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Departments of Obstetrics/Gynecology/Reproductive Sciences and Physiology, Center for Studies in Reproduction, University of Maryland School of Medicine (G.W.A., J.S.B., E.D.A.), Baltimore, Maryland 21201; Department of Physiology, Eastern Virginia Medical School (W.A.D., G.J.P.), Norfolk, Virginia 23501
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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ACTH receptor messenger RNA (mRNA) levels, determined by in
situ hybridization, in the fetal zone of the baboon fetal
adrenal were approximately 2-fold greater (P <
0.05) at mid (i.e. day 100) than at late
(i.e. day 170) gestation and 3-fold greater
(P < 0.01) in the definitive zone than in the
fetal zone in late gestation (term = 184 days). Both ACTH receptor
and low density lipoprotein receptor mRNA levels, determined by
Northern blot in the whole fetal adrenal, also decreased
(P < 0.001) by approximately 50%, whereas the
mRNA levels for the definitive zone-specific
5-3ß-hydroxysteroid dehydrogenase/isomerase (3ß-HSD)
enzyme required for cortisol biosynthesis increased over 13-fold
(P < 0.001) between mid and late gestation. In
contrast, mRNA expression of the steroidogenic enzymes P-450
cholesterol side-chain cleavage and 17
-hydroxylase/17–20 lyase were
unchanged throughout gestation. We conclude that the decrease in ACTH
receptor mRNA expression and ACTH-stimulable dehydroepiandrosterone
formation in the second half of gestation reflect a decline in
functional capacity of the fetal zone, whereas the increase in 3ß-HSD
mRNA expression and cortisol production results from the ACTH
receptor-mediated development and enhanced functional capacity of the
definitive zone.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-hydroxylase/17–20 lyase (P-450170H; see Ref. 1 and 2
for reviews). In contrast, the definitive zone develops relatively late
in pregnancy and is the sole site of the
5-3ß-hydroxysteroid dehydrogenase/isomerase (3ß-HSD)
enzyme (3, 4) that catalyzes cortisol production (5). It seems that
expression of 3ß-HSD (6, 7) and receptor-mediated uptake of low
density lipoprotein (LDL) cholesterol substrate for steroidogenesis
(8, 9, 10, 11) in the fetal adrenal is critically dependent upon ACTH, whereas
an exclusive role for ACTH on P-450scc and
P-450170H is equivocal (7, 12, 13). Although ACTH-dependent zone-specific steroidogenesis has been established in the fetal adrenal, relatively little is known about the developmental regulation of this process. Because of the unique functional interaction between the fetal adrenal and placenta for estrogen production and maturation of the hypothalamic-pituitary-adrenocortical axis of the fetus during primate (5), but not rodent pregnancy, it is important to elucidate the developmental regulation of this important endocrine gland in the primate. We have recently shown that ACTH receptor messenger RNA (mRNA) levels determined by Northern analysis in the baboon fetal adrenal increased from a minimum in early gestation to a maximum at midgestation, then declined late in pregnancy (14). Because the fetal adrenal cortex is made up primarily of the fetal zone throughout gestation, the increase in ACTH receptor expression at midgestation presumably occurred in this cortical zone, possibly in response to ACTH, which has been shown in vitro to stimulate ACTH receptor formation in vitro (15, 16, 17). In the second half of baboon pregnancy, there was a marked increase in definitive zone-specific 3ß-HSD expression, de novo cortisol production (14, 18, 19), and responsivity of fetal adrenal cells to produce cortisol in response to ACTH (20). However, coinciding with the decline in ACTH receptor expression, there was a decrease in ACTH-stimulable DHA formation by baboon fetal adrenocortical cells (20, 21). Therefore, it appears that the fetal adrenal cortical zones develop a divergence in functional capacity in the second half of pregnancy. We propose that the decrease in fetal adrenal ACTH receptor mRNA expression and DHA formation between mid and late gestation occurs in the fetal zone and signals a selective decline in tropic responsivity of this zone to ACTH. To examine this possibility, in the present study the mRNA levels for the ACTH receptor were determined by quantitative in situ hybridization in the baboon fetal adrenal gland at mid and late gestation and compared with the developmental expression of specific components of the steroidogenic pathway.
