Pre-inc
51
27 88
NT
Labeled steroid production from [3H]cholesterol minced adrenals in the medium
with minced adrenals B (fig/dl. 100 mg)
Inc (ACTH)
[3H]A5-P
Inc (ACTH)
ACTH
(fmol/lOO
only
mg)
ACTH + Cyclohex
by
[3H]B (fmol/lOO
Base
Pre-inc
ACTH
only
77
1.25
0.83
3.64
3, 5
Vol. 131, No. 1 I’rmted in USA.
Society
Inherited Congenital Adrenal Absent Cholesterol Side-Chain P450 Gene Expression* SONGYA PANG, XIMING YANG, MICHELL JOSE MANALIGOD, LESLIE P. BULLOCK,
Hyperplasia Cleavage
WANG, ROBERT TISSOT, J. IAN MASON
MARLIN
in the Rabbit: Cytochrome NINO,
AND
Departments of Pediatrics (S.P., X. Y., M. W., M.N.), Genetics (S.P., X. Y., R.T.), and Pathology (J.M.), University of Illinois College of Medicine, Chicago, Illinois 60612; the University of California (L.P.B.), Riuerside, California 92521; and the Cecil H. and Ida Green Center for Reproductive Biology Sciences, and the Departments of Biochemistry and Obstetrics and Gynecology, University of Texas Southwestern Medical Center (J.I.M.), Dallas, Texas 75235 ABSTRACT We investigated adrenal steroidogenic enzymes, their activity and mRNA expression, and in uitro biosynthesis of an enzyme in rabbits with congenital adrenal hyperplasia (CAH; weight: CAH, 19 f 5 mg/ adrenal; normal, 2.7 f 1.0 mg/adrenal). Serum pregnenolone (LV-P) levels in CAH newborn rabbits (12-36 h) were normal (mean/range, 438/51-2191 ng/dl), but corticosterone levels were low [0.05 ? 0.05 pg/ dl; P < 0.001 us. normal (0.66 f 0.57)]. Serum Na’ levels in CAH newborn rabbits were in the normal range (143 + 30 meq/liter), but K’ levels were elevated [7 f 1.1 meq/liter; P < 0.05 us. normal (5.9 * 0.6 meq/liter)]. Minced normal adrenal tissue incubated with [“HI cholesterol (30-100 pmol/flask) and ACTH (100 mu/flask) produced [:‘H]E-P (newborn, 21 and 45 fmol/lOO mg; adult, 3 and 5 fmol/lOO mg) and [“Hlcorticosterone (newborn, 23 fmol/lOO mg; adult, 11.3 fmol/lOO mg), but CAH adrenals produced no product (cl.3 fmol/lOO mg). Adrenal mitochondria from normal newborn rabbits produced L%P (4.4-7 nmol/mg protein), but CAH adrenals did not, while CAH adrenal mitochondria demonstrated over 4 times greater 11/j-hydroxylase activity. A Western blot of adrenal homogenate from normal
newborn rabbits revealed a cholesterol side-chain cleavage cytochrome P450 (P450scc)-immunoreactive species (mol wt, 53 x lo:‘), but this species was absent in CAH adrenals; CAH adrenals had a normal adrenodoxin and intensified 17n-hydroxylase cytochrome P450 (P450,,,,) band compared to normal adrenals. In vitro translation of RNA in a cell-free rabbit reticulocyte lysate system containing [““S] methionine yielded a precursor P45Oscc protein (mol wt, 58.5 x 10”) with normal adrenal RNA, but not with CAH adrenal RNA. P45Oscc mRNA was detected in all normal adrenals, but was not detected in all CAH adrenals. 21.Hydroxylase cytochrome P450 mRNA expression was detected at a similar level in both normal and CAH adrenals. We conclude that CAH in the rabbit is caused by inherited absent P45Oscc gene expression. The clinical, pathological, and biochemical manifestations of P45Oscc deficiency in the rabbit are nearly identical to the human disorder. Increased llfl-hydroxylase activity and increased P45017,, on Western blot of CAH adrenals indicate altered gene expression of other steroidogenic enzymes due to CAH. Further molecular analysis of the P45Oscc gene in this animal CAH model will facilitate understanding of P45Oscc deficiency CAH. (Endocrinology 131: 181186,1992)
C
cytochrome P450 (P45Oscc) in a mitochondrial enzyme complex that also involves adrenodoxin and NADPH adrenodoxin reductase (4, 6). Deficient P45Oscc activity in the conversion of cholesterol to A5-P results in the accumulation of cholesterol in the adrenal cortex, causing lipoid adrenal hyperplasia, a salt-wasting and feminizing disorder in humans (7-10). In animals, an inherited feminizing lethal form of CAH was first reported in rabbits by Fox and Crary (11). Markedly hypertrophied zona fasciculata containing large lipid droplets were described, but the biochemical defect causing CAH was not investigated. In this report we have further characterized CAH in this animal model; the clinical, pathological, and biochemical abnormalities appear identical to those in human P45Oscc deficiency CAH (4, 7-10). We have determined that CAH in affected animals is due to absent P45Oscc gene expression.
