Estrogen Elicits Cortical Zone-Specific Effects on Development of the Primate Fetal Adrenal Gland
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内分泌学杂志 2005年第4期
Departments of Obstetrics, Gynecology and Reproductive Sciences, and Physiology (E.D.A., G.W.A.), Center for Studies in Reproduction, University of Maryland School of Medicine, Baltimore, Maryland 21201; and Department of Physiological Sciences (G.J.P.), Eastern Virginia Medical School, 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. E-mail: ealbrech@umaryland.edu.
Abstract
In the present study, we determined whether endogenous estrogen, the levels of which increase with advancing pregnancy, regulates growth and development of the baboon fetal adrenal cortex. Fetal adrenal glands were obtained at mid- (d 100) and late (d 165, term is 184 d) gestation from untreated baboons and on d 165 from animals in which endogenous estrogen production was suppressed by administration of aromatase inhibitor CGS 20267 between d 100 and 165. Volumes of the respective cortical zones were determined by zone-specific immunocytochemical staining of steroidogenic enzymes and image analysis. Fetal adrenal weight and volume increased (P < 0.01) 3-fold between mid- and late gestation and an additional 70% (P < 0.01) by administration of CGS 20267, which decreased (P < 0.001) fetal serum estradiol levels by more than 95%. Mean ± SE volume (x10–10 μm3) of the fetal cortical zone increased from 3.45 ± 0.28 at midgestation to 6.64 ± 0.69 at late gestation in untreated baboons and to 12.55 ± 0.99 (P < 0.01) in baboons in which estrogen production was suppressed by CGS 20267 administration. The levels of umbilical artery serum dehydroepiandrosterone sulfate, which is secreted primarily by the fetal zone, were increased almost 3-fold (P < 0.01) by administration of CGS 20267. Concomitant administration of CGS 20267 and estradiol returned fetal cortical zone volume and serum dehydroepiandrosterone sulfate levels to normal. In contrast to the effect of estrogen deprivation on the fetal zone, the volumes of the definitive and transitional zones in untreated baboons late in gestation (3.18 ± 0.63 and 2.62 ± 0.43, respectively) and levels of fetal serum cortisol, a steroid secreted from the transitional zone, were not altered by estrogen suppression. The changes in fetal zone growth were not associated with alterations in fetal pituitary proopiomelanocortin mRNA levels. We propose that estrogen acts directly on the fetal adrenal cortex to selectively repress the morphological and functional development of the fetal zone, potentially as a feedback system to maintain physiological secretion of estrogen precursors and thus placental estrogen production to promote normal primate fetal and placental development.
Introduction
THROUGHOUT THE COURSE of human and nonhuman primate pregnancy, the fetal adrenal cortex is comprised primarily of the fetal zone, which expresses the P-450 17-hydroxylase, 17–20 lyase (P-450C17) enzyme catalyzing synthesis of C19-steroids, e.g. dehydroepiandrosterone (DHA) and DHA sulfate (DHAS), used as precursors for estrogen synthesis by the placenta (for reviews, see Refs. 1, 2, 3). The definitive zone, the site of the 5-3?-hydroxysteroid dehydrogenase (3?-HSD) and synthase enzymes that produce aldosterone, is also present at mid- and late gestation. However, the transitional zone, which expresses both the 3?-HSD and P-450C17 enzymes (4), which catalyze the production of cortisol, develops only relatively late in gestation. In addition to functional maturation, the human fetal adrenal cortex, particularly the fetal zone, grows rapidly during the second and third trimesters (5, 6, 7); however, regulation of growth and functional development of the individual fetal adrenal cortical zones has not been clearly established.
ACTH has a pivotal role in the growth and functional maturation of the human and nonhuman primate fetal adrenal gland (8, 9, 10, 11, 12, 13). However, because each of the fetal adrenal cortical zones expresses the ACTH receptor (12, 14) and presumably is exposed to comparable concentrations of ACTH with advancing gestation (15, 16), we have proposed that regulation of the differential zone-specific maturation of the adrenal cortex is multifactorial and involves placental estrogen as well as ACTH (17). Estrogen receptors- and -? are expressed in all zones of the baboon fetal adrenal cortex (18), providing a mechanism for mediating the action of estrogen on fetal adrenal development. Moreover, we have shown that the amount of fetal zone-specific DHA formed per cell in baboon fetal adrenal incubates declined between mid- and late gestation in association with the rise in estrogen (19, 20). Also, at midgestation estrogen suppressed responsivity of the baboon fetal adrenal to ACTH with respect to the formation of DHA but not cortisol in vitro (21, 22) and in vivo (23). Therefore, we have postulated that estrogen feeds back to regulate function of the fetal cortical zone with respect to the production of DHA.
However, the potential role that the increasing levels of endogenous estrogen have on development and growth of the fetal adrenal cortical zones in vivo in the primate has not been investigated. Therefore, we employed the baboon as a nonhuman primate model in which placental estrogen production was suppressed by administration of a highly specific aromatase inhibitor during pregnancy to determine whether zone-specific development of the baboon fetal adrenal cortex is regulated by estrogen.
Materials and Methods
Animals
Female baboons (Papio anubis) weighing 12–16 kg were housed in aluminum stainless-steel large primate cages in air-conditioned rooms under a 12-h light, 12-h dark cycle. Baboons received high-protein monkey chow and fresh fruit twice daily, vitamins daily, and water ad libitum. Females were paired with male baboons for a period of 5 d during the ovulatory phase of the menstrual cycle, and pregnancy was determined by palpation. Animals were cared for and used strictly in accordance with U.S. Department of Agriculture regulations and the Guide for the Care and Use of Laboratory Animals (National Research Council, National Academy Press, 1996). The experimental protocol employed in the present study was approved by the Institutional Animal Care and Use Committees of the University of Maryland School of Medicine and Eastern Virginia Medical School.
Ten baboons received the aromatase inhibitor CGS 20267 [Letrozole; 4, 4'-(1,2,4-triazol-1-yl-methylene)-bis-benzonitrite, Norvartis Pharma AG, Basel, Switzerland] administered sc to the mother (increasing from 0.005 mg/kg body weight per day to a maximum of 0.115 mg/kg body weight per day in 1 ml sesame oil) on d 100–165 of gestation (term = 184 d). Seven additional baboons were injected sc daily on d 100–165 with CGS 20267 plus estradiol benzoate at doses (maximum of 0.115 mg/kg body weight per day each) designed to replicate the normal pattern of maternal serum estradiol. Ten baboons served as untreated controls and were studied at mid- (d 100, n = 4) or late (d 165, n = 6) gestation. Blood samples (4 ml) were taken at 1- to 3-d intervals during the study period from a maternal saphenous vein after brief sedation of baboons with ketamine HCl (10 mg/kg body weight, im) for analysis of serum estradiol levels.
On d 100 or d 165 of gestation, baboons underwent cesarean section under halothane-nitrous oxide anesthesia, and blood samples (3–4 ml) were obtained from a maternal saphenous and umbilical vein and artery and serum stored at –20 C until assayed for estradiol (24), estrone, DHAS (13), and cortisol (25) by immunoassay (Immulite, Diagnostic Products Corp., Los Angeles, CA) as described previously. The fetuses were immediately killed with an overdose of sodium pentobarbital and the adrenals and pituitary removed and weighed, embedded in OCT (Miles Scientific, Elkhart, IN), and snap frozen in isopentane precooled in liquid nitrogen for subsequent immunocytochemical localization of 3?-HSD and P-450C17, volumetric analysis, and proopiomelanocortin (POMC) mRNA analysis by in situ hybridization, respectively.
Fetal adrenal 3?-HSD and P-450C17 immunocytochemistry
Frozen serial sections (10 μm) were cut with a cryostat through the entire adrenal gland and mounted onto Superfrost/Plus microscope slides (Fisher Scientific, Pittsburgh, PA). Every 10th and 11th section were used for immunocytochemical staining using rabbit polyclonal antibodies to human 3?-HSD (generously supplied by Dr. Ian Mason, University of Edinburgh, Scotland, UK) and human P450C17 (generously supplied by Dr. Michael Waterman, Vanderbilt University School of Medicine, Nashville, TN), respectively.