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Materials and Methods |
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Fetuses were removed from pregnant baboons via caesarean section under halothane (1.0–1.5%):nitrous oxide (0.5 liters/min):oxygen (2.5 liters/min) anesthesia on days 58–65 (early), 98–104 (mid), and 164–170 (late) of gestation (term = 184 days). Fetal adrenals were immediately collected under sterile conditions, weighed, a portion placed in 10% formalin/PBS fixative, and the remainder flash frozen and stored in liquid nitrogen.
ACTH receptor in situ hybridization
In situ hybridization histochemistry was used to
determine localization and to compare zone specific levels of ACTH
receptor mRNA in fetal adrenals of mid and late gestation. Using
standard methods to prevent RNAase contamination, 4 µm sections of
paraffin-embedded fetal adrenals were equilibrated (10 min) to room
temperature, rinsed in 2 x SSC (1 x SSC = 0.15
M NaCl, 0.015 M sodium citrate buffer, pH 7.2),
and placed (10 min) in freshly prepared 0.25% acetic anhydride in 0.1
M triethanoloamine hydrochloride-0.9% NaCl. After
dehydration through an ethanol series, delipidation in chloroform (5
min), and rehydration in ethanol (100%, 1 min; 95%, 1 min), sections
were prehybridized for 3 h (37 C) in 50 µl buffer consisting of
4 x SSC, 50% (vol/vol) formamide, 10% (wt/vol) dextran sulfate,
250 µg/ml yeast transfer RNA (Life Technologies, Gaithersburg, MD),
500 µg/ml sheared single-stranded salmon sperm DNA (Life
Technologies), 1 x Denhardt’s solution (Sigma, St. Louis, MO),
1% Na2S2O3, 5 mM NaPPi
and 50 mM freshly prepared dithiothreitol (Sigma). Slides
were rinsed in 2 x SSC, and incubated overnight (45 C) with
either oligodeoxynucleotide antisense or sense probes complementary to
bases 373–401 of the baboon ACTH receptor (14), that were end-labeled
with [35-S] dATP (DuPont-NEN, Boston, MA) and terminal
deoxynucleotidyl transferase (20 U; Promega, Madison, WI) to a specific
activity of 5000 Ci/mmol (22). Sections were rinsed with 2 x SSC
and washed three times (2 x SSC, 50% formamide, 30 min each
time) at 55 C and once in 1 x SSC (40 C), 30 min. After the last
wash, slides were rinsed with 2 x SSC (30 min; 22 C), dipped in
double-distilled water twice, then in ethanol (70% and 95%) and
air-dried. Slides were dipped in fresh Kodak NTB-2 nuclear track
emulsion (Eastman Kodak, Rochester, NY) diluted 1:1 with distilled
water, placed in light tight boxes and exposed for 7–14 days. Slides
were developed in Kodak D-19 (4 min; 15 C), rinsed in distilled water,
fixed (Kodak Fixer; 5 min, 15 C), rinsed and lightly counterstained
with hematoxylin.
Specific localization of ACTH receptor mRNA expression in the definitive and fetal zones of the baboon fetal adrenal cortex was confirmed by light microscopy and dark-field optics. Quantification of fetal adrenal ACTH receptor mRNA expression was performed by counting the specific number of silver grains per 0.025 mm2 using an Optiphot 2 microscope attached to a Video-Based Image-1 Analysis System (Universal Imaging Corp., West Chester, PA). Spatial calibration of the imaging system using a 40x objective was 512 pixels along the horizontal axis and 480 pixels along the vertical axis. For each animal, an average of ten different areas (140 µm x 180 µm) of the fetal zone and definitive zone were examined. After establishing a grey level, cells with a grain density 10-fold greater than background were considered ACTH receptor positive. All grain counts (number/0.025 mm2) were averaged and a single value for each animal determined and used to calculate overall group mean values.
