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Endocrinology Vol. 138, No. 7 2705-2712
Copyright © 1997 by The Endocrine Society

ARTICLES

Inhibition of Fetal Adrenal Adrenocorticotropin Receptor Messenger Ribonucleic Acid Expression by Betamethasone Administration to the Baboon Fetus in Late Gestation1

Maria G. Leavitt, Graham W. Aberdeen, Marcia G. Burch, Eugene D. Albrecht and Gerald J. Pepe

Department of Physiology, Eastern Virginia Medical School, Norfolk, Virginia 23501 (M.G.L., M.G.B., G.J.P.); Departments of Obstetrics/Gynecology/ Reproductive Studies and Physiology (G.W.A., E.D.A.), Center for Studies in Reproduction, The University of Maryland School of Medicine, Baltimore, Maryland 21201

Address all correspondence and requests for reprints to: Gerald J. Pepe, Ph.D., Department of Physiology, Eastern Virginia Medical School, P.O. Box 1980, Norfolk, Virginia 23501-1980. E-mail: gjp{at}borg.evms.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Throughout the majority of intrauterine development, the primate fetal adrenal gland is comprised primarily of fetal zone cells and only late in gestation do definitive zone cells, which express the enzyme {Delta}5-3ß-hydroxysteroid dehydrogenase/isomerase (3ß-HSD) emerge to produce cortisol. The present study was designed to determine whether the induction of definitive zone ACTH receptor messenger RNA (mRNA) levels and components of the steroidogenic pathway known to be expressed specifically in the definitive zone, e.g. the 3ß-HSD enzyme, are dependent upon fetal pituitary ACTH. Fetal pituitaries and adrenal glands were obtained on day 165 (term = day 184) from untreated controls (n = 7) and from baboons in which betamethasone was administered im to the fetus (0.6 mg/100 µl; n = 4) or to the fetus (0.6 mg) and mother (6 mg/ml; n = 4) every other day between days 150 and 164 of gestation. Although fetal pituitary weight was not altered by betamethasone, POMC mRNA levels determined by in situ hybridization were lower (P < 0.05) in betamethasone-treated (0.34 ± 0.07 arbitrary densitometric units) than in untreated controls (0.63 ± 0.04). Associated with this decline in pituitary POMC, levels of the major 3.4-kb mRNA transcript for the ACTH receptor expressed as a ratio of ß-actin were approximately 80% lower (P < 0.05) in fetal adrenals of betamethasone-treated baboons (0.12 ± 0.02) than in untreated controls (0.84 ± 0.05). In situ hybridization indicated that ACTH receptor mRNA expression in the definitive zone exceeded that in the fetal zone and was reduced by betamethasone. Associated with the decrease in ACTH receptor expression, fetal adrenal weight was suppressed (P < 0.05) by 50% and reflected a marked reduction (P < 0.05) in the size of the cells of the definitive and fetal zones. Betamethasone treatment also induced a decrease (P < 0.05) in the width (µm) of the definitive zone (183 ± 14 vs. 128 ± 7; determined by immunohistochemical expression of 3ß-HSD), as well as the levels of the mRNA and protein for 3ß-HSD. Levels of the mRNA for the LDL-receptor and the enzymes 17{alpha}-hydroxylase-C17,20 lyase and P450 cholesterol side chain cleavage were also suppressed in adrenals of betamethasone-treated baboons. These findings indicate that treatment of the baboon fetus with betamethasone in late gestation suppressed fetal pituitary POMC mRNA expression and ACTH receptor mRNA levels in the fetal adrenal gland, as well as the hypertrophy and ACTH receptor mRNA and 3ß-HSD mRNA/protein levels in the cells comprising the newly emerging definitive zone. We conclude that ACTH is necessary for the up-regulation of the mRNAs for the ACTH receptor and steroidogenic enzymes in the definitive zone of the primate fetal adrenal gland in late gestation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
WE HAVE previously demonstrated that messenger ribonucleic acid (mRNA) levels for the ACTH receptor in the baboon fetal adrenal at midgestation (1), a time when the gland is composed almost exclusively of fetal zone cells (2, 3), are greater than respective values in adrenals of late gestation, a time during which there is development of definitive zone cells (4, 5, 6). The increase in ACTH receptor mRNA expression in the fetal adrenal at midgestation would provide a mechanism to mediate ACTH-dependent dehydroepiandrosterone (DHA) production (7, 8). We have proposed, however, that the decrease in ACTH receptor mRNA with advancing gestation reflected a decline in receptor in the fetal zone cells and its appearance in the developing definitive zone (1). In support of this hypothesis, we have recently shown using in situ hybridization (9) that there was a decrease in ACTH receptor mRNA concentrations in the fetal zone between mid and late gestation. Moreover, expression of ACTH receptor mRNA in definitive zone cells late in gestation exceeded respective values in fetal zone cells. Our present studies are aimed at elucidating the mechanisms regulating this biphasic developmental pattern of ACTH receptor mRNA expression in zone-specific cells of the fetal adrenal cortex.

