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Endocrinology Vol. 140, No. 7 3354-3362
Copyright © 1999 by The Endocrine Society

ARTICLES

Prenatal Dexamethasone Treatment Does Not Prevent Alterations of the Hypothalamic Pituitary Adrenal Axis in Steroid 21-Hydroxylase Deficient Mice

Toshihiro Tajima1, Xin-Ming Ma, Stefan R. Bornstein2 and Greti Aguilera

Section on Endocrine Physiology, Developmental Endocrinology Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892

Address all correspondence and requests for reprints to: Greti Aguilera, M.D., Section on Endocrine Physiology, Developmental Endocrinology Branch, NICHD, NIH, Building 10, Room 10n262, 10 Center Drive MSC 1862, Bethesda, Maryland 20892-1862. E-mail: aguilerg{at}exchange.nih.gov


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
A major difficulty in the clinical management of congenital adrenal hyperplasia (CAH) is adjustment of glucocorticoid doses to suppress ACTH and androgens without causing iatrogenic hypercortisolism. The possibility that structural alterations of the adrenal or a dysfunction of the hypothalamic pituitary adrenal (HPA) axis caused by glucocorticoid deficiency during fetal life contribute to this problem was studied in 21-hydroxylase deficient mice caused by deletion of the cytochrome P-450 21-hydroxylase gene. Homozygotes showed about 200-fold elevations in plasma progesterone, hyperplastic adrenal cortices lacking zonation, and structural alterations of adrenocortical mitochondria. Histochemical studies showed increases in hypothalamic CRH messenger RNA (mRNA) and immunoreactive (ir) CRH, and pituitary POMC mRNA in homozygous mice. VP mRNA levels in PVN perikarya were normal, but irVP in parvicellular terminals of the median eminence was increased in homozygotes. Prenatal dexamethasone treatment (0.5 to 2 µg/day) prevented the increases in CRH mRNA, whereas dexamethasone only partially decreased POMC mRNA levels, and had no effect on serum progesterone levels. The data suggest that intrauterine glucocorticoid deficiency in CAH causes hyperactivity of the hypothalamic-pituitary-corticotroph axis and insensitivity to glucocorticoid feedback. These studies in 21-hydroxylase deficient mice may provide new insights on the mechanism, clinical manifestations and management of some types of human CAH.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CONGENITAL adrenal hyperplasia (CAH) is a congenital disorder caused by a defect in one of the enzymes of the steroidogenic pathway leading to synthesis of glucocorticoids (1, 2, 3). The decreased glucocorticoid production results in increased POMC and ACTH secretion from the pituitary, and subsequent hyperplasia of the adrenal cortex. The most common type of CAH (comprising over 90% of cases) is the 21-hydroxylase deficiency caused by mutations, gene conversion or deletions of the cytochrome 21-hydroxylase gene (CYP21) (1, 2, 3). The incidence of the disease is relatively high, 1:14,000 of live births (1). The defective or absent enzyme causes various degrees of glucocorticoid and mineralocorticoid deficiency, in some cases resulting in severe salt wasting, which can be lethal if not diagnosed and treated (1, 2, 3). The decreased glucocorticoid production activates the hypothalamic pituitary adrenal (HPA) axis causing hypersecretion of pituitary ACTH, adrenal hyperplasia, and overproduction of 17-hydroxy progesterone and androgens (1, 2, 3).

The conventional treatment consists in replacing both glucocorticoids and mineralocorticoids to reduce the excessive secretion of ACTH and adrenal androgens (1, 2, 3, 4, 5, 6, 7, 8). However, current treatment regimes often fail to normalize plasma ACTH through the entire day (4, 5, 6, 7, 8). While suppression of elevated ACTH and androgen levels to the normal range can be achieved by increasing the dose of hydrocortisone, such doses expose patients to supraphysiological levels of glucocorticoids causing iatrogenic hypercortisolism (4, 5, 6, 7, 8). Part of the difficulties in suppressing ACTH secretion may be due to HPA axis dysfunction due to chronic lack of glucocorticoids. Moreover, intrauterine glucocorticoid deficiency may affect the sensitivity of feedback inhibition, thus blunting the central effects of treatment.