Isolation of RNA
Fetal adrenal RNA was extracted according to the modified method
originally described by Chirgwin et al. (23). In brief,
tissues were homogenized in 4 M guanidine isothiocyanate,
the homogenate extracted with chloroform:isoamyl alcohol, and RNA
isolated by 5.7 M cesium chloride gradient centrifugation
(174,000 x g for 21 h at 20 C). RNA pellets were
solubilized and resuspended in 0.3 M sodium acetate, and
precipitated in ethanol and the integrity of the RNA confirmed by
electrophoresis. Polyadenylated [poly(A)+]-enriched RNA
was prepared by centrifugation of total RNA over preequilibrated
columns of oligo (deoxythymidine) cellulose according to the
manufacturer’s instructions (Pharmacia Biotech, Inc., Piscataway,
NJ).
Northern analysis
Poly(A+)-enriched RNA (10 µg) was denatured in
50% formamide, 2.2 M formaldehyde, and 20 mM
3-[N-morpholino] propane sulfonic acid (MOPS), pH 7.0, and
size-fractioned by electrophoresis in 1.0% agarose gels containing
0.66 M formaldehyde and 20 mM MOPS, pH 7.0. RNA
was transferred overnight by capillary action in 10 x SSC buffer
(pH 7.0) onto nylon membranes (Gene Screen, DuPont-NEN), which were UV
cross-linked and baked in a vacuum oven at 80 C for 2 h. Membranes
were prehybridized in buffer containing 50% formamide, 0.1%
polyvinylpyrrolidone, 0.1% BSA, 0.1% Ficoll, 2.5 x SSPE (0.375
M NaCl, 0.025 M sodium phosphate, 2.5
mM EDTA-Na2, pH 7.4), 1.0% sodium dodecyl
sulfate (SDS), 10% dextran sulfate, and 0.1 mg/ml denatured salmon
sperm DNA for 18–24 h at 42 C before addition of labeled complementary
DNA (cDNA) probe.
The cDNA for the baboon ACTH receptor was cloned by us and described
previously (14). Bam H1 0.4-kb restriction fragments of the
previously described (24) human LDL receptor cDNA (pLDLR3, No. 57004),
and human ß-actin cDNA (No. 65128) were obtained from American Type
Culture Collection (ATCC, Rockville, MD). The human
EcoRI/SstI 1.1-kb 3ß-HSD cDNA (25) was
generously provided by Dr. J. Ian Mason of the University of Edinburgh,
Edinburgh, Scotland. The human EcoRI-PST 
h SCC-36 0.8-kb
P-450scc (26) and human EcoRI hac17–21.7-kb
P-450170H (27) cDNAs were generously provided by Dr. Walter
Miller of the University of California, San Francisco, CA.
cDNAs were labeled with [-32P] deoxy(d)-cytidine
triphosphate (3,000 Ci/mmol, Amersham Corp., Arlington Heights, IL) to
a specific activity of approximately 109 dpm/µg DNA using
the High Prime DNA labeling kit (Boehringer Mannheim, Indianapolis,
IN). After hybridization, which proceeded for 18–24 h at 42 C with
approximately 106 cpm/ml [32P] labeled cDNA,
the membrane was washed twice at room temperature for 5 min in 2
x SSC (0.3 M NaCl, 0.03 M sodium citrate, pH
7.0), once at 65 C for 30 min in 2 x SSC/1% SDS, and twice in
0.1 x SSC at 22 C for 10 min. Film exposure (Kodak X-OMAT Film,
Eastman Kodak) for autoradiography was for various lengths of time at
-80 C in the presence of intensifying screens. Between hybridizations
with different probes, membranes were stripped by washing in 10
mM Tris, pH 8.0, 1 mM Na2EDTA, pH
8.0, and 1% SDS, at 100 C for 30 min. Autoradiograms were analyzed
using a Bio-Rad Model 620 video densitometer (Bio-Rad, Richmond, CA).