Under in vitro conditions, ACTH up-regulates ACTH binding (10, 11) or ACTH receptor mRNA expression in human and bovine adult (12, 13, 14) and human fetal (15, 16) adrenocortical cells. Recently, we demonstrated that treatment of baboon fetuses at midgestation with ACTH enhanced ACTH receptor mRNA levels in fetal adrenal glands obtained from baboons in which ACTH receptor expression was depleted by betamethasone (17). These findings indicate that ACTH up-regulates its own receptor in fetal zone cells in vivo. However, because ACTH receptor mRNA levels in the fetal zone decline with advancing gestation in the baboon, it is not known whether the concomitant induction of ACTH receptor mRNA in the adrenal definitive zone cells of near term baboon fetuses is regulated by ACTH. Therefore, the present study was designed to determine whether the induction of definitive zone ACTH receptor mRNA levels and components of the steroidogenic pathway known to be expressed specifically in the definitive zone, e.g. the {Delta}5-3ß-hydroxysteroid dehydrogenase-isomerase (3ß-HSD) enzyme, are dependent upon fetal pituitary ACTH.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Female baboons (Papio anubis) weighing 10–15 kg were housed individually in stainless steel cages in air-conditioned quarters and fed Purina monkey chow (Ralston Purina, St. Louis, MO) and fresh fruit and/or carrots daily and water ad libitum. Females were paired with males for 5 days at the anticipated time of ovulation and pregnancy confirmed 30 days post ovulation (18). Baboons 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 this study was approved by the Institutional Animal Care and Use Committee of the Eastern Virginia Medical School.

Experimental protocol
Fetal adrenal glands and pituitaries were collected on day 165 from untreated controls (n = 7) and from baboons in which betamethasone (Celestone Soluspan, Schering Corp., Chicago, IL) was administered to the fetus (0.6 mg/100 µl; n = 4) or to the fetus (0.6 mg/100 µl) and the mother (6.0 mg/ml, im; n = 4) every other day between days 150 and 164 of gestation (term = day 184). Baboons were sedated with ketamine-HCl (10 mg/kg BW; Parke-Davis, Detroit, MI), anesthetized with halothane:nitrous oxide, and betamethasone administered im to the fetus via a 25 gauge needle and maternal transabdominal injection under ultrasound. At 1- to 2-. day intervals between days 140 and 165 of gestation, all baboons were sedated with ketamine-HCl and a maternal saphenous vein blood sample (4–7 ml) collected. Two of the four baboons in which betamethasone was administered only to the fetus delivered spontaneously on days 160 and 164 or approximately 96 and 1 h before their last injection of steroid. Because labor (vaginal bleeding) was initiated between 1 and 2 pm, neonates were recovered from ketamine-sedated mothers during or 30 min after delivery. The neonates were sedated with ketamine and after collecting a blood sample, a lethal dose of Beuthanasia (Butler Corp., Fredericksburg, VA) was administered and tissues obtained within 60 min. In all other animals, umbilical venous blood samples were obtained at the time of cesarean section and samples stored at -20 C until assayed for estradiol and cortisol by solid phase 125I RIAs (Coat-A-Count, Diagnostic Products Corp., Los Angeles, CA) as described previously (19, 20).