ONGENITAL adrenal hyperplasia (CAH) is a family of well known human genetic disorders transmitted by an autosomal recessive trait that causes deficient steroidogenie enzyme activity in glucocorticoid and/or gonadal steroid biosynthesis (l-3). Inadequate glucocorticoid production, in turn, results in excess ACTH secretion, causing adrenocortical hyperplasia. The deficient steroidogenic enzyme activity may also cause under- or overproduction of mineralocorticoid, adrenal androgens, and gonadal sex steroids depending on the particular defect in steroidogenesis. Cholesterol is converted to pregnenolone (A5-P) by successive hydroxylation reactions at C-22 and C-20, followed by cleavage of the side-chain at C-20-22 (4-6). The conversion is mediated by a single cholesterol side-chain cleavage Received December 13, 1991. Address all correspondence and requests for reprints to: Songya Pang, M.D., Department of Pediatrics (M/C 856), 840 South Wood Street, Chicago, Illinois 60612. * This work was supported in part by grants from the USPHS (ROlHD-24360 and P50-HD-11149), the Washington Square Health Foundation at Chicago, and a Biomedical Research Grant to the University of Illinois College of Medicine. Presented in part at the 95th Annual Meeting of the Society for Pediatric Research, Anaheim, CA, May 7-11, 1990.
Materials Animal
and Methods
history and tissue collection
A strain of rabbits carrying the gene for CAH was derived by multiple out-crossings with carriers from the original strain (III vo/ah J) (11).
181
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 12 November 2015. at 15:07 For personal use only. No other uses without permission. . All rights reserved.
182
INHERITED
CAH IN THE RABBIT
Newborn animals affected with CAH invariably died within the first 3 days of life. Blood samples were drawn from the ear median artery in live rabbits between 12-36 h of age. Adrenals were obtained at 1-3 days of age immediately after death by a method approved by the Animal Care Committee of the University of Illinois. The glands were freed of adhering fat, immediately frozen with liquid nitrogen, and stored at -70 C. The weight of adrenals was 2.7 & 1.0 mg/gland in phenotypically normal newborn litters (n = 50) and 195 mg/gland in CAH newborn litters (n = 20). Adrenal histological examination of CAH animals in our laboratory confirmed the hypertrophied zona fasciculata cells with multiple vacuolization as previously described (11). A pedigree study of our animals confirmed autosomal recessive transmission of the mutant trait for CAH (11).
Sexing of newborn
animals
The sex of phenotypically normal and CAH newborn rabbits was determined by a previously reported technique of examining both external and internal genitalia (12). Initially, sex was determined phenotypitally by gently exerting pressure on either side of the urogenital papillae of the external genital orifice. Male phenotype was assigned if a penis was everted from the genital orifice, and female if the slit-like vulva was present after protrusion of the genital orifice (Fig. 1). Ultimately, the sex of normal newborn rabbits and newborns affected with CAH was confirmed by examination of internal genitalia, including testes, ductus deferens, and gubernaculum in males, and ovaries, uterus, and chorda gubernaculi in females. The external genitalia of all male and female newborn rabbits with CAH were not distinguishable from normal female external genitalia (Fig. 1).