Tissues were fixed in either 70% ethanol for 1 min (3?-HSD) or 4% buffered paraformaldehyde for 10 min (P450C17). Slides were then washed thoroughly in PBS and incubated in 1% hydrogen peroxide in PBS for 10 min to remove endogenous peroxidase. After washing in PBS, tissue was blocked using reagents in the avidin/biotin blocking kit (Vector Laboratories, Burlingame, CA) diluted in 5% normal goat serum for 1 h to inhibit nonspecific binding.
Sections were incubated 24–48 h at 4 C with either 3?-HSD (1:7500) or P450C17 (1:2000) antibody using PBS/5% normal goat serum diluent, washed in PBS, and incubated for 1 h in biotinylated goat antirabbit IgG (1:600, Vector Laboratories). After repeated washes in PBS (3 x 5 min), slides were incubated for 1 h with avidin/biotin complex reagent kit (Vector Laboratories).
Sections were then colorized for 15 min using diaminobenzidine-HCl (Sigma-Aldrich, St. Louis, MO) in 50 mM Tris-HCl buffer as a chromagen. Slides were washed in 50 mM Tris-HCl buffer and PBS and rinsed thoroughly in distilled water. Tissue sections were lightly counterstained with Harris’ hematoxylin (Fisher Scientific), dehydrated in several changes of graded ethanol, cleared in two to three changes of xylene, and mounted in Permount mounting media (Fisher Scientific).
Volumetric analysis of fetal adrenal cortical zones
Fetal adrenal cortical zone volumes were determined using an Eclipse E1000/video-based Image 1 analysis system (Nikon, New York, NY) with analytical Scion Image software (Scanalytics, Inc., Fairfax, VA). Using 3?-HSD and P-450C17 immunocytochemistry to demarcate the cortical zones, volumes of the 3?-HSD-negative fetal zone, 3?-HSD-positive and P-450C17-negative definitive zone, and 3?-HSD-positive and P-450C17-positive transitional zone were quantified and integrated for the entire gland using adrenal cortical zone areas and section thickness.
In situ hybridization of fetal pituitary POMC mRNA
The methods for detection of POMC mRNA expression in the baboon fetal pituitary by in situ hybridization (16) were modified using procedures recently developed in our laboratories (16, 26) and a 717-bp riboprobe to the rat POMC gene (27) generously provided by Dr. James L. Roberts (University of Texas Medical Center, San Antonio, TX). Briefly, frozen fetal pituitary sections (10 μm) were treated (15 min; 37 C) with 5 μg/ml proteinase K (Sigma-Aldrich), incubated (10 min) with 10% buffered formalin, rinsed in PBS, and treated with 0.25 M acetic anhydride in 0.1 M triethanolamine hydrochloride-0.9% NaC1. After dehydration in ethanol, delipidation in chloroform, and rehydration, sections were hybridized overnight at 55 C with 33P-labeled antisense POMC riboprobe (106 dpm/slide) in 50 μl hybridization buffer consisting of 10 ml formamide, 1 ml 1 M Tris (pH 8.0), 0.1 ml 0.5 M EDTA, 500 μl yeast tRNA (Sigma-Aldrich), 500 μl 50x Denhardt’s solution (Sigma-Aldrich), 20 μl polyadenylic acid (0.1 μg/μl), 20 μl salmon sperm DNA (500 μg/ml; Life Technologies, Inc., Grand Island, NY), 2 g dextran sulfate, 0.8 ml 5 M NaCl, and diethylpyrocarbonate-H2O. After incubation, sections were washed repeatedly in 4x saline sodium citrate (SSC) [1x SSC = 0.15 M NaCl and 0.015 sodium citrate buffer (pH 7.2)]; serially dehydrated; and washed in 50% formamide, 300 mM NaCl, 25 mM Tris, and 1 mM EDTA. After a 10-min rinse in 2x SSC, nonspecific hybridized probe was removed by incubation (10 min, 37 C) with RNase A (25 μg/ml, Promega Corp., Madison, WI) and RNase T1 (2000 U, Promega) in RNase buffer consisting of 500 mM NaCl, 10 mM Tris, and 1 mM EDTA (pH 8.0).
Subsequently tissues were washed twice in RNase buffer at 50 C for 15 min, four times in 2x SSC for 15 min at 50 C, and once in 0.2x SSC at 60 C for 2 h before serial dehydration and vacuum drying. Slides were placed against X-OMAT film (Kodak, Rochester, NY) in x-ray film holders for 48 h and areas of POMC mRNA expression circumscribed and quantified by densitometric analysis using an LKB Bromma Ultroscan XL enhanced laser densitometer (Pharmacia LKB, Piscataway, NY) as described previously (16). For each animal, six to 10 sections of fetal pituitary were analyzed. Applicability of the probe for use on baboon tissue was confirmed by binding to baboon pituitary RNA as determined by Northern blot and specificity verified by absence of expression over the posterior pituitary (data not shown). To ensure that densitometric analysis of film was not compromised by tissue folding, pituitary sections were also examined by light microscopy.
Statistical analysis
Fetal adrenal and pituitary parameters and hormone levels were analyzed using one-way ANOVA and Newman-Keuls multiple comparison test to assess significant differences between individual group means (Instat, GraphPad Software, San Diego, CA).
Results
Serum estradiol and estrone levels
Serum estradiol concentrations (mean ± SE) in the maternal saphenous (1514 ± 321 pg/ml) and umbilical (331 ± 57 pg/ml, Fig. 1) veins on d 100 of gestation (i.e. midgestation) increased (P < 0.01) to 3493 ± 467 and 590 ± 72 pg/ml (Fig. 1), respectively, on d 165 (i.e. late) of gestation. The administration of CGS 20267 to baboons decreased serum estradiol levels in the maternal saphenous (96 ± 12 pg/ml) and umbilical (47 ± 4 pg/ml) veins on d 165 of gestation to values that were approximately 3 (P < 0.001) and 8% (P < 0.001), respectively, of the values observed in the untreated controls (Fig. 1). Concomitant administration of CGS 20267 and estradiol restored maternal serum estradiol levels (4796 ± 614 pg/ml) to normal and increased estradiol concentrations in the umbilical vein to a value (152 ± 24 pg/ml) similar to that observed after CGS 20267 alone but lower than in untreated baboon fetuses in late gestation (Fig. 1).
FIG. 1. Mean (±SE) maternal saphenous and umbilical vein serum estradiol levels on d 100 (mid, n = 4) and d 165 (late, n = 6) of gestation in baboons that were untreated and on d 165 in animals treated with antiestrogen CGS 20267 (maximum of 0.115 mg/kg body weight per day sc, n = 10) or CGS 20267 plus estradiol benzoate (each at maximum of 0.115 mg/kg body weight per day sc, n = 7) daily on d 100–165 of gestation (term = 184 d). Values indicated by different letter superscripts are significantly different at P < 0.01, ANOVA and Newman-Keul’s multiple comparison test.
Serum estrone levels in the maternal saphenous vein were 596 ± 96 pg/ml at midgestation and 1252 ± 190 pg/ml at late gestation (Fig. 2). The administration of CGS 20267 decreased (P < 0.001) estrone to a value (85 ± 20 pg/ml) that was approximately 7% of that in untreated baboons near term. After simultaneous CGS 20267 administration, maternal estrone levels (924 ± 241) were restored to normal. In the umbilical vein, serum estrone levels appeared higher than estradiol (Fig. 1), consistent with the somewhat greater secretion of placental estrone into the fetal compartment during primate pregnancy (28). Umbilical vein serum estrone levels increased (P < 0.01) between mid- (1111 ± 152 pg/ml) and late (2049 ± 329 pg/ml) gestation and also were decreased (P < 0.001) by CGS 20267 treatment (306 ± 48 pg/ml). Concomitant administration of CGS 20267 and estradiol increased umbilical vein estrone to a level (602 ± 112 pg/ml) that was 2-fold, but not significantly, greater than in baboons that received CGS 20267 alone.