Because ß-actin mRNA expression was constant during the course of
gestation, densitometric readings (relative arbitrary units) for
components of the steroidogenic pathway were corrected for ß-actin to
determine specific effects of gestational age on these parameters.
3ß-HSD immunocytochemistry
Sections (4 µm) of paraffin embedded fetal
adrenal glands were mounted onto Superfrost microscope slides (Fisher
Scientific Co., Arlington, VA), heat fixed and endogenous peroxidase
blocked with 0.4% H2O2 in methanol. After
incubation (4 C) overnight with polyclonal antibody to rabbit antihuman
3ß-HSD (generously supplied by Dr. Ian Mason, University of
Edinburgh, UK), tissue sections were washed, incubated with
biotinylated goat antirabbit IgG (Boehringer Mannheim, Indianapolis,
IN), Avidin DH and horseradish peroxidase H (Vectastain Elite Kit,
Vector Laboratories, Burlingame, CA), and lightly counterstained with
Gill’s hematoxylin (Fisher).
Statistical analysis
The mRNA levels were analyzed by Northern blot in two different
sets of tissues obtained in early, mid, and late gestation and the data
combined. Data were analyzed using one-way ANOVA and Newman-Keuls
multiple comparison test ( = 0.05) to identify significant
differences between individual group means (Instat, Graphpad Software,
San Diego, CA).
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Results |
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depicts representative photomicrographic
sections of mid (A–C) and late (D–F) gestation fetal adrenal glands
hybridized with 35S-labeled ACTH receptor antisense (A, B,
D, and E) or sense (C and F) probes. Specificity of ACTH receptor
labeling was documented by the relative absence of silver grains over
cells of the fetal (C) and definitive (F) zones after incubation with
the sense probes, the low number of grains associated with the medulla
of an adult adrenal hybridized with antisense probe (data not shown),
and the selectively greater distribution of silver grains over cells of
the definitive (D) vs. the fetal (E) zone in fetal adrenals
of late gestation.
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) than in cells of the central core of the
fetal zone (Fig. 1B). The mean (±SE) number of silver
grains/0.025 mm2 in the fetal zone at midgestation
(200 ± 21, Figs. 1, A and B, and 2) was approximately 2-fold
greater (P < 0.05) than in late gestation (115 ±
24, Figs. 1E and 2). In adrenals of late gestation, the number of
silver grains associated with cells of the emerging definitive zone
(309 ± 18, Figs. 1D and 2), which comprises
approximately 20% of the fetal adrenal gland at this time in
development, exceeded (P < 0.01) by approximately
3-fold the number in the fetal zone (Fig. 1E). This difference in ACTH
receptor mRNA expression between the definitive and fetal zones near
term is also illustrated in Fig. 3 by dark-field low
magnification microscopy.
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). ACTH receptor
mRNA levels then declined (P < 0.001) by 62% in late
gestation (0.54 ± 0.10), a pattern of expression consistent with
our previously reported observations (14).
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). LDL receptor
mRNA expression was extremely low in the fetal adrenal early in
gestation (0.03 ± 0.01, Fig. 5, A and C). However, LDL receptor
mRNA levels, expressed as a ratio of ß-actin (Fig. 5B), increased
(P < 0.001) approximately 30-fold to a maximum at
midgestation (0.94 ± 0.04), then declined (P <
0.001) by 50% (0.47 ± 0.11) in the second half of gestation.
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). The
3ß-HSD protein, determined by immunocytochemistry, was localized
specifically in the definitive zone that increased from a very thin rim
of cells at midgestation (Fig. 7A) to a wide band of
positive cells in late gestation (Fig. 7B).