Laboratory
1992 No 1
tane-ethyl acetate, 99:l for A5-I’ and 60:40 for B), TLC (CHC13-methanol. 92:8). HPLC-1 (methanol-H1O, 60:40 for A5-P and 50:50 for B), and HPLC-2 (acetonitrile-H20, 60:40 for A5-I’ and 50:50 for B). The radioactivity of 3H and 14C was assessed using dual channels of a Packard scintillation counter (Downers Grove, IL). The constant ratio of [‘H]A5P to [“CIA5-P and of [3H]B to 5i4C ] B from the fractions of consecutive HPLC indicated the purity of H-labeled steroids produced from [‘HI cholesterol. [3H]A5-P and [3H]B were estimated by correcting for the loss of added “C-labeled steroids.
Adrenal
mitochondrial
steroid formation
Mitochondria were prepared as previously described (16). The protein concentration was determined by the method of Lowry et al. (17). Mitochondrial A5-I’ formation was determined using a RIA previously described (15). Medium (1 ml) containing approximately 1 mg mitochondrial protein was preincubated at 37 C with and without exogenous cholesterol (100 PM) for 10 min. o,L-Isocitrate (10 mM) was added to initiate the sterol side-chain cleavage reaction. Aliquots of the reaction mixture (0.2 ml at 0, 5, and 10 min) were assayed for A5-P by RIA. Mitochondrial 1 l/3-hydroxylase (P4501ip) activity was determined by preincubating mitochondrial protein (0.5 mg protein) in buffers, as previously described (15), supplemented with 1 mM CaC12, 10 PM [“Cl deoxycorticosterone (DOC) at 37 C for 10 min. The assay was initiated by the addition of 10 ~1 NADPH (7.5 mg/ml). Aliquots from the reaction mixture at 5 and 30 min were extracted with methanol, 30 pg DOC, and B, and the extracts were subjected to TLC (CHQmethanol, 95:5). P450,,p activity (picomoles per min/mg protein) was estimated from the fractional (percent) conversion of [r4C]DOC to [“C]B.
Western immunoblot
method
Na+ and K+ were determined by flame photometry. A5-I’ and corticosterone (B) concentrations were determined by specific RIA after extraction with an organic solvent and purification using Celite column chromatography (13). The inter- and intraassay variations in the RIA for A5-P and B were 12-15% and 8-lo%, respectively.
In vitro minced adrenal
Endo. Voll31.
incubation
and steroidogenesis
study
Adrenal glands from each animal group were pooled and minced with fine scissors. Incubation of the minced adrenals (48-100 mg/flask) was carried out according to a previously described method (14), with an initial oreincubation in an atmosphere of 95% 02-S% CO, at 37 C for 1 h. The preincubation medium was replaced in all flasks with fresh medium (5 ml). ACTH (100 mu/50 ~1 H20) was added to a flask; ACTH (100 mu) and [3H]cholesterol (30-100 pmol/20 PL ethanol; [1,2,6,7-N‘Hlcholesterol; 93.8 Ci/mmol) were added to a second flask; while in the third flask, ACTH, [3H]cholesterol, and cycloheximide (50 pg/20 ~1 H,O) were added. All flasks were incubated for 2 h. The medium was collected from the flask containing only ACTH. The media and rinses from the second and third flasks were extracted twice with equal volumes (10 ml) of methvlene chloride (15). A5-I’ and B compounds (100 fig/liter ethanol) were first added to the extracts as carriers, and additionally, 7000 cpm (100 ~1 ethanol) [4-‘4C]A5-P (NEC-375 A5-I’; 60 mCi/mmol) and [4-Y]B (NEC-421; 60 mCi/mmol) were added to each tube as internal markers. The dried residues were subjected to four consecutive chromatographic purifications using Celite columns (isooc-
The adrenal homogenate (50 pg protein/lane) was resolved by onedimensional electrophoresis on a 7.5% polyacrylamide gel containing 0.1% sodium dodecyl sulfate (SDS) at 4 C and 12 mamp for 18 h; followed bv transblottine. to a nitrocellulose filter at 4 C and 100 V for 1 h (18, 19). The nitrovcellulose filter was incubated in a sequential manner with a solution of BLOTTO buffer containing antibovine adrenal P45Oscc immunoglobulin G (IgG), antibovine adrenal adrenodoxin, or antiporcine testicular 17cu-hvdroxvlase cytochrome I’450 (I’45017a) IgG (20 ig/ml) for 1 h at room temperature (18, 19). The filter wasthen washed five times with BLOTTO buffer. For immunoblots of P45Oscc and adrenodoxin, the filter was incubated with the buffer containing antirabbit IgG alkaline phosphatase for 2 h at room temperature. After washing with BLOTTO buffer, the filter was treated with an alkaline phosphatase color reagent kit (Bio-Rad, Richmond, CA). For the immunoblot of P450 rTn, the filter was incubated with [rz51]protein-A (lo6 dpm/ ml) for 2 h at room temperature. After washing with buffer, the filter was analyzed using autoradiography.