FIG. 2. Maternal saphenous and umbilical vein serum estrone concentrations for the baboons in which serum estradiol levels are shown in Fig. 1. Values indicated by different letter superscripts are significantly different at P < 0.01, ANOVA and Newman-Keul’s multiple comparison test.
Fetal adrenal weight and volumes
Mean (±SE) fetal adrenal weight in untreated baboons increased (P < 0.001) approximately 3-fold from 127 ± 5 mg on d 100 to 352 ± 14 mg on d 165 (Fig. 3A). Fetal adrenal weight in CGS 20267-treated animals on d 165 (475 ± 28 mg) was approximately 35% greater (P < 0.001) than in the untreated controls. Whole adrenal gland volume assessed by image analysis also increased (P < 0.01) approximately 3-fold between mid- (4.29 ± 0.42 x 10–10 μm3) and late (11.97 ± 1.20 x 10–10 μm3) gestation (Fig. 3B) and an additional 70% (P < 0.01) by administration of CGS 20267 (20.28 ± 1.58 x 10–10 μm3) when compared with untreated animals in late gestation. In baboons that received CGS 20267 plus estradiol, fetal adrenal weight (342 ± 30 mg) and volume (13.37 ± 1.72 x 10–10 μm3) were similar to respective values in the untreated controls. Although fetal body and organ weights increased (P < 0.05) between mid- and late gestation (Table 1), respective values at late gestation were similar in untreated and CGS 20267 ± estradiol-treated baboons.
FIG. 3. Fetal adrenal weight (two glands, A) and volume assessed by image analysis (B) in untreated baboons on d 100 (mid, n = 4) and 165 (late, n = 6) of gestation and on d 165 in animals that received CGS 20267 (maximum of 0.115 mg/kg body weight per day, n = 10) or CGS 20267 and estradiol (maximum of 0.115 mg/kg body weight per day each, n = 7) daily on d 100–165 of gestation. Values indicated by different letters are significantly different at P < 0.001 (A) and P < 0.01 (B), ANOVA and Newman-Keul’s multiple comparison test.
TABLE 1. Fetal body and organ weights in baboons untreated or treated with CGS 20267 with or without estradiol1
Figure 4 illustrates in a representative fetal adrenal gland of an untreated baboon at late gestation, hematoxylin and eosin histology (Fig. 4A) and 3?-HSD (Fig. 4B) and P-450C17 (Fig. 4C) immunocytochemistry, which was used to mark and thus determine by image analysis volumes of the cortical zones. The cortical cell layer not expressing 3?-HSD is the fetal zone (Fig. 4B), whereas the layer expressing 3?-HSD (Fig. 4B) but not P-450C17 (Fig. 4C) is the definitive zone. The transitional zone was identified by overlapping 3?-HSD and P-450C17 immunostaining.
FIG. 4. Photomicrographs of hematoxylin and eosin histology (A) and 3?-HSD (B) and P-450C17 (C) immunocytochemistry (brown precipitate) of a representative fetal adrenal gland of an untreated baboon on d 165 of gestation. The 3?-HSD-negative fetal zone (FZ), 3?-HSD-positive, P-450 C17-negative definitive zone (DZ), and 3?-HSD-positive, P-450 C17-positive transitional zone (TZ) are evident. m, medulla. Magnification bar, 320 μm.
As assessed by image analysis, the volume of the fetal zone in untreated baboons increased from 3.45 ± 0.28 x 10–10 μm3 at midgestation to 6.64 ± 0.69 x 10–10 μm3 at late gestation (Fig. 5A). In baboons in which estrogen production was suppressed by CGS 20267 administration, the volume of the fetal zone (12.55 ± 0.99 x 10–10 μm3) on d 165 was approximately 2-fold greater (P < 0.01) than that of untreated animals in late gestation. Concomitant administration of CGS 20267 and estradiol returned fetal cortical zone volume (8.23 ± 1.37 x 10–10 μm3) to normal. The levels of umbilical artery serum DHAS, which is secreted primarily by the fetal zone of the adrenal, were not significantly different at mid- (14 ± 4 μg/100 ml) and late (25 ± 4 μg/100 ml) gestation (Fig. 5B) but were increased almost 3-fold (P < 0.01) by administration of CGS 20267 (68 ± 13 μg/100 ml) and restored to normal by administration of CGS 20267 and estradiol (37 ± 10 μg/100 ml).
FIG. 5. A, Volume (x 10–10 μm3) of the fetal zone (A) determined by image analysis of 3?-HSD-negative and P-450 C17-positive cells of the fetal adrenal cortex in untreated baboons on d 100 (mid, n = 4) and 165 (late, n = 6) of gestation and on d 165 in animals that were treated with aromatase inhibitor CGS 20267 (n = 10) or CGS 20267 plus estradiol (n = 7) as detailed in the footnote of Table 1. B, Umbilical artery serum DHAS levels of the baboons in which fetal cortical zone volumes are shown in A. Values indicated by different letter superscripts are different at P < 0.05 to P < 0.01 from each other (ANOVA and Newman-Keul’s multiple comparison test).
The fetal adrenal at midgestation exhibited only a narrow rim of 3?-HSD-positive/P-450C17-negative immunoreactivity indicative of the definitive zone. Late in gestation, however, this band of 3?-HSD-positive, P-450C17-negative cells became more prominent, reflecting a marked increase in size of the definitive zone. When analyzed by image analysis, the volume of the definitive zone increased markedly (P < 0.05) between mid- (0.52 ± 0.11 x 10–10 μm3) and late (3.18 ± 0.63 x 10–10 μm3) gestation (Fig. 6A). A narrow band of overlapping 3?-HSD/P-450C17-positive cells, reflecting development of the transitional zone, also was present in late but not midgestation (Fig. 6B). In contrast to the observations on the fetal zone, the volumes of the definitive and transitional zones were not significantly different in untreated (3.18 ± 0.63 and 2.62 ± 0.43 x 10–10 μm3, Fig. 6, A and B, respectively), CGS 20267-treated (3.94 ± 0.61 and 3.11 ± 0.67 x 10–10 μm3, respectively), or CGS 20267 plus estradiol-treated (2.31 ± 0.48 and 1.60 ± 0.25 x 10–10 μm3, respectively) baboons. The levels of umbilical serum cortisol, the primary steroid produced by the transitional zone, were increased (P < 0.01) between mid- (13 ± 2 μg per 100 ml) and late (24 ± 2 μg per 100 ml) gestation (Fig. 6C). However, cortisol levels were not altered by treatment with CGS 20267 (20 ± 2 μg per 100 ml) or CGS 20267 plus estradiol (20 ± 2 μg per 100 ml).
FIG. 6. Volumes of the fetal adrenal definitive (A) and transitional (B) zones and umbilical artery serum cortisol levels (C) of the baboons on which fetal zone volumes are shown in Fig. 5, except for data of the transitional zone in which the number of baboon fetal adrenals analyzed was three (untreated, late), seven (CGS 20267), and five (CGS 20267 plus estradiol).
Fetal adrenal capsule volumes also were similar in value on d 165 of gestation in untreated baboons (0.63 ± 0.12 x 10–10 μm3) or those treated with CGS 20267 (0.81 x 0.14 x 10–10 μm3) or CGS 20267 plus estradiol (0.59 ± 0.10 x 10–10 μm3).
Fetal pituitary POMC mRNA
Fetal pituitary POMC mRNA levels determined by quantitative in situ hybridization increased (P < 0.05) from 2.51 ± 0.28 arbitrary units in untreated baboons at midgestation to 5.74 ± 0.56 arbitrary units in late gestation (Fig. 7A). However, fetal pituitary POMC mRNA levels late in gestation were not significantly altered by the administration of CGS 20267 or CGS 20267 plus estradiol when compared with the untreated controls.