The main regulators of pituitary ACTH secretion are CRH and vasopressin (VP), both produced by parvicellular neurons of the hypothalamic paraventricular nucleus (PVN) (9, 10). Adrenal glucocorticoids exert their negative feedback effect at multiple levels by acting upon glucocorticoid receptors, mainly type I in the hippocampus and type II in the PVN and in the pituitary corticotroph (11, 12). In humans, pregnancies at risk for 21-hydroxylase deficiency may by treated prenatally to reduce or eliminate virilization in the female fetus, although this is controversial (13, 14, 15, 16). Dexamethasone (20 µg/kg·day) is administered at a gestational age of 6–7 weeks before external genitalia begin to differentiate (13, 14, 15, 16). Presumably prenatal dexamethasone treatment will normalize the HPA axis of affected fetuses, but so far no data are available from in vivo studies of either human or experimental animals showing the interrelation between hypothalamic regulators, ACTH, and corticosteroids in 21-hydroxylase deficient individuals.

In this study, the 21-hydroxylase deficient mouse was used as a model to investigate the effect of intrauterine glucocorticoid deficiency on HPA axis activity. The Japanese strain of mice with 21-hydroxylase (H-2 aw18 haplotype) has a deletion of the CYP21 gene and has been reported to have completely impaired 21-hydroxylase activity. As in the human disease, the lack of glucocorticoids results in adrenocortical hyperplasia and accumulation of precursor steroids. In mice, which lack of 17-{alpha}-hydroxylase in the adrenal, the enzymatic blockade results mainly in accumulation of progesterone. The majority of affected mice die within a week if not treated with gluco- and mineralocorticoids (17, 18, 19). Although, the 21-OH deficient mouse is not strictly comparable with human CAH, it may provide a useful model to study the pathophysiology of the disease.

In these studies, in situ hybridization and immunohistochemical techniques were employed to investigate the alterations of the HPA axis in 21-OH deficient mice. Levels of messenger RNA (mRNA) and immunoreactive CRH and VP in the hypothalamus, and POMC mRNA in the pituitary were determined in newborn wild-type and homozygous 21-OH deficient mice, with and without prenatal glucocorticoid replacement. Adrenocortical changes were analyzed by light and electron microscopy. The results demonstrate that homozygous 21-hydroxylase deficient mice show hyperactivity of the hypothalamic-pituitary corticotroph axis and adrenal abnormalities, and that these changes were only partially prevented by prenatal treatment with high doses of dexamethasone.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and experimental protocols
Heterozygous 21-deficient mice (H-2 aw18 haplotype), kindly provided by Dr. Toshihiro Shiroishi, Institute of Genetics, Japan, and wild-type C5BL10J mice purchased from The Jackson Laboratory (Bar Harbor, ME), were maintained according to the NIH guidelines with a 12-h light, 12-h dark cycle and free access to food and water. The presence of vaginal plug on the morning after mating was set as day 0.5 of gestation. Because in initial litters most homozygous pups died soon after birth, all dams were treated with 5 µg of dexamethasone in 50 µl of peanut oil, sc, at gestational day 20 to prevent loss of experimental material. In a first set of experiments, litters were killed immediately after birth, blood was collected for progesterone measurement, and the heads frozen in dry ice for in situ hybridization or fixed in paraformaldehyde for immunohistochemistry. In a second set of experiments, pregnant mice received injections of dexamethasone (50 µl in peanut oil, sc), 0.5 and 1 µg/day (16.5 and 33 µg/kg), which are in the range of doses used in human therapy, and higher doses from 2 to 5 µg/day (66 and 165 µg/kg). Experiments by our laboratory in the rat have shown that this mode of administration of dexamethasone provides steroid levels able to suppress corticosterone responses to immobilization stress up to 24 h after injection (Rabadan-Diehl, C., and G. Aguilera, unpublished observation). Litters were killed immediately after birth for blood and tissue collection. All animal protocols were approved by the Animal Users Care Committee of the NICHD, NIH.

