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Original Studies |
Departments of Obstetrics/Gynecology/Reproductive Sciences and Physiology, Center for Studies in Reproduction, University of Maryland School of Medicine (G.W.A., E.D.A.), Baltimore, Maryland 21201; and the Department of Physiology, Eastern Virginia Medical School (M.G.L., G.J.P.), Norfolk, Virginia 23507
Address all correspondence and requests for reprints to: Eugene D. Albrecht, Ph.D., Department of Obstetrics, Gynecology, and Reproductive Sciences, University of Maryland School of Medicine, Bressler Research Laboratories 11–019, 655 West Baltimore Street, Baltimore, Maryland 21201.
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Abstract |
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5-3ß-hydroxysteroid dehydrogenase expression. The
concomitant administration of ACTH and betamethasone restored these
aspects of adrenal integrity to normal. Moreover, there was
approximately a 95% decrease (P < 0.01) in fetal
adrenal expression of ACTH receptor, P-450 cholesterol side-chain
cleavage, and P-450 17
-hydroxylase 17/20-lyase mRNA levels after
betamethasone administration. We conclude that fetal pituitary ACTH is
necessary for the growth and development of fetal and definitive
cortical zones and the marked coordinated increase in ACTH receptor and
maintenance of P-450 cholesterol side-chain cleavage/P-450
17-hydroxylase 17/20-lyase expression in the baboon fetal adrenal
gland during the first half of gestation. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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5-3ß-hydroxysteroid
dehydrogenase (3ßHSD) enzyme required for cortisol synthesis (see
Refs. 1 and 2 for reviews). The fetal zone makes up the majority of the
adrenal cortex throughout gestation, whereas the definitive zone does
not undergo extensive maturation until late in gestation. Studies
conducted in vitro with human fetal adrenal cells (3, 4, 5) and
in vivo in the rhesus monkey (6, 7, 8, 9) indicate that ACTH has a
major role in regulating cellular proliferation, maturation, and
steroidogenesis within the fetal adrenal during the second half of
gestation. Although the human fetal adrenal develops normally in
anencephalic fetuses through the first trimester, but not thereafter
(10), the importance of fetal pituitary ACTH on adrenal development and
function in the first half of gestation has not been established
in vivo in the primate. ACTH is present at this time,
because human (11) and baboon (12) fetal pituitaries express and/or
secrete POMC and ACTH by midgestation. Moreover, maximal fetal adrenal
ACTH receptor messenger ribonucleic acid (mRNA) levels were exhibited
in the baboon at midgestation (13), indicating that a mechanism for
mediating the action of ACTH exists within the adrenal at this stage of
development. Although the ACTH receptor is up-regulated by ACTH in
cultures of human fetal adrenal cells (14, 15), the role of ACTH in
receptor expression has not been established in vivo in the
primate.
Therefore, in the present study betamethasone, a synthetic
glucocorticoid that readily crosses the placenta and suppresses the
fetal pituitary adrenocortical axis (6, 8), was used to investigate the
role of pituitary ACTH in vivo on fetal adrenal development.
Fetal adrenal growth, development of the fetal and definitive zones,
and expression of the ACTH receptor and the ACTH-dependent
steroidogenic enzymes P-450 cholesterol side-chain cleavage (P-450scc),
P-450 17-hydroxylase-17/20-lyase (P-450C17), and 3ßHSD
were determined in baboons treated with betamethasone and/or ACTH in
the first half of gestation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Pregnant baboons (Papio anubis) were maintained as described previously (13). Animals were cared for and used strictly in accordance with USDA regulations and the NIH Guide for the Care and Use of Laboratory Animals (Publication 85-23, 1985). The experimental protocol employed in the present study was approved by the institutional animal care and use committee of the University of Maryland School of Medicine.