FIG. 7. Fetal pituitary POMC mRNA expression determined by quantitative in situ hybridization in untreated baboons at mid- (n = 5) and late (n = 5) gestation and late in gestation in animals treated with CGS 20267 (n = 6) or CGS 20267 plus estradiol (n = 6).
Discussion
The present study shows that when the formation and thus levels of endogenous estrogen were suppressed by administration of aromatase inhibitor CGS 20267 throughout the second half of baboon pregnancy, the overall size, i.e. volume, of the fetal adrenals was markedly increased. The rise in adrenal growth in estrogen-deprived baboons was due primarily to a striking increase in volume of the fetal cortical zone, whereas definitive and transitional zone volumes were unaltered. The effect of estrogen deprivation on fetal cortical zone development was prevented by simultaneously administering CGS 20267 plus estradiol. A mechanism for the subcellular action of estrogen directly on the primate fetal adrenal cortex exists because we have recently shown expression of the mRNAs and proteins for both the - and ?-isoforms of the estrogen receptor in the baboon fetal adrenal cortex at mid- and late gestation (18). In addition to effects on morphological development, we previously reported that estrogen also altered fetal adrenal function by suppressing the output of fetal zone-specific DHA but not cortisol by the baboon fetal adrenal as assessed by both short-term incubation (19, 20, 21) and in vivo (23) experimental approaches. The 3-fold increase in fetal serum DHAS levels in estrogen-deprived baboons of the current study and restoration of DHAS in CGS 20267-estradiol-treated animals are consistent with the latter observations and indicate that the primary origin of umbilical DHAS was the fetal adrenal. Collectively, based on these studies, we propose that estrogen selectively represses both the morphological and functional development of the fetal zone of the primate fetal adrenal gland in utero during the second half of gestation. The inhibitory effect of estrogen on growth of the fetal zone, however, does not seem to be absolute because there was an increase, albeit nonsignificant, in volume of the latter cortical zone in association with the rise in estrogen during the second half of pregnancy.
Although the physiological relevance of these observations remains to be established, we propose that placental estrogen, which is formed from C19-steroid precursors of fetal cortical zone origin, feeds back on the latter fetal adrenal zone to restrain its growth and development potentially to maintain a physiologically normal level of secretion of precursors such as DHA and consequently placental estrogen production. Normal levels of estrogen are important for optimal fetal and placental development (17); however, abnormally high levels of estrogen and other endocrine disruptors are known to have deleterious effects on fetal maturation (29). Consistent with the suggestion of feedback regulation of estrogen on fetal adrenal steroid precursor secretion, we have reported a significant elevation in endogenous estrogen levels in baboons treated with an estrogen receptor antagonist (30).
The present study further shows that the action of estrogen on fetal adrenal development was cortical zone specific. Thus, in contrast to the marked increase in volume of the fetal cortical zone, neither volumes of the definitive and transitional zones, or output of transitional zone-specific cortisol as assessed by fetal serum cortisol levels, were changed by estrogen suppression. It appears that estrogen acts selectively on the fetal cortical zone, despite the presence of estrogen receptor- and -? in the outer zones of the baboon fetal adrenal cortex (18).
Although estradiol and estrone levels in the baboon mother were fully restored by maternal estradiol/CGS 20267 administration, estradiol and estrone concentrations in the umbilical vein were only partially restored to values not significantly greater than after CGS 20267 treatment alone. Presumably, this resulted from the limited transfer of maternally administered estrogen across the placenta and selective secretion of estrogens into the maternal compartment (28). However, the concentrations of estradiol (152 pg/ml) and estrone (602 pg/ml) achieved in the umbilical vein approximated 10–9 M, a level similar to the dissociation constant for estrogen receptor binding and that exerts marked physiological effects in target tissues, e.g. the uterus during the menstrual cycle. Therefore, considering the latter and the presence of estrogen receptor- and -? in the fetal adrenal (18), we suggest that the effects of estrogen observed in the fetal adrenal in baboons of the present study reflected a direct action of estrogen on fetal adrenocortical development.
The mechanism(s) underlying the cortical zone-specific action of estrogen on the primate fetal adrenal gland is unknown. Although fetal pituitary ACTH is absolutely essential for normal growth and development of the baboon (12, 13), rhesus monkey (8, 10, 31), and human (11) fetal adrenal gland, pituitary POMC levels were not significantly altered by CGS 20267 treatment in baboons of the present study. Therefore, the increase in growth of the fetal zone in estrogen-suppressed baboons does not seem to have resulted from a change in pituitary POMC expression, although plasma ACTH levels need to be measured to determine whether the ACTH drive to the fetal adrenal was altered by estrogen suppression. We previously reported that ACTH receptor mRNA levels decreased within the fetal zone and were elevated in the transitional zone between mid- and late baboon gestation in association with the progressive rise in estrogen (14, 26). It is possible, therefore, that estrogen interferes with the receptor-mediated action of ACTH in the fetal zone without compromising ACTH action on the transitional zone that underwent degeneration after fetal ACTH secretion was suppressed in baboons by betamethasone administration (12, 13). Consistent with this suggestion, we previously showed that estrogen suppressed ACTH-stimulated but not basal DHA formation in incubates of baboon fetal adrenal cells (22).
The significant increase in size of the fetal cortical zone in estrogen-deprived baboons may have resulted from enhanced cell mitosis, i.e. hyperplasia; increased cell size, i.e. hypertrophy; or both. An increase in mitotic index within the human fetal zone was observed between wk 10–14 and 15–19 of gestation, although number of apoptotic nuclei in the fetal zone also increased during the second half of pregnancy (32). Estrogen has a well-established role in regulating cell proliferation in other tissues, including the uterus and breast (33, 34, 35). However, estrogen typically has a stimulatory role in promoting cellular proliferation in the latter tissues, e.g. by enhancing expression of cell cycle-specific cyclins and their kinases (35, 36, 37), whereas estrogen suppressed the growth of the fetal cortical zone. Further investigation is needed to determine whether this involves changes in cell division, cell size, or expression of specific cyclins and/or kinases.
In apparent contrast to the inhibitory effect of estrogen on fetal adrenal development and function as assessed in baboons by our laboratories (21, 22, 23) and the present study, estrogen increased ACTH-induced DHAS and cortisol synthesis in long-term cultures of human fetal adrenal cells (38, 39, 40). Because human fetal adrenal cells rapidly differentiate in culture (41), however, the apparently disparate effects of estrogen observed on androgen production as studied in human fetal adrenal long-term cultures and baboon fetal adrenal short-term incubates or in vivo may reflect an important difference in the qualitative nature of the fetal adrenal cells as studied under these two experimental conditions.
In addition to the direct action of estrogen that we propose exists on fetal adrenal cortical maturation, estrogen also exerts an indirect action on fetal adrenal development by regulating the expression and subcellular localization of 11?-hydroxysteroid dehydrogenase 1 and 2 enzymes within the syncytiotrophoblast (2, 42). This estrogen-dependent event catalyzes the switch in transplacental conversion of bioactive cortisol and bioinactive cortisone between mid- and late gestation that causes the release of fetal pituitary ACTH, the latter of which regulates the maturation and de novo production of cortisol by the transitional zone. Consequently, we suggest that a complex interaction between ACTH and estrogen occurs in utero to regulate directly and indirectly, in a cortical zone-specific manner, growth and functional maturation of the primate fetal adrenal gland.
In summary, the present study shows that in baboons in which estrogen production and levels were suppressed by administration of an aromatase inhibitor, the volume of the fetal zone of the fetal adrenal increased, whereas definitive and transitional zone volumes were unchanged, effects that were reversed by concomitant administration of CGS 20267 and estradiol. It is concluded that estrogen selectively represses growth and development of the fetal zone of the fetal adrenal cortex during the second half of primate pregnancy.