Determination of genotype
Genomic DNA was extracted from livers or tails using standard procedures as previously described (18). A 950-bp complementary DNA (cDNA) fragment encoding exons 3 to 9 of the mouse CYP21 cDNA was prepared by PCR using 500 ng of total adrenal RNA (prepared with TRIzol reagent, Gibco BRL, Gaithersburg, MD) and the GeneAmpRNA PCR kit (Perkin Elmer Corp., Foster City, CA). The upstream primer was 5'-GAAAGATGGACTTGGACCTGTCCT-3', and the downstream primer was 5'-AGGGTAGTCATAGCCGGAGAT-3'. PCR was performed using 500 ng of mouse adrenal RNA as template under the following conditions: 30 cycles, 1 min at 94 C; 1 min at 58 C, and 3 min extension at 72 C. The blunt-ended PCR product was cloned using TA-cloning kit (Invitrogen, Carlsbad, CA) and used for preparation of random primer radiolabeled probes for Southern blot analysis of Bgl-II digests of the genomic DNA (18). As previously shown, wild-type mice showed two bands corresponding to the active CYP21 gene and functionally inactive CYP21 pseudogene. Homozygous mice showed a single smaller band containing the CYP21 pseudogene, whereas heterozygotes showed three bands. In some experiments, genotype was determined by PCR analysis of the genomic DNA using the primers described above under the following conditions: 35 cycles, 1 min 94 C, 2 min at 58 C, and 3 min extension at 72 C.

Adrenal morphology
Adrenal glands were removed, dissected, and fixed for 3 h in 2% paraformaldehyde, 2% glutaraldehyde in 0.1 M phosphate buffer, pH 7.3. Tissue slices were postfixed for 90 min (2%OsO4 in 0.1 M cacodylate buffer pH 7.3), dehydrated in ethanol, and embedded in epoxy resin. Ultrathin sections were stained with lead citrate and examined at 80 kV in a Phillips EM 301 (C Phillips Electronics, Mahway, NJ).

For specific staining of chromaffin cells, paraffin sections of mice adrenals were immunostained with anti-tyrosine hydroxylase antibody (Boehringer Mannheim, Mannheim, Germany). After preincubation for 30 min with 5% normal rabbit serum in 0.1 M Tris-buffered saline, pH 7.4, sections were incubated with tyrosine hydroxylase antibody diluted 1:10 in TBS with 5% normal rabbit serum, at 4 C, overnight, washed in TBS 3 times for 10 min, incubated with rabbit antimouse antiserum (DAKO Corp., Hamburg, Germany) 1:50 for 60 min at room temperature, followed by an additional three washes in TBS and immersion in a rabbit PAP complex 1:50 dilution. Immunostaining was visualized by incubation with 3-amino 9 ethyl carbazole (AEC) chromogen system (Immunotech, Hamburg, Germany) as described by the manufacturer. Slides were counterstained with hematoxylin, dehydrated and mounted with gelatin.

Measurement of progesterone
Serum was collected from newborn mice by decapitation between 0900 and 1100 h using nonheparinized capillary tubes. Serum progesterone concentration was determined using commercial kit reagents from Diagnostics Systems Laboratories, Inc. (Webster, TX).