Adrenals were weighed and representative sections frozen in liquid nitrogen for subsequent analysis of the mRNAs for ACTH receptor, LDL receptor, 17{alpha}-hydroxylase-C17,20 lyase (P450c17), P450 cholesterol side chain cleavage (P450scc), and 3ß-HSD and peptide levels of P450c17 and 3ß-HSD. Additional sections of adrenal tissue were fixed in 10% buffered formalin (Sigma Chemical Co., St. Louis, MO) for subsequent localization of 3ß-HSD and ACTH receptor mRNA by immunohistochemistry and in situ hybridization, respectively, and for determination of the number of fetal zone and definitive zone cells and the number of each cell type expressing the cell cycle marker, proliferating cell nuclear antigen (PCNA). In some instances, fetal adrenal sections were stored at -80 C for subsequent analysis of 3ß-HSD enzyme activity. Fetal pituitaries of four control baboons and of four baboons in which betamethasone was administered to the fetus (n = 3) or to the fetus and mother (n = 1) were placed in sterile cryomolds containing OCT embedding medium (Miles Scientific, Elkhart, IN) and stored at -80 C until analyzed for POMC mRNA.

Adrenal morphometry and immunocytochemistry
Sections (4 µm) of paraffin embedded 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 anti-PCNA/cyclin monoclonal antibody PC-10 diluted 1:200 in 5% normal goat serum (NGS; Boehringer Mannheim, Indianapolis, IN) or with polyclonal antibody to rabbit antihuman 3ß-HSD (generously supplied by Dr. Ian Mason) diluted 1:5000 in 5% NGS, sections were washed and incubated with biotinylated goat antimouse or goat antirabbit IgG (Boehringer Mannheim), Avidin DH, and horseradish peroxidase H (Vectastain Elite Kit, Vector Laboratories, Burlingame, CA). Sections were lightly counterstained with Gill’s hematoxylin (Fisher) and mounted in Biomount (Fisher) and PCNA and 3ß-HSD expression analyzed by Image Analysis on an average of six randomly selected areas (157 µm x 130 µm)/slide of 4–8 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 definitive and fetal cortical cells per 0.025 mm2 was quantified by counting nuclei in six randomly selected sections and results compared with the number of cells in which nuclear expression of PCNA was 5-fold greater than background. The growth of the definitive zone cells was quantified as the width of the cell layer immunostaining for 3ß-HSD and determined by examining six randomly selected regions of each adrenal section.

mRNA for 3ß-HSD, P450c17, P450scc, and the receptors for ACTH and LDL
These were determined by Northern blot essentially as described previously (21). Briefly, approximately 10 µg of fetal adrenal poly(A+) RNA was denatured and size-fractionated by electrophoresis in 1.0% agarose gel containing 0.66 M formaldehyde and 20 mM MOPS. RNA was transferred overnight by capillary action onto nylon membrane (GeneScreen, DuPont-New England Nuclear, Boston, MA), UV cross-linked, baked in a vacuum oven (80 C for 2 h) and 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 NaH2PO4-H2O and 2.5 mM EDTA-Na2, pH 7.4), 1.0% SDS, 10% dextran sulfate, and denatured salmon sperm DNA (100 µg/ml) for 24 h at 42 C before addition of labeled probe. The complementary DNAs for the baboon ACTH receptor prepared by us (1), the 3ß-HSD (provided by Dr. Ian Mason), the P450c17, P450scc (provided by Dr. Walter Miller), and the LDL receptor (pLDLR3 No 57004) and ß-actin (no. 65128) both obtained from the American Type Culture Collection (Rockville, MD) were labeled with approximately 50 µCi [{alpha}-32P]deoxy-CTP (3000 Ci/mmol; Amersham Corp., Arlington Heights, IL) to a specific activity of approximately 109 dpm/µg DNA using the Random-Primed DNA labeling kit (Boehringer-Mannheim) according to the methods of Feinberg and Vogelstein (22). Hybridization was performed in fresh buffer at 42 C for 23 h with 32P-labeled complementary DNA. After washing under stringent conditions, the membranes were exposed to Kodak X-AR film (Eastman Kodak, Rochester, NY) at -80 C and the intensities of the bands on each Northern blot analyzed by densitometric autoradiographic scanning using a model 620 Video Densitometer (Bio-Rad, Hercules, CA).