Acknowledgments
The authors greatly appreciated the secretarial assistance of Mrs. Wanda H. James with the manuscript; the technical assistance of Ms. Donna Suresch with the immunocytochemistry; and William Davies, Ph.D., with the POMC mRNA in situ hybridization. The authors gratefully acknowledge Novartis Pharma AG for providing CGS 20267 (Letrozole) for the conduct of this study.
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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. E-mail: ealbrech@umaryland.edu.
Abstract
In the present study, we determined whether endogenous estrogen, the levels of which increase with advancing pregnancy, regulates growth and development of the baboon fetal adrenal cortex. Fetal adrenal glands were obtained at mid- (d 100) and late (d 165, term is 184 d) gestation from untreated baboons and on d 165 from animals in which endogenous estrogen production was suppressed by administration of aromatase inhibitor CGS 20267 between d 100 and 165. Volumes of the respective cortical zones were determined by zone-specific immunocytochemical staining of steroidogenic enzymes and image analysis. Fetal adrenal weight and volume increased (P < 0.01) 3-fold between mid- and late gestation and an additional 70% (P < 0.01) by administration of CGS 20267, which decreased (P < 0.001) fetal serum estradiol levels by more than 95%. Mean ± SE volume (x10–10 μm3) of the fetal cortical zone increased from 3.45 ± 0.28 at midgestation to 6.64 ± 0.69 at late gestation in untreated baboons and to 12.55 ± 0.99 (P < 0.01) in baboons in which estrogen production was suppressed by CGS 20267 administration. The levels of umbilical artery serum dehydroepiandrosterone sulfate, which is secreted primarily by the fetal zone, were increased almost 3-fold (P < 0.01) by administration of CGS 20267. Concomitant administration of CGS 20267 and estradiol returned fetal cortical zone volume and serum dehydroepiandrosterone sulfate levels to normal. In contrast to the effect of estrogen deprivation on the fetal zone, the volumes of the definitive and transitional zones in untreated baboons late in gestation (3.18 ± 0.63 and 2.62 ± 0.43, respectively) and levels of fetal serum cortisol, a steroid secreted from the transitional zone, were not altered by estrogen suppression. The changes in fetal zone growth were not associated with alterations in fetal pituitary proopiomelanocortin mRNA levels. We propose that estrogen acts directly on the fetal adrenal cortex to selectively repress the morphological and functional development of the fetal zone, potentially as a feedback system to maintain physiological secretion of estrogen precursors and thus placental estrogen production to promote normal primate fetal and placental development.
Introduction
THROUGHOUT THE COURSE of human and nonhuman primate pregnancy, the fetal adrenal cortex is comprised primarily of the fetal zone, which expresses the P-450 17-hydroxylase, 17–20 lyase (P-450C17) enzyme catalyzing synthesis of C19-steroids, e.g. dehydroepiandrosterone (DHA) and DHA sulfate (DHAS), used as precursors for estrogen synthesis by the placenta (for reviews, see Refs. 1, 2, 3). The definitive zone, the site of the 5-3?-hydroxysteroid dehydrogenase (3?-HSD) and synthase enzymes that produce aldosterone, is also present at mid- and late gestation. However, the transitional zone, which expresses both the 3?-HSD and P-450C17 enzymes (4), which catalyze the production of cortisol, develops only relatively late in gestation. In addition to functional maturation, the human fetal adrenal cortex, particularly the fetal zone, grows rapidly during the second and third trimesters (5, 6, 7); however, regulation of growth and functional development of the individual fetal adrenal cortical zones has not been clearly established.
ACTH has a pivotal role in the growth and functional maturation of the human and nonhuman primate fetal adrenal gland (8, 9, 10, 11, 12, 13). However, because each of the fetal adrenal cortical zones expresses the ACTH receptor (12, 14) and presumably is exposed to comparable concentrations of ACTH with advancing gestation (15, 16), we have proposed that regulation of the differential zone-specific maturation of the adrenal cortex is multifactorial and involves placental estrogen as well as ACTH (17). Estrogen receptors- and -? are expressed in all zones of the baboon fetal adrenal cortex (18), providing a mechanism for mediating the action of estrogen on fetal adrenal development. Moreover, we have shown that the amount of fetal zone-specific DHA formed per cell in baboon fetal adrenal incubates declined between mid- and late gestation in association with the rise in estrogen (19, 20). Also, at midgestation estrogen suppressed responsivity of the baboon fetal adrenal to ACTH with respect to the formation of DHA but not cortisol in vitro (21, 22) and in vivo (23). Therefore, we have postulated that estrogen feeds back to regulate function of the fetal cortical zone with respect to the production of DHA.
However, the potential role that the increasing levels of endogenous estrogen have on development and growth of the fetal adrenal cortical zones in vivo in the primate has not been investigated. Therefore, we employed the baboon as a nonhuman primate model in which placental estrogen production was suppressed by administration of a highly specific aromatase inhibitor during pregnancy to determine whether zone-specific development of the baboon fetal adrenal cortex is regulated by estrogen.
Materials and Methods
Animals
Female baboons (Papio anubis) weighing 12–16 kg were housed in aluminum stainless-steel large primate cages in air-conditioned rooms under a 12-h light, 12-h dark cycle. Baboons received high-protein monkey chow and fresh fruit twice daily, vitamins daily, and water ad libitum. Females were paired with male baboons for a period of 5 d during the ovulatory phase of the menstrual cycle, and pregnancy was determined by palpation. Animals were cared for and used strictly in accordance with U.S. Department of Agriculture regulations and the Guide for the Care and Use of Laboratory Animals (National Research Council, National Academy Press, 1996). The experimental protocol employed in the present study was approved by the Institutional Animal Care and Use Committees of the University of Maryland School of Medicine and Eastern Virginia Medical School.
Ten baboons received the aromatase inhibitor CGS 20267 [Letrozole; 4, 4'-(1,2,4-triazol-1-yl-methylene)-bis-benzonitrite, Norvartis Pharma AG, Basel, Switzerland] administered sc to the mother (increasing from 0.005 mg/kg body weight per day to a maximum of 0.115 mg/kg body weight per day in 1 ml sesame oil) on d 100–165 of gestation (term = 184 d). Seven additional baboons were injected sc daily on d 100–165 with CGS 20267 plus estradiol benzoate at doses (maximum of 0.115 mg/kg body weight per day each) designed to replicate the normal pattern of maternal serum estradiol. Ten baboons served as untreated controls and were studied at mid- (d 100, n = 4) or late (d 165, n = 6) gestation. Blood samples (4 ml) were taken at 1- to 3-d intervals during the study period from a maternal saphenous vein after brief sedation of baboons with ketamine HCl (10 mg/kg body weight, im) for analysis of serum estradiol levels.
On d 100 or d 165 of gestation, baboons underwent cesarean section under halothane-nitrous oxide anesthesia, and blood samples (3–4 ml) were obtained from a maternal saphenous and umbilical vein and artery and serum stored at –20 C until assayed for estradiol (24), estrone, DHAS (13), and cortisol (25) by immunoassay (Immulite, Diagnostic Products Corp., Los Angeles, CA) as described previously. The fetuses were immediately killed with an overdose of sodium pentobarbital and the adrenals and pituitary removed and weighed, embedded in OCT (Miles Scientific, Elkhart, IN), and snap frozen in isopentane precooled in liquid nitrogen for subsequent immunocytochemical localization of 3?-HSD and P-450C17, volumetric analysis, and proopiomelanocortin (POMC) mRNA analysis by in situ hybridization, respectively.
Fetal adrenal 3?-HSD and P-450C17 immunocytochemistry
Frozen serial sections (10 μm) were cut with a cryostat through the entire adrenal gland and mounted onto Superfrost/Plus microscope slides (Fisher Scientific, Pittsburgh, PA). Every 10th and 11th section were used for immunocytochemical staining using rabbit polyclonal antibodies to human 3?-HSD (generously supplied by Dr. Ian Mason, University of Edinburgh, Scotland, UK) and human P450C17 (generously supplied by Dr. Michael Waterman, Vanderbilt University School of Medicine, Nashville, TN), respectively.