In situ hybridization
Frozen heads were stored at -80 C until sectioned in a cryostat at -18 C. Twelve-micrometer coronal sections comprising the hypothalamic region and pituitary gland were thaw mounted onto poly-L-lysine- (Sigma Chemical Co., St. Louis, MO) coated slides, and frozen at -80 C until used for in situ hybridization. Tissue sections were processed for in situ hybridization as described previously (20). Antisense and sense CRH riboprobes were transcribed from a mouse CRH 578-bp PstI fragment subcloned into pGem4Z kindly provided by Dr. Audrey Seasholtz (University of Michigan, Ann Arbor, MI) (21). For VP, a 230-bp fragment of exon 3 of the rat cDNA cloned into pGem4Z, kindly provided by Drs. Susan Wray and Harold Gainer (NINDS, NIH), was used to transcribe an antisense cRNA probe. High specific activity antisense cRNA probes were synthesized using 35S-UTP and 35S-CTP as previously described (20). Slide mounted sections were fixed, acetylated and hybridized overnight in a humidified chamber at 55 C. After hybridization, sections were washed, treated with RNase-A to remove nonspecifically bound probe, dehydrated and exposed to Kodak BIOMAX-film (Eastman Kodak Co., Rochester, NY) for 48–72 h, and then dipped in Kodak NTB-2 nuclear emulsion (diluted 1:1 with water), and exposed for 4–7 weeks. POMC hybridization was performed as previously described using a 48-mer oligonucleotide directed toward the carboxy-terminal amino acids of rat POMC (22). Sections from control and experimental groups were processed in the same hybridization. Relative mRNA abundance was semiquantitated from the optical densities of the autoradiographies using computerized image analysis system (Imaging Research, Inc., Ontario, Canada) using the public domain NIH Image program (developed at the NIH, and available on the Internet at http://rsb.info.nih.gov/nih-image). Values for each animal were calculated from the optical densities in three sections after subtracting the background, and the results of each experimental group was obtained from the average of the values in at least five mice per group. No hybridization was observed using sense probes.

Immunohistochemistry for CRH and VP
Immediately after decapitation, brains were carefully removed, fixed in 4% paraformaldehyde in PBS for 48 h at 4 C, transferred to 5% sucrose for an additional 48 h at 4 C, and frozen in powdered dry ice after being placed in a cryomold containing embedding medium (TissueTeck OCT, Sakura Finetek USA, Torrance, CA). Thirty-micrometer coronal cryostat sections were mounted onto poly-lysine-coated slides and used for immunohistochemical detection of CRH and VP using the rCRH antibody GA-13 at a dilution of 1:2,000 (23) or rVP antibody kindly provided by Dr. Harold Gainer, NINDS, NIH, at a dilution of 1:1,500, and Vectostain ABC kit reagents (Vector Laboratories, Inc., Burlingame, CA), as previously described (23).

Data analysis
Data are presented as the mean and SE of the values for the number of observations indicated in Results. Unless otherwise indicated, statistical significance of the differences between experimental groups was determined by unpaired t test or one way-ANOVA, followed by Fisher’s least significant difference procedure test for multiple group comparisons.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Adrenal morphology
21-hydroxylase deficient mice showed marked changes in adrenal structure compared with wild-type mice. Under light microscopy, adrenals of newborn mutant mice were markedly enlarged with hyperplasia of adrenocortical cells. A regular zona glomerulosa was absent, and fasciculata-like cells reached the capsule. The formation of the adrenal medulla in the center of the gland was incomplete, with single cells and islets of chromaffin cells remaining within the adrenal cortex as demonstrated by the presence of tyrosine hydroxylase stained cells (Fig. 1Go, A and B).