Western analysis of 3ß-HSD and P450c17
Analysis of 3ß-HSD and P450c17 peptides in fetal adrenal extracts was performed using procedures developed previously in our laboratories (23). Briefly, adrenals were homogenized on ice in 2.5 ml buffer composed of 1% cholic acid (Sigma), 0.1% SDS, 1 mM EDTA in PBS to which had been added 0.1 mg/ml phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, and 0.1 mg/ml trypsin inhibitor and centrifuged at 800 x g to remove cell debris. After determination of protein concentrations using the bicinchoninic acid procedure (Sigma), 5x Laemmli buffer (24) was added to a final concentration of 1x and samples boiled for 2 min, centrifuged (1,000 x g for 10 min) and loaded (30 µg protein/lane) onto preformed 10% SDS-polyacrylamide minigels maintained in Bio Rad Mini-Protean II electrophoresis chambers, electrophoresed at 100 V, and transferred to Immobilon P (Life Technologies, Bethesda, MD). After blocking with 3% BSA in 50 mM Tris, pH 7.5, containing 150 mM NaCl and 0.05% Tween 20 (Sigma), samples were incubated (37 C, 1 h) with polyclonal antibodies to rabbit antihuman placental 3ß-HSD or rabbit antiporcine testicular microsomal P450c17 (generously supplied by Dr. Ian Mason) diluted 1:10,000 or 1:2,000 respectively in 50 mM Tris buffer (pH 7.5) containing 150 mM NaCl, 0.05% Tween 20, 0.05% Nonidet P-40 (Sigma) and 1.5% BSA. Membranes were washed and then incubated with donkey antirabbit IgG horseradish peroxidase conjugated second antibody (Amersham) at dilutions recommended by the manufacturer and which contributed no nonspecific bands at the concentrations employed. After washing, equal amounts of enhanced chemiluminescent reagent (ECL; Amersham) were applied to membranes for 1 min, the membranes wrapped in plastic and then placed against Kodak X-Omat film (Kodak) in x-ray film cassettes and exposed for 15–60 sec. Samples were developed and quantified by 1 dimensional densitometry using an LKB Bromma Ultroscan XL Enhanced Laser densitometer.

3ß-HSD enzyme activity
The activity of 3ß-HSD was determined essentially as described previously (6). Briefly, adrenals were homogenized in 2.5 ml 0.05 M NaH2PO4 buffer (pH 7.4) and microsomes (or buffer blank) incubated in duplicate (37 C) for 2, 5, and 10 min with 125,000 cpm [7-3H] pregnenolone (SA, 25 Ci/mmol; DuPont-New England Nuclear), 125 ng radioinert pregnenolone (Sigma) and 2.5 mg NAD+ (Sigma). Samples were extracted with ethyl acetate, radiolabeled product progesterone isolated by paper chromatography and [3H] concentrations, corrected for procedural losses estimated by recovery of [4-14C] progesterone (SA 35 mCi/mmol; Dupont-New England Nuclear) added before extraction, determined by liquid scintillation spectrometry.

In situ hybridization histochemistry of POMC and ACTH receptor mRNAs
In situ hybridization detection and quantification of POMC mRNA expression were performed using our previously published methods (25). Briefly, 0.1 µmol purified POMC antisense (and sense) oligodeoxynucleotide probes were 3' end-labeled with [35S]dATP (SA > 1000 Ci/mmol; NEN) and terminal deoxynucleotidyl transferase (20 U; Promega, Madison, WI) to a specific activity of approximately 5000 Ci/mmol. Sections of the fetal pituitary were selected, incubated overnight (50 C) with 40 µl labeled antisense or sense probe and then washed at 60 C, rinsed and placed against Kodak X-Omat film in x-ray film holders and exposed 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).

Localization of ACTH receptor mRNA was determined using our previously published procedures (9). Briefly, sections of paraffin-embedded fetal adrenal glands were cleared in xylene, rehydrated in graded ethanols, rinsed in PBS, and hybridized overnight at 45 C with an oligodeoxynucleotide antisense (sense) probe complementary to bases 373–401 of the baboon ACTH receptor (1) and end-labeled with [35S] dATP (Dupont-New England Nuclear). Slides were washed at 60 C, rinsed, dipped in Kodak NTB-2 nuclear track emulsion diluted 1:1 with distilled water, placed in light tight boxes, exposed for 12–17 days, and then developed in Kodak D-19. The cellular distribution of silver grains was determined using an Optiphot 2 microscope attached to a Video Based Image-1 Analysis System (Universal Imaging Corp., West Chester, PA).