Tissues were fixed in either 70% ethanol for 1 min (3?-HSD) or 4% buffered paraformaldehyde for 10 min (P450C17). Slides were then washed thoroughly in PBS and incubated in 1% hydrogen peroxide in PBS for 10 min to remove endogenous peroxidase. After washing in PBS, tissue was blocked using reagents in the avidin/biotin blocking kit (Vector Laboratories, Burlingame, CA) diluted in 5% normal goat serum for 1 h to inhibit nonspecific binding.
Sections were incubated 24–48 h at 4 C with either 3?-HSD (1:7500) or P450C17 (1:2000) antibody using PBS/5% normal goat serum diluent, washed in PBS, and incubated for 1 h in biotinylated goat antirabbit IgG (1:600, Vector Laboratories). After repeated washes in PBS (3 x 5 min), slides were incubated for 1 h with avidin/biotin complex reagent kit (Vector Laboratories).
Sections were then colorized for 15 min using diaminobenzidine-HCl (Sigma-Aldrich, St. Louis, MO) in 50 mM Tris-HCl buffer as a chromagen. Slides were washed in 50 mM Tris-HCl buffer and PBS and rinsed thoroughly in distilled water. Tissue sections were lightly counterstained with Harris’ hematoxylin (Fisher Scientific), dehydrated in several changes of graded ethanol, cleared in two to three changes of xylene, and mounted in Permount mounting media (Fisher Scientific).
Volumetric analysis of fetal adrenal cortical zones
Fetal adrenal cortical zone volumes were determined using an Eclipse E1000/video-based Image 1 analysis system (Nikon, New York, NY) with analytical Scion Image software (Scanalytics, Inc., Fairfax, VA). Using 3?-HSD and P-450C17 immunocytochemistry to demarcate the cortical zones, volumes of the 3?-HSD-negative fetal zone, 3?-HSD-positive and P-450C17-negative definitive zone, and 3?-HSD-positive and P-450C17-positive transitional zone were quantified and integrated for the entire gland using adrenal cortical zone areas and section thickness.
In situ hybridization of fetal pituitary POMC mRNA
The methods for detection of POMC mRNA expression in the baboon fetal pituitary by in situ hybridization (16) were modified using procedures recently developed in our laboratories (16, 26) and a 717-bp riboprobe to the rat POMC gene (27) generously provided by Dr. James L. Roberts (University of Texas Medical Center, San Antonio, TX). Briefly, frozen fetal pituitary sections (10 μm) were treated (15 min; 37 C) with 5 μg/ml proteinase K (Sigma-Aldrich), incubated (10 min) with 10% buffered formalin, rinsed in PBS, and treated with 0.25 M acetic anhydride in 0.1 M triethanolamine hydrochloride-0.9% NaC1. After dehydration in ethanol, delipidation in chloroform, and rehydration, sections were hybridized overnight at 55 C with 33P-labeled antisense POMC riboprobe (106 dpm/slide) in 50 μl hybridization buffer consisting of 10 ml formamide, 1 ml 1 M Tris (pH 8.0), 0.1 ml 0.5 M EDTA, 500 μl yeast tRNA (Sigma-Aldrich), 500 μl 50x Denhardt’s solution (Sigma-Aldrich), 20 μl polyadenylic acid (0.1 μg/μl), 20 μl salmon sperm DNA (500 μg/ml; Life Technologies, Inc., Grand Island, NY), 2 g dextran sulfate, 0.8 ml 5 M NaCl, and diethylpyrocarbonate-H2O. After incubation, sections were washed repeatedly in 4x saline sodium citrate (SSC) [1x SSC = 0.15 M NaCl and 0.015 sodium citrate buffer (pH 7.2)]; serially dehydrated; and washed in 50% formamide, 300 mM NaCl, 25 mM Tris, and 1 mM EDTA. After a 10-min rinse in 2x SSC, nonspecific hybridized probe was removed by incubation (10 min, 37 C) with RNase A (25 μg/ml, Promega Corp., Madison, WI) and RNase T1 (2000 U, Promega) in RNase buffer consisting of 500 mM NaCl, 10 mM Tris, and 1 mM EDTA (pH 8.0).
Subsequently tissues were washed twice in RNase buffer at 50 C for 15 min, four times in 2x SSC for 15 min at 50 C, and once in 0.2x SSC at 60 C for 2 h before serial dehydration and vacuum drying. Slides were placed against X-OMAT film (Kodak, Rochester, NY) in x-ray film holders for 48 h and areas of POMC mRNA expression circumscribed and quantified by densitometric analysis using an LKB Bromma Ultroscan XL enhanced laser densitometer (Pharmacia LKB, Piscataway, NY) as described previously (16). For each animal, six to 10 sections of fetal pituitary were analyzed. Applicability of the probe for use on baboon tissue was confirmed by binding to baboon pituitary RNA as determined by Northern blot and specificity verified by absence of expression over the posterior pituitary (data not shown). To ensure that densitometric analysis of film was not compromised by tissue folding, pituitary sections were also examined by light microscopy.
Statistical analysis
Fetal adrenal and pituitary parameters and hormone levels were analyzed using one-way ANOVA and Newman-Keuls multiple comparison test to assess significant differences between individual group means (Instat, GraphPad Software, San Diego, CA).
Results
Serum estradiol and estrone levels
Serum estradiol concentrations (mean ± SE) in the maternal saphenous (1514 ± 321 pg/ml) and umbilical (331 ± 57 pg/ml, Fig. 1) veins on d 100 of gestation (i.e. midgestation) increased (P < 0.01) to 3493 ± 467 and 590 ± 72 pg/ml (Fig. 1), respectively, on d 165 (i.e. late) of gestation. The administration of CGS 20267 to baboons decreased serum estradiol levels in the maternal saphenous (96 ± 12 pg/ml) and umbilical (47 ± 4 pg/ml) veins on d 165 of gestation to values that were approximately 3 (P < 0.001) and 8% (P < 0.001), respectively, of the values observed in the untreated controls (Fig. 1). Concomitant administration of CGS 20267 and estradiol restored maternal serum estradiol levels (4796 ± 614 pg/ml) to normal and increased estradiol concentrations in the umbilical vein to a value (152 ± 24 pg/ml) similar to that observed after CGS 20267 alone but lower than in untreated baboon fetuses in late gestation (Fig. 1).
FIG. 1. Mean (±SE) maternal saphenous and umbilical vein serum estradiol levels on d 100 (mid, n = 4) and d 165 (late, n = 6) of gestation in baboons that were untreated and on d 165 in animals treated with antiestrogen CGS 20267 (maximum of 0.115 mg/kg body weight per day sc, n = 10) or CGS 20267 plus estradiol benzoate (each at maximum of 0.115 mg/kg body weight per day sc, n = 7) daily on d 100–165 of gestation (term = 184 d). Values indicated by different letter superscripts are significantly different at P < 0.01, ANOVA and Newman-Keul’s multiple comparison test.
Serum estrone levels in the maternal saphenous vein were 596 ± 96 pg/ml at midgestation and 1252 ± 190 pg/ml at late gestation (Fig. 2). The administration of CGS 20267 decreased (P < 0.001) estrone to a value (85 ± 20 pg/ml) that was approximately 7% of that in untreated baboons near term. After simultaneous CGS 20267 administration, maternal estrone levels (924 ± 241) were restored to normal. In the umbilical vein, serum estrone levels appeared higher than estradiol (Fig. 1), consistent with the somewhat greater secretion of placental estrone into the fetal compartment during primate pregnancy (28). Umbilical vein serum estrone levels increased (P < 0.01) between mid- (1111 ± 152 pg/ml) and late (2049 ± 329 pg/ml) gestation and also were decreased (P < 0.001) by CGS 20267 treatment (306 ± 48 pg/ml). Concomitant administration of CGS 20267 and estradiol increased umbilical vein estrone to a level (602 ± 112 pg/ml) that was 2-fold, but not significantly, greater than in baboons that received CGS 20267 alone.