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  		Figure 1. Structural changes of adrenals of 21-OH-deficient mice compared with wild-type animals at 7 days of age. A, Histological section of wild-type adrenals demonstrate a regular zonation with a thick capsule (CAP), zona glomerulosa (ZG), zona fasciculata (ZF); zona reticularis (ZR), and an inner zona medullaris (ZM) characterized by tyrosine hydroxylase immunostaining of chromaffin cells (x200). B, The adrenal cortex of the 21-OH-deficient mice is markedly enlarged showing hyperplastic adrenocortical cells (small arrows). There is no regular zonation. A normal zona glomerulosa is absent and strings of fasciculata-like cells reach to the outer capsule (large arrows). Chromaffin tissue characterized by immunostaining for tyrosine hydroxylase formed a medulla in the center of the gland but islets and single chromaffin cells (CC) are located within the cortex (x200) C, Electron micrograph of adrenocortical cell of newborn mice reveals characteristic round mitochondria with dense vesicular inner membranes (x16900). D, 21-OH-deficient mice show conspicuous ultrastructural changes with large polymorphic mitochondria (MIT) and sparse inner membranes. Intermitochondrial herniations can be seen (arrowheads); NUC, nucleus; x16,900.

 
On the ultrastructural level, adrenocortical cells of wild-type mice demonstrated smooth endoplasmic reticulum, characteristic vesicular mitochondria and liposomes. Cellular and mitochondrial structure allowed differentiation of glomerulosa cells with elongated tubulolamellar mitochondria from fasciculata/reticularis cell type with round tubulovesicular mitochondria. The subcellular structure of adrenocortical cells of 21-hydroxylase deficient mice was markedly altered showing a particularly conspicuous enlargement of the mitochondria. The giant mitochondria showed poorly developed internal membranes, some of them with myelin-type figures and lipidic inclusions. Also, intermitochondrial herniations with finger-like projections from one mitochondrion into an invagination of a neighboring mitochondrion were frequently detected (Fig. 1Go, C and D).

Serum progesterone
Serum progesterone levels were markedly elevated (>200-fold) in homozygous newborn mice, whereas in heterozygous mice progesterone levels were similar to wild-type controls (Table 1Go). To suppress adrenocortical hyperstimulation in homozygous fetuses, dexamethasone (0.5 to 5 µg) was injected daily to pregnant mice from gestational day 11. Weight and number of pups in the litters were normal in mice receiving 0.5 to 2 µg/day dexamethasone, but higher doses of dexamethasone (3–5 µg) decreased pups weight or caused fetal death. Unexpectedly, maternal treatment with up to 2 µg dexamethasone completely failed to suppress serum progesterone levels in homozygous mice (Table 1Go).


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  		Table 1. Effect of prenatal dexamethasone treatment on serum progesterone levels in litters from aw18/b heterozygous mice

 
Expression of hypothalamic CRH and VP and pituitary POMC
To determine whether prenatal glucocorticoid deficiency in 21-hydroxylase deficient mice caused compensatory changes at the hypothalamic and pituitary levels, the relative abundance of CRH mRNA and VP mRNA in the PVN, and POMC mRNA in the pituitary were analyzed at birth in homozygous and wild-type mice. As shown in Fig. 2Go, A and B, CRH mRNA optical density levels in homozygous mice were about 2-fold those in wild-type mice (193.4 ± 14.7%). The elevated CRH mRNA density levels in the PVN of homozygous mice were accompanied by increased irCRH content. As expected in the absence of colchicine treatment (23), ir CRH was almost undetectable in the PVN of wild-type pups, whereas clear immunostaining was observed in PVN perikarya of all homozygous mice analyzed (Fig. 2CGo). No consistent differences in CRH immunostaining were observed in the external zone of the median eminence of homozygous mice (Fig. 2DGo).



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  		Figure 2. CRH mRNA levels in the PVN (A and B), and immunoreactive CRH in the PVN (C) of wild-type (WT) and homozygous new-born 21-hydroxylase deficient mice (HZ). A, Representative PVN sections from WT and HZ mice hybridized for CRH mRNA and exposed to photographic emulsion for 65 days. B, CRH mRNA levels in the PVN in WT and HZ mice. Bars are the mean and SE of the values obtained in five to six mice per group. *, P < 0.01. C, Representative hypothalamic sections from WT and HZ mice showing the increase in irCRH staining in 21-hydoxylase deficient mice.