In vitro RNA translation
in a cell-free system
Adrenal RNA was isolated as previously described (20). A lo-pg sample of RNA was translated using a commercially available rabbit reticulocyte lysate kit containing [35S]methionine (DuPont-New England Nuclear, Boston, MA), as previously described (21). [35S]Methionine translation products (6 X lo6 cpm) were immunoisolated after incubation with antibovine P45Oscc IgG (20 rg) overnight at 4 C and then immu-
FIG. 1. External genitalia of newborn rabbits: phenotypically normal male, CAH male, and phenotypically normal female. Note that the external genitalia of the CAH male is not distinguishable from that of the normal female. The arrows indicate a penile structure in a normal newborn male and the slit-like vulva in a CAH newborn male (center) and a normal female.
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INHERITED
CAH
IN THE
RABBIT
183
noprecipitated with Pansorbin-A (20 ~1) at room temperature for 10 min. The washed Pansorbin pellet was suspended in an electrophoresis sample buffer, incubated at 100 C for 3 h, and centrifuged. The supernatant was subjected to 7.5-12% gradient SDS-polyacrylamide gel electrophoresis with subsequent flourography.
exogenous cholesterol substrate. CAH adrenal mitochondria did not produce detectable A5-I’. CAH adrenal mitochondrial P450,,,, activity was 3.5-fold greater than that in normal adrenals.
Northern
Immunoblots of P45Oscc, adrenodoxin, of newborn rabbits (Fig. 2)
RNA analysis
Adrenal RNA was loaded (15 pg/lane) on a 1.2% denaturing agarose gel for electrophoresis and stained with acridine for RNA quantification. RNA was extracted from the adrenals (n = l/lane) of individual proven homozygous normal adult rabbits (wt/wt alleles), pooled adrenals (n = 6&8/lane) of phenotypically normal newborn rabbits born from proven heteroLygous parents, and adrenals (n = I-2/lane) of individual CAIi newborn rabbits. RNA was transferred to a Nytran membrane (Schleicher and Schuell, Keene, NH) and hybridized to human 1’450s~~ (a gift from Dr. W. L. Miller, Umversity of Cahforma, San Francisco, CA) (22) or human 1’450~21 cDNA vrobes (American Tvue Culture Coliection, Rockvrlle, MD) (23) in the $esence~ of 50% formamide. The final washing was performed using IX SCC buffer-0.1% SDS at 55 C and autoradiographed.