Eight baboons were left untreated, and six baboons were treated with 3 mg betamethasone (Celestone Soluspan, Schering Corp., Chicago, IL) administered im to the mothers daily after ketamine HCl (10 mg/kg BW; Aveco Co., Ft. Dodge, IA) sedation between days 60 and 99 of gestation (term = 184 days). To determine whether the effects of betamethasone could be overcome by concomitant treatment with ACTH, the fetuses of four additional betamethasone-treated (3 mg/day; days 60–99) animals and five additional untreated baboons were administered 25 µg ACTH (Cortrosyn, Organon, West Orange, NJ), im, in 100 µL saline on days 95–99 via maternal transabdominal injection after anesthetization with halothane.
Maternal saphenous vein blood samples were obtained daily on days 95–100 of gestation, and on day 100, baboons were anesthetized with halothane, the fetuses were delivered by cesarean section, and an umbilical artery blood sample was obtained. Serum ACTH, estradiol, DHAS, and cortisol concentrations were determined by solid phase 125I RIA (Coat-A-Count, Diagnostic Products Corp., Los Angeles, CA) as described previously (12). One of the fetal adrenal glands was immediately frozen in liquid nitrogen for Northern analysis of mRNAs. The other gland was fixed in 10% buffered formalin and embedded in paraffin for immunocytochemical analysis of 3ßHSD, quantification of fetal cortical cells, and histological determination of cellular integrity, including apoptosis. Fetal pituitaries were placed in cryomolds and stored at -80 C until analyzed for POMC mRNA.
Pituitary POMC mRNA
Localization and quantification of POMC mRNA were performed by
in situ hybridization using our published methods (12).
Briefly, 0.1 µmol purified POMC antisense (and sense)
oligodeoxynucleotide probes complimentary to 30 bases of the human POMC
mRNA (16) was 3'-end labeled with [35S]deoxy-ATP
(DuPont-New England Nuclear, Boston, MA). Fetal pituitary sections were
incubated with approximately 750,000 cpm 35S-labeled
antisense or sense probe, washed at 60 C (
19 C below calculated Tm),
and placed against Kodak X-Omat film (Eastman Kodak, Rochester, NY) for
5–7 days. POMC mRNA expression was determined by densitometric
analysis using an LKB Bromma Ultroscan XL Enhanced Laser Densitometer
(Pharmacia LKB, Piscataway, NY).
Adrenal morphology and immunocytochemistry
Sections (4 µm) of paraffin-embedded fetal adrenal glands were heat fixed and incubated overnight with polyclonal antibody to rabbit antihuman 3ßHSD (supplied by Dr. Ian Mason, University of Edinburgh, Edinburgh, UK). Sections were incubated with biotinylated goat antirabbit IgG (Boehringer Mannheim, Indianapolis, IN), avidin DH, and horseradish peroxidase H (Vectastain Elite Kit, Vector Laboratories, Burlingame, CA) and counterstained with hematoxylin. 3ßHSD expression was analyzed on six randomly selected areas (157 x 130 µm/slide of four to eight fetal adrenal sections per animal) using an Optiphot-2 microscope attached to a Video-Based Image 1 Analysis System (Universal Imaging Corp., West Chester, PA). The number of fetal zone cells per 0.025 mm2 was quantified by counting nuclei in six randomly selected sections. The width of the definitive cell layer was quantified by examining 3ßHSD-positive cells in six randomly selected regions of each adrenal section.
Evaluation of adrenal cells for apoptosis was performed using an Apoptag Plus in situ apoptosis detection kit (Oncor, Gaithersburg, MD) and procedures for use with paraffin-embedded tissues supplied by the manufacturer.
Northern analysis of adrenal mRNA
Fetal adrenal mRNA levels were determined by Northern blot,
using methods implemented in our laboratories (17). Approximately 2.5
µg polyadenylated RNA were size-fractioned by agarose gel
electrophoresis, transferred onto nylon membrane (GeneScreen,
DuPont-New England Nuclear), and prehybridized in 50% formamide
buffer for 18 h at 42 C. The complementary DNAs (cDNAs) for the
baboon ACTH receptor (13) and human ß-actin (no. 65128, American Type
Culture Collection, Rockville, MD), P-450scc, and P-450C17
(provided by Dr. Walter Miller, University of California-San Francisco)
were labeled with [-32P]deoxy-CTP (Amersham Corp.,
Arlington Heights, IL). Hybridization was performed in fresh buffer at
42 C for 23 h with approximately 106 cpm/mL
32P-labeled cDNA. After hybridization, the membrane was
washed, then exposed to Kodak X-AR film (Eastman Kodak) at -80 C. The
intensities of the bands were analyzed by densitometric
autoradiography, using a model 620 video densitometer (Bio-Rad,
Hercules, CA).