 
Optical density levels of VP mRNA and irVP in the PVN reflecting mostly magnocellular neurons were similar in wild-type and homozygous mice (not shown). However, irVP content in the external zone of the median eminence containing axons from parvicellular neurons was increased in all sections from homozygous mice compared with almost undetectable immunostaining in wild-type mice (see Fig. 5Go). POMC mRNA levels were markedly increased by 4-fold (404.2 ± 29.2%) in the anterior pituitary lobe of homozygous mice (Fig. 3Go).



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  		Figure 5. Immunoreactive VP in the median eminence of wild-type (A) homozygous new-born 21-hydroxylase deficient without (B) and with (C) prenatal treatment with 2 µg/day dexamethasone. Figure is representative of the results in five WT, four untreated HZ, and three treated HZ.

 


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  		Figure 3. POMC mRNA levels in wild-type (WT) and homozygous new-born 21-hydroxylase deficient (HZ) mice. A, Representative pituitary sections of wild-type (WT) and homozygous 21-hydroxylase deficient mice hybridized for POMC mRNA after exposure to photographic emulsion for 30 days. B, Mean and SE of the values obtained in five to six mice per group. *, P < 0.001.

 
Effect of prenatal dexamethasone treatment
Daily injection of doses of dexamethasone similar to those used in humans on a per kg basis (0.5 µg/mice) into pregnant mice reduced CRH mRNA in the PVN of homozygous mice from optical density values of 223 ± 7.5 without treatment to 152 ± 7.5. These values were significantly higher than those in wild-type mice (31.6 ± 4.7%, P < 0.05). Prenatal treatment with higher doses of dexamethasone, 1.0 and 2.0 µg, suppressed CRH mRNA density levels in the PVN to those observed in wild-type mice (103.2 ± 13.5%, and 96.5 ± 13.7%, respectively) (Fig. 4Go). Consistent with the changes in CRH mRNA, prenatal treatment with 2 µg dexamethasone reduced CRH immunostaining in homozygous mice to undetectable levels as observed in wild-type mice (not shown).



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  		Figure 4. Effect of prenatal dexamethasone treatment on CRH mRNA levels in the PVN of newborn wild-type and homozygous 21-hydroxylase deficient mice. Bars are the mean and SE of the values obtained in five mice per group. *, P < 0.001 vs. wild-type; #, P < 0.05 vs. wild-type; o, P < 0.01 vs. untreated homozygote.

 
Prenatal treatment with dexamethasone had no effect on VP mRNA or irVP in the PVN of homozygous mice. However, analysis of irVP in three mice receiving prenatal dexamethasone treatment showed a reduction in immunostaining in the external zone of the median eminence to levels similar to those seen in wild-type mice (Fig. 5Go).

In contrast to the complete inhibitory effect of prenatal dexamethasone on the expression of hypothalamic CRH and VP, this treatment only partially decreased pituitary POMC mRNA. Values decreased from 404.2 ± 29.2% over wild-type controls with no prenatal treatment to 345.6 ± 30.5%, 278.2 ± 6.0% and 240 ± 7.1% with 0.5, 1.0 and 2.0 µg/day (Fig. 6Go).



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  		Figure 6. Effect of prenatal dexamethasone treatment on POMC mRNA levels in the anterior pituitary of newborn wild-type and homozygous 21-hydroxylase deficient mice. Bars are the mean and SE of the values obtained in five mice per group. *, P < 0.001 vs. wild-type; #, P < 0.05 vs. untreated homozygotes; o, P < 0.05 vs. 0.5 µg dexamethasone-treated homozygotes.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study in mice shows that lack of fetal glucocorticoid production due to congenital 21-hydroxylase deficiency causes profound alterations of the HPA axis that cannot be prevented by prenatal dexamethasone treatment. In humans with CAH, prenatal treatment is given under the assumption that the HPA axis is functional in the fetus and that exogenous glucocorticoids will prevent its hyperactivity caused by the lack of endogenous glucocorticoid feedback (13, 14, 15, 16). The main regulators of pituitary ACTH secretion are the hypothalamic peptides CRH and VP produced in parvicellular neurons of the hypothalamic paraventricular nucleus (9, 10). The increases in CRH mRNA and irCRH in the PVN in 21-hydroxylase deficient mice clearly show an overproduction and presumably increased release of the peptide to the hypophyseal portal circulation. Similar increases in CRH mRNA in the PVN have been observed in glucocorticoid receptor knockout mice (24).