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). In contrast to
the developmental changes in the steroidogenic components described
above, however, P-450scc mRNA levels were similar in early
(0.79 ± 0.04), mid (0.94 ± 0.02), and late (0.83 ±
0.07) gestation (Fig. 8B). As observed for P-450scc,
expression of the 2.0-kb P-450170H transcript (Fig. 9A) was relatively constant in early (1.69 ±
0.30), mid (2.07 ± 0.16), and late (1.80 ± 0.13) gestation
(Fig. 9B).
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). In contrast, there was a progressive decrease
(P < 0.001) in the ratio of fetal adrenal:body weight
with advancing gestation.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Jaffe and co-workers (17) have recently shown that ACTH receptor mRNA expression was greater in the 3ß-HSD-positive definitive/transitional zones than in more central areas of the fetal zone of the human fetal adrenal at midgestation. These authors suggested that the cells in the outermost zones are more sensitive to ACTH than cells in the center of the cortex. The results of the present study in fetal baboons near term are consistent with the latter observations. However, the current study further shows that the fetal zone cells undergo a decline in ACTH receptor expression between mid and late gestation.
We propose that the divergent pattern of DHA and cortisol production within the fetal adrenal gland in the latter part of gestation may be the result of an inhibitory effect of the increasing levels of estrogen specifically on the fetal zone. Thus, we have shown that estrogen suppressed the ability of the baboon fetal adrenal to respond to ACTH with respect to the production of DHA, but not cortisol, as assessed in vitro (34, 35) and in vivo (36). Consequently, we have proposed that a negative feedback system may be operable within the fetal-placental unit, in which placental estrogen feeds back on the fetal adrenal to down-regulate the formation of C19-steroid precursors, resulting in a physiologically normal balance of estrogen production during primate pregnancy. The regulatory effect of estrogen may be upon the ACTH receptor and/or ACTH-dependent components of the C19-steroid biosynthetic pathway.
The present study also shows that there was a biphasic developmental change in expression of the LDL receptor in the baboon fetal adrenal gland, in which mRNA levels increased to a maximum by midgestation, then declined in the second half of pregnancy. Because the fetal adrenal is comprised almost entirely of fetal zone cells at midgestation, the increase in LDL receptor mRNA levels presumably occurred within this cortical zone, possibly as a result of a stimulatory effect of ACTH at this time in development. The increase in LDL receptor expression, coupled with a high level of P-450scc and P-450170H expression throughout pregnancy, may provide the necessary components for the uptake of cholesterol substrate and its utilization for C19-steroid synthesis at this time in pregnancy. The protein levels for these components of the steroid biosynthetic pathway must be quantified to definitively confirm this possibility. Additional study is also needed to determine if the decline in LDL receptor expression with advancing pregnancy occurs within the fetal zone. However, zone-specific changes in expression of the LDL cholesterol pathway, as well as the ACTH receptor, could also contribute to the divergent pattern of steroidogenesis observed between the adrenocortical zones during development of the fetal adrenal gland.
In summary, the present study shows that ACTH receptor mRNA levels decreased in the fetal zone and became elevated in the definitive zone of the baboon fetal adrenal cortex in the second half of pregnancy. The fetal adrenal exhibited a marked increase in definitive zone-specific 3ß-HSD mRNA expression late in gestation. We conclude that the decrease in ACTH receptor mRNA expression and previously reported ACTH stimulable DHA formation in the second half of gestation reflect a decline in functional capacity of the fetal zone, whereas the increase in 3ß-HSD mRNA expression and de novo cortisol formation result from the development and functional capacity of the definitive zone. Consequently, we propose that there is an uncoupling of ACTH receptor-mediated adrenocortical zone-specific steroidogenesis within the primate fetal adrenal gland during the second half of intrauterine development.
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Footnotes |
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Received November 13, 1996.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-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]
5-3ß-hydroxysteroid dehydrogenase and
17-hydroxylase-17,20-lyase activity. Endocrinology 128:2395–2401[Abstract]
-hydroxylase/17, 20 lyase): cloning of human adrenal and
testis cDNAs indicates the same gene is expressed in both tissues. Proc
Natl Acad Sci USA 84:407–411This article has been cited by other articles:
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