Statistics
Because fetal adrenal weight and fetal pituitary POMC mRNA expression was not different after maternal and/or fetal betamethasone, values for the various parameters examined in these two treatment groups were combined and expressed as an overall mean for the effects of betamethasone. Data were analyzed by analysis of variance with post hoc comparison of the means by the Student-Newman-Keul’s statistic or were compared by Student’s t tests for independent or dependent observations.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Maternal serum cortisol and estradiol concentrations in untreated control baboons were not significantly changed during the study period (Fig. 1Go, A and B). In contrast, betamethasone administration to the mother and fetus resulted in a rapid and sustained decrease in maternal serum estradiol and cortisol concentrations (Fig. 1Go, E and F). However, in baboons in which betamethasone was administered only to the fetus, reductions in maternal serum estradiol and cortisol concentrations, although significant, were much less marked (Fig. 1Go, C and D). Moreover, umbilical venous serum concentrations (mean ± SE) of cortisol and estradiol obtained at the time of delivery were decreased (P < 0.05) when betamethasone was administered to the fetus and further reduced in animals in which both the mother and fetus received betamethasone (Table 1Go), although the latter was not confirmed statistically due to heterogeneity of variance between the groups.



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  		Figure 1. Serum estradiol (E2) and cortisol (F) concentrations in maternal saphenous vein blood samples obtained between days 140 and 164 of gestation (term = day 184) from representative baboons untreated (A and B) or treated with betamethasone administered to the fetus (C and D) or to the mother and fetus (E and F) every other day between days 150 and 164 of gestation. See footnote of Table 1Go for details.

 

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  		Table 1. Effects of administration of betamethasone during baboon pregnancy on umbilical vein serum levels of cortisol and estradiol and on fetal adrenal, pituitary and body weights

 
Although fetal pituitary and body weights were not altered by betamethasone (Table 1Go), POMC mRNA levels (Fig. 2Go) were lower (P < 0.05) in betamethasone-treated (0.34 ± 0.07 arbitrary densitometric units) than in untreated control (0.63 ± 0.04) baboons. Associated with the decline in pituitary POMC mRNA, levels of the major 3.4-kb mRNA transcript for the ACTH receptor (Fig. 3Go) expressed as a ratio of ß-actin, were approximately 80% lower (P < 0.05) in the fetal adrenal of baboons in which betamethasone was administered to the fetus and/or to the mother and fetus (0.12 ± 0.02) than in untreated controls (0.84 ± 0.05). However, in the two animals in which the fetus was delivered spontaneously, ACTH receptor mRNA levels (1.06 and 0.63) were similar to those of the untreated controls. In situ hybridization indicated that in control baboons ACTH receptor mRNA levels, expressed as the number of silver grains/0.025 mm2, were greater (P < 0.01) in the definitive zone than in the corresponding fetal zone and that the reduction of ACTH receptor mRNA expression elicited by betamethasone occurred primarily in the definitive zone (Fig. 4Go). Associated with the decrease in ACTH receptor expression, fetal adrenal weight was reduced (P < 0.05) approximately 50% by the administration of betamethasone to the fetus (Table 1Go) but was not further suppressed by administration of betamethasone to both the mother and fetus. The decrease in adrenal weight was associated with an increase (P < 0.05) in the number of fetal and definitive zone cells per unit area (Table 2Go), indicating that the size of these cells was reduced by betamethasone administration. The number of fetal adrenal cells expressing PCNA, however, was similar in control and betamethasone-treated animals (Table 2Go).



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  		Figure 2. Effect of administration of betamethasone during baboon pregnancy on fetal pituitary POMC mRNA levels (mean ± SE) determined by in situ hybridization. Pituitary glands were obtained on day 165 of gestation (term = day 184) from three baboons in which the fetus alone was injected with betamethasone and from one animal in which the fetus and mother were treated with betamethasone between days 150 and 164 of gestation. POMC mRNA in the fetal pituitary of the one baboon in which both the mother and fetus received betamethasone (0.32) was similar to the mean value (0.36) in the three animals in which betamethasone was administered only to the fetus. *, Mean value differs from that in the control (n = 5) at P < 0.05 (Student’s t test).