FIG. 2. Maternal saphenous and umbilical vein serum estrone concentrations for the baboons in which serum estradiol levels are shown in Fig. 1. Values indicated by different letter superscripts are significantly different at P < 0.01, ANOVA and Newman-Keul’s multiple comparison test.
Fetal adrenal weight and volumes
Mean (±SE) fetal adrenal weight in untreated baboons increased (P < 0.001) approximately 3-fold from 127 ± 5 mg on d 100 to 352 ± 14 mg on d 165 (Fig. 3A). Fetal adrenal weight in CGS 20267-treated animals on d 165 (475 ± 28 mg) was approximately 35% greater (P < 0.001) than in the untreated controls. Whole adrenal gland volume assessed by image analysis also increased (P < 0.01) approximately 3-fold between mid- (4.29 ± 0.42 x 10–10 μm3) and late (11.97 ± 1.20 x 10–10 μm3) gestation (Fig. 3B) and an additional 70% (P < 0.01) by administration of CGS 20267 (20.28 ± 1.58 x 10–10 μm3) when compared with untreated animals in late gestation. In baboons that received CGS 20267 plus estradiol, fetal adrenal weight (342 ± 30 mg) and volume (13.37 ± 1.72 x 10–10 μm3) were similar to respective values in the untreated controls. Although fetal body and organ weights increased (P < 0.05) between mid- and late gestation (Table 1), respective values at late gestation were similar in untreated and CGS 20267 ± estradiol-treated baboons.
FIG. 3. Fetal adrenal weight (two glands, A) and volume assessed by image analysis (B) in untreated baboons on d 100 (mid, n = 4) and 165 (late, n = 6) of gestation and on d 165 in animals that received CGS 20267 (maximum of 0.115 mg/kg body weight per day, n = 10) or CGS 20267 and estradiol (maximum of 0.115 mg/kg body weight per day each, n = 7) daily on d 100–165 of gestation. Values indicated by different letters are significantly different at P < 0.001 (A) and P < 0.01 (B), ANOVA and Newman-Keul’s multiple comparison test.
TABLE 1. Fetal body and organ weights in baboons untreated or treated with CGS 20267 with or without estradiol1
Figure 4 illustrates in a representative fetal adrenal gland of an untreated baboon at late gestation, hematoxylin and eosin histology (Fig. 4A) and 3?-HSD (Fig. 4B) and P-450C17 (Fig. 4C) immunocytochemistry, which was used to mark and thus determine by image analysis volumes of the cortical zones. The cortical cell layer not expressing 3?-HSD is the fetal zone (Fig. 4B), whereas the layer expressing 3?-HSD (Fig. 4B) but not P-450C17 (Fig. 4C) is the definitive zone. The transitional zone was identified by overlapping 3?-HSD and P-450C17 immunostaining.
FIG. 4. Photomicrographs of hematoxylin and eosin histology (A) and 3?-HSD (B) and P-450C17 (C) immunocytochemistry (brown precipitate) of a representative fetal adrenal gland of an untreated baboon on d 165 of gestation. The 3?-HSD-negative fetal zone (FZ), 3?-HSD-positive, P-450 C17-negative definitive zone (DZ), and 3?-HSD-positive, P-450 C17-positive transitional zone (TZ) are evident. m, medulla. Magnification bar, 320 μm.
As assessed by image analysis, the volume of the fetal zone in untreated baboons increased from 3.45 ± 0.28 x 10–10 μm3 at midgestation to 6.64 ± 0.69 x 10–10 μm3 at late gestation (Fig. 5A). In baboons in which estrogen production was suppressed by CGS 20267 administration, the volume of the fetal zone (12.55 ± 0.99 x 10–10 μm3) on d 165 was approximately 2-fold greater (P < 0.01) than that of untreated animals in late gestation. Concomitant administration of CGS 20267 and estradiol returned fetal cortical zone volume (8.23 ± 1.37 x 10–10 μm3) to normal. The levels of umbilical artery serum DHAS, which is secreted primarily by the fetal zone of the adrenal, were not significantly different at mid- (14 ± 4 μg/100 ml) and late (25 ± 4 μg/100 ml) gestation (Fig. 5B) but were increased almost 3-fold (P < 0.01) by administration of CGS 20267 (68 ± 13 μg/100 ml) and restored to normal by administration of CGS 20267 and estradiol (37 ± 10 μg/100 ml).
FIG. 5. A, Volume (x 10–10 μm3) of the fetal zone (A) determined by image analysis of 3?-HSD-negative and P-450 C17-positive cells of the fetal adrenal cortex in untreated baboons on d 100 (mid, n = 4) and 165 (late, n = 6) of gestation and on d 165 in animals that were treated with aromatase inhibitor CGS 20267 (n = 10) or CGS 20267 plus estradiol (n = 7) as detailed in the footnote of Table 1. B, Umbilical artery serum DHAS levels of the baboons in which fetal cortical zone volumes are shown in A. Values indicated by different letter superscripts are different at P < 0.05 to P < 0.01 from each other (ANOVA and Newman-Keul’s multiple comparison test).
The fetal adrenal at midgestation exhibited only a narrow rim of 3?-HSD-positive/P-450C17-negative immunoreactivity indicative of the definitive zone. Late in gestation, however, this band of 3?-HSD-positive, P-450C17-negative cells became more prominent, reflecting a marked increase in size of the definitive zone. When analyzed by image analysis, the volume of the definitive zone increased markedly (P < 0.05) between mid- (0.52 ± 0.11 x 10–10 μm3) and late (3.18 ± 0.63 x 10–10 μm3) gestation (Fig. 6A). A narrow band of overlapping 3?-HSD/P-450C17-positive cells, reflecting development of the transitional zone, also was present in late but not midgestation (Fig. 6B). In contrast to the observations on the fetal zone, the volumes of the definitive and transitional zones were not significantly different in untreated (3.18 ± 0.63 and 2.62 ± 0.43 x 10–10 μm3, Fig. 6, A and B, respectively), CGS 20267-treated (3.94 ± 0.61 and 3.11 ± 0.67 x 10–10 μm3, respectively), or CGS 20267 plus estradiol-treated (2.31 ± 0.48 and 1.60 ± 0.25 x 10–10 μm3, respectively) baboons. The levels of umbilical serum cortisol, the primary steroid produced by the transitional zone, were increased (P < 0.01) between mid- (13 ± 2 μg per 100 ml) and late (24 ± 2 μg per 100 ml) gestation (Fig. 6C). However, cortisol levels were not altered by treatment with CGS 20267 (20 ± 2 μg per 100 ml) or CGS 20267 plus estradiol (20 ± 2 μg per 100 ml).
FIG. 6. Volumes of the fetal adrenal definitive (A) and transitional (B) zones and umbilical artery serum cortisol levels (C) of the baboons on which fetal zone volumes are shown in Fig. 5, except for data of the transitional zone in which the number of baboon fetal adrenals analyzed was three (untreated, late), seven (CGS 20267), and five (CGS 20267 plus estradiol).
Fetal adrenal capsule volumes also were similar in value on d 165 of gestation in untreated baboons (0.63 ± 0.12 x 10–10 μm3) or those treated with CGS 20267 (0.81 x 0.14 x 10–10 μm3) or CGS 20267 plus estradiol (0.59 ± 0.10 x 10–10 μm3).
Fetal pituitary POMC mRNA
Fetal pituitary POMC mRNA levels determined by quantitative in situ hybridization increased (P < 0.05) from 2.51 ± 0.28 arbitrary units in untreated baboons at midgestation to 5.74 ± 0.56 arbitrary units in late gestation (Fig. 7A). However, fetal pituitary POMC mRNA levels late in gestation were not significantly altered by the administration of CGS 20267 or CGS 20267 plus estradiol when compared with the untreated controls.
FIG. 7. Fetal pituitary POMC mRNA expression determined by quantitative in situ hybridization in untreated baboons at mid- (n = 5) and late (n = 5) gestation and late in gestation in animals treated with CGS 20267 (n = 6) or CGS 20267 plus estradiol (n = 6).