Statistical analysis
Data were analyzed by ANOVA, with post-hoc comparisons by Newman-Keuls multiple comparison test. Comparison of untreated and betamethasone-treated fetuses was performed using Student’s unpaired t test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Fetal pituitary POMC mRNA levels were decreased (P
< 0.01) by 54% after betamethasone treatment (Fig. 1A
). However, umbilical artery serum
(comparable to fetal peripheral serum) ACTH concentrations were
decreased (P < 0.05) to undetectable values after
betamethasone administration (Fig. 1B).
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).
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Fetal adrenal absolute and relative (to body) weights were
decreased 50–60% (P < 0.01) by betamethasone (Fig. 2 and Table 2). This was associated with a 2-fold
increase (P < 0.05) in the number of adrenal cortical
cells per unit area (Table 2), indicating that cell size was decreased.
Although there was no evidence of apoptosis in fetal adrenals of the
untreated controls (Fig. 3A), DNA
oligonucleosomes indicative of programmed cell death were extensive
after betamethasone administration (Fig. 3B).
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). Betamethasone virtually eliminated
the definitive zone (Fig. 5) with its immunocytochemical expression of
definitive zone-specific 3ßHSD (Fig. 4B), and the fetal zone
exhibited cellular disorganization (Fig. 4B).
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and Table 2).
Moreover, ACTH consistently reversed apoptosis induced by long term
administration of betamethasone (Fig. 3C), uniformly restored the
cellular integrity of and 3ßHSD expression within the adrenal cortex
(Fig. 4C), and returned definitive zone width to normal (Fig. 5). ACTH alone increased
(P < 0.01) fetal adrenal weight by 43% (Fig. 2) and
enhanced (P < 0.01) definitive zone width by
approximately 2-fold (Fig. 5). ACTH receptor, P-450scc and P-450C17 mRNAs
The baboon ACTH receptor cDNA hybridized with a major 3.4-kilobase
(kb) and two lesser 4.0- and 1.8-kb mRNA transcripts in the fetal
adrenal gland. Although the relative changes in each of the transcripts
appeared similar with treatment, only the primary 3.4-kb transcript
(Fig. 6A) was used for statistical
analysis (Fig. 6C). ACTH receptor mRNA levels, not corrected for
ß-actin, in the fetal adrenal were decreased by approximately 95%
(P < 0.001) in all four animals by betamethasone
administration (Fig. 6C). This was accompanied by a loss
(P < 0.001) of ß-actin mRNA expression (Fig. 6B).
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). However, ACTH
administration alone did not alter ACTH receptor or ß-actin mRNA
expression.
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). The administration of ACTH to
betamethasone-suppressed animals restored P-450scc and
P-450C17 mRNA levels in two of the four fetuses, whereas
ACTH alone had no effect.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although the definitive zone of the fetal adrenal cortex only begins to emerge and express 3ßHSD at midgestation in the baboon (19) and human (20, 21), the administration of betamethasone to baboons of the present study virtually eliminated this cellular zone, as demonstrated by the loss of immunocytochemically demonstrable 3ßHSD enzyme, and this effect was also reversed by ACTH. Consequently, it seems that fetal pituitary ACTH is also responsible for the initial development of the definitive cortical zone and the onset in expression of enzymes critical to steroidogenesis.