 


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  		Figure 3. Representative Northern blot of the mRNA for ACTH receptor (A) and ß-actin (B) in fetal adrenal glands obtained on day 165 of gestation by cesarean section from untreated control baboons (lanes 1–3) and from animals in which the mother and/or fetus were administered betamethasone (ßm) between days 150 and 164 of gestation and the fetus delivered by cesarean section (CS; lanes 5, 6, 8, 9) or spontaneously (SD; lanes 4 and 7) 96 or 1 h before day 165. C, Mean (±SE) levels of the 3.4-kb mRNA transcript for the ACTH receptor determined by Northern analysis in fetal adrenal glands of control (n = 5) and betamethasone-treated baboons delivered by cesarean section (n = 4) or spontaneously (n = 2). *, Value differs from that in the control at P < 0.05 (Student’s t test).

 


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  		Figure 4. Representative darkfield photomicrographs of sections of baboon fetal adrenal glands hybridized with 35S-labeled ACTH receptor antisense (A and B) or sense (C) oligonucleotide probes (magnification = x100; magnification bar = 100 µm). Adrenal glands obtained on day 165 of gestation from untreated (A and C) baboons and from animals in which the mother and fetus were treated with betamethasone between days 150 and 164 of gestation (B). The arrowhead denotes the demarcation of the definitive (top) and fetal cortical zones. A, Brightfield photograph (magnification = x 1000) of representative cells of the definitive cortex of sections of near term fetal adrenal from untreated baboons hybridized with 35S-labeled ACTH receptor antisense probe. D, ACTH receptor mRNA levels determined by in situ hybridization expressed as the mean (±SE) number of silver grains/0.025 mm2 of definitive zone and fetal zone of fetal adrenals from untreated (n = 4) and betamethasone-treated (ßm) baboons (n = 3) of late gestation. *, Different from values in control (P < 0.01; t test for dependent observations); {dagger}, different from respective control value (P < 0.02; t test for independent observations).

 

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  		Table 2. Effect of administration of betamethasone during baboon pregnancy on fetal and definitive zone cell number and expression of proliferating cell nuclear antigen (PCNA)

 
Betamethasone treatment also induced a significant decrease (P < 0.05) in the width (µm) of the definitive zone (183 ± 14 vs. 128 ± 7), as determined by immunohistochemical expression of 3ß-HSD (Fig. 5AGo). The decrease in definitive zone width was associated with a reduction (P < 0.05) in expression of the single 2.0-kb 3ß-HSD mRNA transcript (Fig. 5BGo), the 36-kDa 3ß-HSD peptide (Fig. 5CGo), and 3ß-HSD enzyme activity (2.2 pmol/mg; Fig. 5DGo), which was 20-fold lower than that previously measured by us in adrenals of untreated baboons (41 ± 11 pmol/mg) at the same time in gestation (6).



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  		Figure 5. Effect of betamethasone on baboon fetal adrenal definitive zone: A, width in microns (determined by 3ß-HSD immunocytochemical expression); B, mRNA levels (ratio to ß-actin); C, protein levels (arbitrary units); and D, enzyme activity (pmol pregnenolone converted to progesterone/min/mg tissue). Adrenals obtained on day 165 of gestation from untreated controls and from baboons in which the mother and/or fetus were treated with betamethasone every other day between days 150 and 164 of gestation. For each parameter, the number of adrenals analyzed ranged from four to seven for the controls and three to five for the betamethasone-treated group. Because fetal adrenal weight was not different after maternal and/or fetal betamethasone (See Table 1Go), 3ß-HSD in this figure represents analyses from both treatment groups, pooled and presented as the overall effects of betamethasone. *, Value (mean ± SE) differs from the control at P < 0.05 (Student’s t test).