Discussion
The present study shows that when the formation and thus levels of endogenous estrogen were suppressed by administration of aromatase inhibitor CGS 20267 throughout the second half of baboon pregnancy, the overall size, i.e. volume, of the fetal adrenals was markedly increased. The rise in adrenal growth in estrogen-deprived baboons was due primarily to a striking increase in volume of the fetal cortical zone, whereas definitive and transitional zone volumes were unaltered. The effect of estrogen deprivation on fetal cortical zone development was prevented by simultaneously administering CGS 20267 plus estradiol. A mechanism for the subcellular action of estrogen directly on the primate fetal adrenal cortex exists because we have recently shown expression of the mRNAs and proteins for both the - and ?-isoforms of the estrogen receptor in the baboon fetal adrenal cortex at mid- and late gestation (18). In addition to effects on morphological development, we previously reported that estrogen also altered fetal adrenal function by suppressing the output of fetal zone-specific DHA but not cortisol by the baboon fetal adrenal as assessed by both short-term incubation (19, 20, 21) and in vivo (23) experimental approaches. The 3-fold increase in fetal serum DHAS levels in estrogen-deprived baboons of the current study and restoration of DHAS in CGS 20267-estradiol-treated animals are consistent with the latter observations and indicate that the primary origin of umbilical DHAS was the fetal adrenal. Collectively, based on these studies, we propose that estrogen selectively represses both the morphological and functional development of the fetal zone of the primate fetal adrenal gland in utero during the second half of gestation. The inhibitory effect of estrogen on growth of the fetal zone, however, does not seem to be absolute because there was an increase, albeit nonsignificant, in volume of the latter cortical zone in association with the rise in estrogen during the second half of pregnancy.
Although the physiological relevance of these observations remains to be established, we propose that placental estrogen, which is formed from C19-steroid precursors of fetal cortical zone origin, feeds back on the latter fetal adrenal zone to restrain its growth and development potentially to maintain a physiologically normal level of secretion of precursors such as DHA and consequently placental estrogen production. Normal levels of estrogen are important for optimal fetal and placental development (17); however, abnormally high levels of estrogen and other endocrine disruptors are known to have deleterious effects on fetal maturation (29). Consistent with the suggestion of feedback regulation of estrogen on fetal adrenal steroid precursor secretion, we have reported a significant elevation in endogenous estrogen levels in baboons treated with an estrogen receptor antagonist (30).
The present study further shows that the action of estrogen on fetal adrenal development was cortical zone specific. Thus, in contrast to the marked increase in volume of the fetal cortical zone, neither volumes of the definitive and transitional zones, or output of transitional zone-specific cortisol as assessed by fetal serum cortisol levels, were changed by estrogen suppression. It appears that estrogen acts selectively on the fetal cortical zone, despite the presence of estrogen receptor- and -? in the outer zones of the baboon fetal adrenal cortex (18).
Although estradiol and estrone levels in the baboon mother were fully restored by maternal estradiol/CGS 20267 administration, estradiol and estrone concentrations in the umbilical vein were only partially restored to values not significantly greater than after CGS 20267 treatment alone. Presumably, this resulted from the limited transfer of maternally administered estrogen across the placenta and selective secretion of estrogens into the maternal compartment (28). However, the concentrations of estradiol (152 pg/ml) and estrone (602 pg/ml) achieved in the umbilical vein approximated 10–9 M, a level similar to the dissociation constant for estrogen receptor binding and that exerts marked physiological effects in target tissues, e.g. the uterus during the menstrual cycle. Therefore, considering the latter and the presence of estrogen receptor- and -? in the fetal adrenal (18), we suggest that the effects of estrogen observed in the fetal adrenal in baboons of the present study reflected a direct action of estrogen on fetal adrenocortical development.
The mechanism(s) underlying the cortical zone-specific action of estrogen on the primate fetal adrenal gland is unknown. Although fetal pituitary ACTH is absolutely essential for normal growth and development of the baboon (12, 13), rhesus monkey (8, 10, 31), and human (11) fetal adrenal gland, pituitary POMC levels were not significantly altered by CGS 20267 treatment in baboons of the present study. Therefore, the increase in growth of the fetal zone in estrogen-suppressed baboons does not seem to have resulted from a change in pituitary POMC expression, although plasma ACTH levels need to be measured to determine whether the ACTH drive to the fetal adrenal was altered by estrogen suppression. We previously reported that ACTH receptor mRNA levels decreased within the fetal zone and were elevated in the transitional zone between mid- and late baboon gestation in association with the progressive rise in estrogen (14, 26). It is possible, therefore, that estrogen interferes with the receptor-mediated action of ACTH in the fetal zone without compromising ACTH action on the transitional zone that underwent degeneration after fetal ACTH secretion was suppressed in baboons by betamethasone administration (12, 13). Consistent with this suggestion, we previously showed that estrogen suppressed ACTH-stimulated but not basal DHA formation in incubates of baboon fetal adrenal cells (22).
The significant increase in size of the fetal cortical zone in estrogen-deprived baboons may have resulted from enhanced cell mitosis, i.e. hyperplasia; increased cell size, i.e. hypertrophy; or both. An increase in mitotic index within the human fetal zone was observed between wk 10–14 and 15–19 of gestation, although number of apoptotic nuclei in the fetal zone also increased during the second half of pregnancy (32). Estrogen has a well-established role in regulating cell proliferation in other tissues, including the uterus and breast (33, 34, 35). However, estrogen typically has a stimulatory role in promoting cellular proliferation in the latter tissues, e.g. by enhancing expression of cell cycle-specific cyclins and their kinases (35, 36, 37), whereas estrogen suppressed the growth of the fetal cortical zone. Further investigation is needed to determine whether this involves changes in cell division, cell size, or expression of specific cyclins and/or kinases.
In apparent contrast to the inhibitory effect of estrogen on fetal adrenal development and function as assessed in baboons by our laboratories (21, 22, 23) and the present study, estrogen increased ACTH-induced DHAS and cortisol synthesis in long-term cultures of human fetal adrenal cells (38, 39, 40). Because human fetal adrenal cells rapidly differentiate in culture (41), however, the apparently disparate effects of estrogen observed on androgen production as studied in human fetal adrenal long-term cultures and baboon fetal adrenal short-term incubates or in vivo may reflect an important difference in the qualitative nature of the fetal adrenal cells as studied under these two experimental conditions.
In addition to the direct action of estrogen that we propose exists on fetal adrenal cortical maturation, estrogen also exerts an indirect action on fetal adrenal development by regulating the expression and subcellular localization of 11?-hydroxysteroid dehydrogenase 1 and 2 enzymes within the syncytiotrophoblast (2, 42). This estrogen-dependent event catalyzes the switch in transplacental conversion of bioactive cortisol and bioinactive cortisone between mid- and late gestation that causes the release of fetal pituitary ACTH, the latter of which regulates the maturation and de novo production of cortisol by the transitional zone. Consequently, we suggest that a complex interaction between ACTH and estrogen occurs in utero to regulate directly and indirectly, in a cortical zone-specific manner, growth and functional maturation of the primate fetal adrenal gland.
In summary, the present study shows that in baboons in which estrogen production and levels were suppressed by administration of an aromatase inhibitor, the volume of the fetal zone of the fetal adrenal increased, whereas definitive and transitional zone volumes were unchanged, effects that were reversed by concomitant administration of CGS 20267 and estradiol. It is concluded that estrogen selectively represses growth and development of the fetal zone of the fetal adrenal cortex during the second half of primate pregnancy.
Acknowledgments
The authors greatly appreciated the secretarial assistance of Mrs. Wanda H. James with the manuscript; the technical assistance of Ms. Donna Suresch with the immunocytochemistry; and William Davies, Ph.D., with the POMC mRNA in situ hybridization. The authors gratefully acknowledge Novartis Pharma AG for providing CGS 20267 (Letrozole) for the conduct of this study.
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