The apparent lack of change in VP mRNA and irVP in the PVN is consistent with findings in glucocorticoid receptor and CRH knockout mice (24, 25), and is probably due to the high levels of VP expression in hypothalamic-neurohypophyseal magnocellular neurons that would mask any changes in hypophyseotrophic parvicellular VP containing neurons (9, 10). While the internal zone of the median eminence carries magnocellular axons projecting to the neurohypophysis, the external zone contains parvicellular nerve terminals which interact with capillaries of the pituitary portal circulation (10). Thus, the increase in irVP shown in the external zone of the median eminence strongly suggests that VP expression in parvicellular neurons is enhanced in 21-hydroxylase deficient mice. Also, consistent with the fetal glucocorticoid deficiency, POMC mRNA levels were markedly elevated suggesting hyperproduction of ACTH.

In these experiments, prenatal treatment with dexamethasone initiated at the time of adrenal differentiation failed to have any inhibitory effect on plasma progesterone levels of homozygous 21-hydroxylase deficient mice. This is consistent with reports in humans indicating that 17-hydroxyprogesterone levels in umbilical blood are elevated in spite of reduced virilization (13, 26). While persistence of high progesterone may be due to the adrenal abnormalities as has been suggested for the lack of suppression of 17-hydroxy progesterone and androgens in humans (6), the high doses of dexamethasone required to suppress CRH mRNA and the lack of complete pituitary POMC inhibition indicate a relative insensitivity to glucocorticoid feedback in these mice. The decrease in body weight and fetal death observed in these experiments with doses of dexamethasone higher than 2 µg/day is consistent with previous reports (27) and suggests that the lack of inhibition of the HPA axis is not due to poor passage of the steroid through the placenta. The most intriguing finding was the incomplete pituitary inhibition with doses of dexamethasone capable to prevent increases in hypothalamic CRH and VP. It is unlikely that this dissociation is due to the circadian variations because 1) the increase in CRH mRNA should precede that of POMC mRNA, and 2) the mismatch was found in all litters irrespective of the time of the day in which they were born and killed. Moreover, the synthetic glucocorticoid, dexamethasone, used in these experiments has a higher affinity for glucocorticoid receptors (the type present in the pituitary) than for hippocampal mineralocorticoid receptors responsible for hypothalamic feedback in basal conditions (28, 29). Thus, one would expect a higher effectiveness of the prenatal treatment at the pituitary than at the hypothalamic level. Also, dexamethasone has poor access to the brain; therefore, depending on the efficiency of the fetal blood-brain-barrier, the brain may be exposed to lower concentrations of the steroid than the pituitary (30). This suggest that factors other than CRH and VP, such as cytokines or other compounds capable of directly stimulating the pituitary, may be activated in 21-hydroxylase deficiency (31, 32, 33).

The high POMC levels in 21-hydroxylase-deficient mice suggest that excessive production of ACTH is responsible for hyperstimulation of the adrenal. A number of studies in different species have shown that the fetal adrenal is responsive to ACTH (34, 35, 36, 37). However, it is well established now that factors other than pituitary ACTH contribute to adrenal activation independently, or by interacting with ACTH (38). Peptides with adrenocorticotrophic activity, such as ACTH itself and CRH, are produced in the placenta and other nonpituitary sites, where in contrast to the PVN and pituitary, their expression is stimulated rather than inhibited by glucocorticoids (39, 40, 41). Therefore, it is possible that exogenous glucocorticoids, especially dexamethasone, which has little affinity for glucocorticoid binding globulin (42) or 11–8-dehydrogenase (43), will stimulate the production of these or other adrenocorticotrophic peptides.