 
Associated with the reduction in ACTH receptor mRNA, the levels of the 6.2-kb mRNA for the LDL receptor (Fig. 6AGo), the 2.2-kb mRNA for P450scc (Fig. 6BGo) and the 2.1-kb mRNA for P450c17 (Fig. 6CGo) in adrenals of term controls (1.23 ± 0.20, 1.35 ± 0.07, and 1.61 ± 0.09, respectively) were also decreased (P < 0.05) by approximately 80–90% by betamethasone in cesarean (0.09 ± 0.02, 0.25 ± 0.05, and 0.30 ± 0.06, respectively) but not spontaneously delivered baboons. The levels of the 55-kDa P450c17 protein in adrenals of term controls (3.15 ± 0.19) were also decreased (P < 0.05) by betamethasone in cesarean delivered (0.88 ± 0.18) but not spontaneously delivered (2.1) baboons (data not shown).



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  		Figure 6. Mean (± SE) levels of the 6.2-kb mRNA for the LDL receptor (A), the 2.2-kb mRNA for P450scc (B), and the 2.1-kb mRNA for P450c17 (C) determined by Northern analysis in fetal adrenals obtained by cesarean section from untreated controls baboons (n = 4–7/analysis) and from animals in which the mother and/or fetus were administered betamethasone (ßm) between days 150 and 164 of gestation and the fetus delivered by cesarean section (CS; n = 3–5/analysis) or spontaneously (SD) 96 or 1 h before day 165 (n = 2). *, Value differs from that in the control at P < 0.05 (Student’s t tests). Representative examples of the size and relative intensities of the mRNA for each parameter are indicated above the histogram. Lanes 1–3, controls; lanes 5, 6, 8, 9, ßm (CS); lanes 4 and 7, ßm (SD).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The results of the present study indicate that fetal adrenal ACTH receptor mRNA levels were markedly suppressed in baboons in which fetal pituitary POMC mRNA expression was reduced by treatment with betamethasone during late gestation. We have previously shown that the increase in fetal pituitary POMC mRNA levels with advancing baboon gestation is associated with a concomitant increase in the number of pituitary corticotrophes expressing and presumably secreting ACTH peptide (25). Therefore, we suggest that ACTH of fetal pituitary origin regulates fetal adrenal ACTH receptor mRNA expression in the baboon fetus late in gestation. Previous studies in rhesus monkeys have also shown that maternal or fetal administration of dexamethasone/betamethasone (26, 27) markedly suppressed fetal adrenal function/maturation late in gestation. However, the results of the present study are the first to show that these glucocorticoid-induced changes in late gestation are due to suppression of fetal pituitary POMC mRNA/ACTH and inhibition of fetal adrenal ACTH receptor mRNA expression. Although additional studies in which ACTH is infused into the late gestation fetus remain to be performed, the results of the present study are consistent with in vitro observations demonstrating that ACTH receptor mRNA levels were stimulated by ACTH in cultures of human fetal adrenal cells (12, 13, 15, 16) and with our recent in vivo studies showing that ACTH partially reversed the decline in ACTH receptor mRNA elicited by betamethasone at midgestation (17). Mesiano et al. (16) have also demonstrated that ACTH receptor mRNA expression was maximally enhanced 12–16 h after treatment of cultured human fetal adrenal cells with ACTH in a dose-dependent manner. This apparent relatively rapid increase in ACTH receptor mRNA induction as measured in vitro may also occur in vivo. Thus, ACTH receptor mRNA levels were up-regulated in two animals of the present study that labored and delivered spontaneously and in which ACTH levels were probably elevated (28, 29).

The present results also indicate that the ACTH receptor was primarily expressed in cells of the newly emerging definitive zone of the fetal adrenal and exceeded that in cells of the fetal zone. Mesiano et al. (16) noted a similar pattern of ACTH receptor mRNA expression in the human fetal adrenal. We (9) have previously shown that ACTH receptor mRNA levels declined in the fetal zone with advancing baboon gestation and have suggested that the concomitant developmental increase in ACTH receptor expression in the definitive zone indicates that the definitive cells are more sensitive to ACTH than cells of the fetal zone late in pregnancy. The results of the present study are consistent with this suggestion because ACTH receptor mRNA levels as assessed by in situ hybridization and expressed per adrenal cellular area were decreased in the definitive zone, but not the fetal zone, in baboons in which fetal pituitary POMC mRNA was suppressed. Moreover, we (30) have previously shown that basal as well as ACTH-dependent DHA production by the baboon fetal adrenal in vitro is lower at term than at midgestation, whereas basal and ACTH stimulable cortisol synthesis at term exceeds that at midgestation. Because fetal pituitary POMC mRNA expression and presumably secretion of ACTH increases with advancing gestation in the baboon (25) and perhaps the human (31), it appears that factors in addition to ACTH play a role in modulating ACTH receptor expression in the fetal zone of the primate fetal adrenal gland. Although additional studies utilizing in situ hybridization and molecular approaches are required to address this possibility, we have recently shown that estrogen acts directly on the fetal adrenal gland to modulate ACTH-dependent DHA production (32, 33). However, whether estrogen acts to modulate ACTH receptor mRNA also remains to be determined.