Results Serum electrolyte
levels in newborn
rabbits
Serum Na’ levels in seven CAH rabbits were in the normal newborn range (n = 20; 143 * 30 meq/liter); K’ levels (7 + 1.1 meq/liter) were significantly higher (P < 0.05) than those in normal newborn rabbits (5.9 f 0.6 meq/liter). Serum steroid levels in newborn
rabbits
Serum A5-I’ levels in six CAH newborns (258 f 200 ng/ dl) were lower than those in normal newborns (438 f 655 ng/dl), but were in the normal range; serum B levels (0.05 f 0.05) were unequivocally lower (P < 0.001) than those in the newborn animals (0.66 ? 0.57 +g/dl). Radiolabeled and total steroid levels in medium from incubation with minced adrenals of normal newborn and adult and CAH newborn and term fetal animals (Table 1) [“H]A5-P and [“H]B produced from [3H]cholesterol in the minced adrenal tissue of phenotypically normal newborn and adult animals were detected in the medium. Cycloheximide treatment inhibited the production of [3H]A5-P and [3H]B in normal adrenals, as evidenced by the decreased or undetectable [3H]A5-P or [3H]B levels in the medium. The CAH adrenals of newborn rabbits and fetuses did not produce detectable levels of [3H]A5-P or [3H]B. Total A5-P and B concentrations in the medium of normal newborn and adult adrenals increased 2.4- to 2.8-fold and 43 to 4.7-fold, respectively, after incubation with ACTH. In the medium of term CAH fetal adrenals, the A5-I’ concentration in the basal state was higher, while the B concentration was lower than those in normal adrenals, but the concentrations decreased or were unchanged after incubation with ACTH. Adrenal
mitochondrial
steroidogenic
enzyme activities
(Table 2)
Adrenal mitochondria of normal newborn rabbits produced A5-P in the absence and presence (3-fold increase) of
and P45017,, in adrenals
A P450scc-immunoreactive band (mol wt, 53 x 103) was detected in adrenals of phenotypically normal newborn rabbits. This band was absent in adrenals from CAH newborn rabbits, indicating the absence of P45Oscc (Fig. 2A). A control sample, mouse Leydig tumor cells, revealed the expected immunoreactive P45Oscc band at mol wt 51 X lo1 (Fig. 2A). lmmunoblots of normal and CAH adrenals revealed an adrenodoxin-immunoreactive species band at mol wt 12 x 10’. Immunoblots of normal adrenal demonstrated a P450,,,, band (mol wt, 55 x 10”; Fig. 2B) on autoradiography, which comigrated with bovine P450j7,, (not shown in the figure). This immunoreactive band was markedly intensified in the CAH adrenal, indicating an increased level of P450,,,, in hyperplastic adrenals (Fig. 28). In vitro translation
of
newborn
rabbit adrenal
RNA (Fig. 3)
The adrenal RNA translation products of normal newborn rabbits showed a radiolabeled protein band at mol wt 58.5 X lo1 on autoradiography. This indicated the biosynthesis of a higher mol wt mitochondrial P45Oscc precursor protein from normal newborn rabbit adrenal RNA. The adrenal RNA translation products of CAH newborn rabbits did not yield the labeled protein band at mol wt 58.5 X lo3 on autoradiography, indicating the absence of P45Oscc biosynthesis from CAH adrenal RNA. P45Oscc mRNA
and P45Oc21 mRNA
expression
(Fig. 4)
P45Oscc mRNA [-2.0 kilobases (kb)] was detected from the adrenals of individual proven homozygous normal adult rabbits (lanes 1, 3, and 10) and from the pooled adrenals of phenotypicaily normal newborn rabbits (lanes 4-6, Fig. 4A). P45Oscc mRNA was not detected in all CAH newborn adrenals (lanes 2 and 7-9, Fig. 4A). P45Oscc mRNA of phenotypically normal newborn adrenals showed two levels of expression, similar to (lane 5) and lower than (lanes 4 and 6) those of adult adrenals, These findings were consistently noted in subsequent Northern blot analysis. The integrity of RNA in newborn rabbit adrenals was confirmed by the presence of a control P4502i, mRNA in pooled normal newborn adrenals (lane 11) and a CAH adrenal (lane 12, Fig. 4A). In addition, 28s and 18s ribosomal markers in the corresponding lanes depict the integrity and equal quantities of 28s and 18s RNA markers used for the Northern blot hybridization.
Discussion The presentation of CAH animals, including pedigree data, sexual differentiation, invariable death, and markedly hy-
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INHERITED
184 TABLE
1. In vitro
Endo. Voll31.
1992 No 1
steroidogenic capacity of minced normal newborn, adult adrenals, CAH newborn, and CAH term fetal adrenals Medium
Adrenals pooled, tissue/flask (mg)
Normal adult (n = 4), 96-100 mg Normal newborn (n = 56), 18,” 50-52
CAH IN THE RABBIT
cow. after incubation
5~.P (ng/dl. 100 mg) Base
Pre-inc
51
27 88
NT
Labeled steroid production from [3H]cholesterol minced adrenals in the medium
with minced adrenals B (fig/dl. 100 mg)
Inc (ACTH)
[3H]A5-P
Inc (ACTH)
ACTH
(fmol/lOO
only
mg)
ACTH + Cyclohex
by
[3H]B (fmol/lOO
Base
Pre-inc
ACTH
only
77
1.25
0.83
3.64
3, 5