The decline in fetal adrenal ACTH receptor mRNA expression in baboons caused by betamethasone administration is consistent with a role for pituitary ACTH in generating the marked 13-fold rise in receptor expression that occurs between early and midgestation (13). The present in vivo observations are also consistent with recent in vitro studies, which showed an ACTH-induced increase in ACTH receptor mRNA levels (14, 15) and ACTH binding (22) in cultures of human fetal adrenal cells. The corresponding decline in ACTH-depleted baboons in mRNA expression for P-450scc and P-450C17, enzymes expressed in the fetal zone (21) and stimulated in vitro by ACTH (15, 21, 23, 24), supports the concept of a role for the receptor-mediated action of ACTH in vivo in regulating those enzymes critical to fetal adrenal C19 steroid biosynthesis and, thus, estrogen production at mid-gestation. Indeed, acute administration of ACTH to baboon fetuses at midgestation enhanced fetal adrenal DHA secretion (25). Prior observations of a decrease in fetal adrenal P-450scc and P-450C17 mRNA levels in rhesus monkeys treated with betamethasone (9) and a decrease and subsequent increase in fetal plasma DHAS, estradiol, and cortisol in acutely dexamethasone suppressed/ACTH-stimulated rhesus monkey fetuses in the second half of pregnancy (8) are also consistent with the findings of the present study. Moreover, Mesiano et al. (15) have recently shown that the ACTH receptor and P-450scc are coordinately expressed in human fetal adrenal cells in culture, and they have suggested that the ACTH receptor belongs to a cohort of ACTH-responsive genes required to maintain fetal adrenal differentiation and responsiveness to ACTH. The present study demonstrates that a similar ACTH-dependent coordinated regulation exists in vivo within the baboon fetal adrenal at midgestation.
The mRNA levels for the ACTH receptor were not corrected for those of ß-actin in the fetal adrenals of the present study, because ß-actin, which is typically used as a constitutively expressed gene marker, was also suppressed by betamethasone. It is suggested that the decrease in ß-actin further points to the absolute requirement of ACTH for the structural and functional integrity of the primate fetal adrenal gland at midgestation.
Although fetal adrenal weight/growth, cellular integrity, and 3ßHSD expression were uniformly restored to normal by the administration of ACTH to betamethasone-suppressed baboons, the mRNA levels for the ACTH receptor, P-450scc, and P-450C17 were only restored in half of the fetuses injected with the particular dose of ACTH used in this preliminary study. There is no obvious explanation for this inconsistent response in ACTH receptor mRNA expression to ACTH. It is possible that the level of ACTH required to consistently induce the ACTH receptor and P-450 genes is greater than that needed to maintain growth and differentiation of the fetal adrenal. Because glucocorticoid-type steroids directly suppress ACTH-induced steroidogenesis in isolated rat adrenal cells (26), it is possible that glucocorticoids have a direct inhibitory effect on components of the ACTH receptor signal transduction pathway that is not easily overcome by exogenous ACTH. It is also possible that the turnover of the mRNAs for the receptor and steroidogenic enzymes is rapid, and thus, their inconsistent restoration reflected their short half-life and the single daily injection of ACTH administered to animals of the present study. Clearly, further study is needed to sort out these possibilities and to more precisely define the absolute levels of ACTH as well as ACTH receptor required in vivo to fully achieve normal fetal adrenal function at midgestation.
In summary, suppression of fetal pituitary POMC expression and serum ACTH levels in baboons by betamethasone administration at midgestation resulted in apoptosis, cellular disorganization, loss of 3ßHSD expression, and a decline in ACTH receptor and P-450 steroidogenic enzyme mRNA levels in the fetal adrenal gland. It is concluded that ACTH is necessary for the growth and development of the fetal and definitive zones of as well as the coordinated increase in the expression of the ACTH receptor and maintenance of the P-450 enzyme components critical to steroidogenesis within the primate fetal adrenal cortex at midgestation.
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Footnotes |
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Received June 13, 1997.
Revised November 7, 1997.
Accepted November 21, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-melanocyte-stimulating
hormone on fetal and maternal plasma steroids. Endocrinology. 104:1805–1813.[Medline]
-hydroxylase/17,20-lyase, and 3ß-hydroxysteroid dehydrogenase
isomerase steroidogenic enzymes in human and rhesus monkey fetal
adrenal glands: reappraisal of functional zonation. J Clin
Endocrinol Metab. 77:1184–1189.[Abstract]
-hydroxylase/17,20-lyase) in cultured human fetal adrenal cells. J Clin Endocrinol Metab. 65:170–175.[Abstract]
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