The present study also indicates that the mRNA and/or protein levels of the enzymes 3ß-HSD, P450c17 and P450scc and the membrane receptor for LDL were decreased in adrenals of betamethasone-treated animals near term. The activities and/or mRNAs for the 3ß-HSD and the P450scc/P450c17 enzymes were regulated by ACTH in cultures of human adrenal cells (34, 35, 36) and in vivo in the sheep fetus (37). Moreover, expression of P450c17 mRNA is very sensitive to ACTH because the mRNA for this enzyme rapidly declines under culture conditions in the absence of ACTH (35). Similarly, LDL receptor binding is lower in adrenals of anencephalic fetuses and increased after treatment of fetal adrenal cells in vitro with ACTH (38). As with ACTH receptor expression, the mRNA and protein levels of P450c17 and mRNA levels for P450scc and the LDL receptor were similar in adrenals of control cesarean delivered and betamethasone-treated spontaneously delivered baboons. Collectively, these observations indicate that the ACTH receptor mRNA is regulated coordinately with other ACTH-dependent genes, in utero in the primate fetus as recently suggested by Mesiano et al. (16) based on studies of human adrenal cells in culture.

Our findings also indicate that after suppression of ACTH receptor mRNA there was a reduction in cell size of both the fetal and definitive zones. Thus, the decrease in width of the definitive zone, as assessed by immunohistochemical expression of zone-specific 3ß-HSD (39), apparently reflected the smaller size of these cells. Therefore, one of the actions of ACTH may be to stimulate hypertrophy of the fetal and definitive zone cells as previously demonstrated in the fetal rhesus monkey (40, 41). ACTH also stimulated proliferation of adrenocortical cells in the rat, but this effect occurred only after stimulation of cellular hypertrophy (42). Although it remains to be determined whether the latter sequence also occurs in the fetal adrenal, in baboons of the present study the percentage of fetal and definitive zone cells expressing PCNA was not altered by betamethasone. Therefore, it is possible that the capacity of these cells to undergo replication if appropriately stimulated was not compromised. Indeed, Jaffe and colleagues (43) have shown that fetal adrenal cells in culture proliferate in the continuous presence of physiological levels of ACTH.

In animals of the present study in which the fetal pituitary-adrenal axis was maximally suppressed by fetal administration of betamethasone, estradiol levels were only reduced by 50%. This suggests that the maternal adrenal is also a contributor of adrenal C-19 steroid precursors for placental estrogen production in nonhuman primate pregnancy as in humans (44). Thus, only when the maternal pituitary-adrenal axis was blocked was there a more marked suppression of estrogen synthesis.

In summary, the present study shows that treatment of baboon fetuses with betamethasone in late gestation suppressed fetal pituitary POMC mRNA expression and ACTH receptor mRNA levels and 3ß-HSD expression in the definitive zone of the fetal adrenal gland. Moreover, in association with these changes in ACTH receptor expression, there was a significant decrease in fetal adrenal size and growth of the definitive zone. It is concluded that ACTH is necessary for the up-regulation of the mRNAs for the ACTH receptor and steroidogenic enzymes in the definitive zone of the primate fetal adrenal in late gestation.


   Acknowledgments
 
The authors sincerely appreciate the secretarial assistance of Ms. Sandra Huband with the preparation of the manuscript and figures and the generous gift of antibodies to 3ß-HSD and P450c17 supplied by Dr. Ian Mason of the University of Edinburgh, Edinburgh, Scotland.


   Footnotes
 
1 This work was supported by NIH Research Grant R01-HD-13294. Back

Received December 11, 1996.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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