A novel finding was the marked alterations in adrenal structure in 21-hydroxylase deficient mice. In contrast to the normal adrenal zonation observed in wild-type mice, adrenals of 21-hydroxylase deficient mice lacked a differentiated zona glomerulosa, and showed striking changes in mitochondrial structure. In vitro and in vivo experiments in the rat have shown that exposure of the adrenal to high concentrations of ACTH induces differentiation of glomerulosa to fasciculata type cells (44), suggesting that high ACTH levels are responsible for the lack of a normal zona glomerulosa. Mitochondrial changes including enlargement and poorly developed internal membranes, as seen in 21-hydroxylase deficient mice, have been described in humans with CAH (45), and are in contrast to the increase in mitochondrial vesicular membranes occurring following ACTH or CRH administration (46, 47). This suggests that the ultrastructural changes are related to lack of CYP21 or accumulation of abnormal steroids due to the blockade in steroidogenesis rather than ACTH excess. Allmann et al. (48) have reported changes in mitochondrial structure associated with uncoupling of steroidogenesis, but the mechanism of this relationship is unknown. Glucocorticoids play an important role in the expression of enzymes involved in catecholamine synthesis and differentiation of chromaffin cells (49, 50). Therefore, the lack of glucocorticoids in 21-hydroxylase deficient mice could account for the incomplete chromaffin cell migration and formation of the adrenal medulla observed in these mice.

The consequences of glucocorticoid deficiency and hyperstimulation of the HPA axis during fetal life is currently under investigation in our laboratory. It is well recognized that challenges during fetal life or early postnatal development can have long-term effects on the HPA axis (51, 52, 53, 54). For example, immune challenge or ethanol administration during pregnancy, or maternal separation of pups during the first 2 weeks of life in rats have been reported to induce hyperactivity of the HPA axis in adulthood. The mechanism of these alterations appear to involve decreases in central glucocorticoid receptors with decreased sensitivity to glucocorticoid feedback (54). Similar mechanisms could operate in CAH and contribute to the difficulties in adjusting glucocorticoid replacement doses in patients affected with the disorder (4, 5, 6, 7, 8).

In conclusion, the data suggest that intrauterine glucocorticoid deficiency in 21-hydroxylase-deficient mice causes hyperactivity of the hypothalamic-pituitary corticotroph axis with insensitivity to glucocorticoid feedback. The dissociation between hypothalamic and pituitary inhibition following prenatal dexamethasone treatment suggests that ACTH production in this disorder is not solely dependent on hypothalamic hyperactivity. In addition, the adrenal abnormalities in this animal model cannot be explained by overproduction of ACTH. These experiments in 21-hydroxylase-deficient mice indicate that the current approach of glucocorticoid administration for prenatal treatment in humans may not be sufficient to suppress the fetal HPA axis and may provide a useful model to understand the clinical manifestations and management of CAH.


   Acknowledgments
 
The authors would like to thank Dr. T. Shiroishi (Department of Cell Genetics, Institute of Genetics, Mishima, Japan) for the mutant mice, Dr. A. Seasholtz (University of Michigan, Ann Arbor, MI) for the mouse CRH probe, and Dr. Susan Wray and Harold Gainer (NIMH, NIH) for the VP probe and antibody.


   Footnotes
 
1 Present address: Department of Pediatrics, School of Medicine, Hokkaido University, Saporo Kita-ku N15 W7, Japan. Back

2 Supported by a Heisenberg grant of the Deutsche Forschungs Gemeinschaft. Back

Received October 2, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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