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Endocrinology Vol. 140, No. 12 5953-5961
Copyright © 1999 by The Endocrine Society

ARTICLES

Identification and Developmental Expression of the Estrogen Receptor {alpha} and ß in the Baboon Fetal Adrenal Gland1

Eugene D. Albrecht, Jeffery S. Babischkin, William A. Davies, Maria G. Leavitt and Gerald J. Pepe

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

Address all correspondence and requests for reprints to: Eugene D. Albrecht, Ph.D., Department of Obstetrics, Gynecology, and Reproductive Sciences, University of Maryland School of Medicine, Bressler Research Laboratories 11–019, 655 West Baltimore Street, Baltimore, Maryland 21201. E-mail: ealbrech{at}umaryland.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We have previously shown that estrogen regulates the development and function of the fetal and definitive/transitional zones of the primate fetal adrenal gland. Thus, during baboon pregnancy estrogen acts directly on the fetal zone to suppress ACTH-stimulated dehydroepiandrosterone (DHA) formation, potentially to modulate C19-steroid production and consequently placental estrogen synthesis. It is proposed that this action of estrogen is mediated by the estrogen receptor. Therefore, in the present study a developmental approach was used to determine whether the messenger RNA (mRNA) and protein for the estrogen receptor were expressed in the fetal and definitive/transitional zones of the baboon fetal adrenal gland at mid (day 100) and late (day 170) gestation (term = 184 days). Estrogen receptor {alpha} mRNA levels, determined by competitive RT-PCR, were approximately 7-fold greater (P < 0.02) in the fetal adrenal of late (187.8 ± 40.3 attomoles/µg RNA) compared with mid (27.4 ± 5.4 attomoles/µg RNA) gestation. Moreover, estrogen receptor {alpha} mRNA expression, determined by quantitative in situ hybridization, was approximately 2.5-fold greater (P < 0.05) in the definitive/transitional zones (21.6 ± 0.5 silver grains/0.025 mm2) than in the fetal zone (8.3 ± 1.5 grains/0.025 mm2) late in gestation. The mRNA for the ß-isoform of the estrogen receptor was also expressed in the baboon fetal adrenal cortex. There was a gradient of immunocytochemical staining for the estrogen receptor {alpha} and ß proteins, with extensive immunoreactivity for both isoforms in the definitive zone and lower staining in the transitional zone and the fetal zone. In summary, the results of the present study show that estrogen receptor {alpha} and ß were expressed in the fetal and definitive/transitional zones of the baboon fetal adrenal cortex at mid and late gestation. The presence of the estrogen receptor provides a mechanism for mediating the action of estrogen in modulating ACTH-dependent and cortical zone-specific development and function of the primate fetal adrenal gland.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THROUGHOUT gestation the primate fetal adrenal cortex is comprised primarily of an inner fetal zone, which is a major source of the C19 steroids, e.g. dehydroepiandrosterone (DHA) and DHA sulfate (DHAS), that are precursors for placental estrogen production (reviewed in Refs. 1, 2). The outer definitive and transitional cortical zones develop relatively late in gestation and express the {Delta}5-3ß-hydroxy-steroid dehydrogenase (3ßHSD) enzyme needed for the formation of aldosterone and cortisol, respectively, hormones required for neonatal maturation and homeostasis. The regulation of the maturation and function of this important endocrine gland during fetal and neonatal stages of development, however, is incompletely understood.

We have demonstrated, as illustrated in Fig. 1Go, that estrogen has two different actions on fetal adrenal development and function in the primate. Estrogen has an indirect action on fetal adrenal development in the second half of baboon pregnancy by regulating the placental 11ßHSD-1 and -2 enzyme system and thus the increase in transplacental oxidation of cortisol to cortisone that results in fetal pituitary ACTH release and maturation of the fetal adrenal transitional zone for cortisol synthesis (reviewed in Refs. 1, 3, 4). In contrast, estrogen elicits a direct action on the baboon fetal adrenal by inhibiting the responsivity of the fetal zone to ACTH with respect to the production of DHA (Fig. 1Go), as shown in vitro (5, 6) and in vivo (7), possibly to maintain a physiologically normal balance of estrogen production. These two effects of estrogen on fetal adrenal function are consistent with the observation that in incubates of dispersed baboon fetal adrenal cells the formation of cortisol increased, whereas that of DHA decreased, when expressed on a per cell basis between mid- and late gestation (8, 9). Therefore, we have hypothesized that there is an estrogen-dependent divergence in cortical zone-specific development and function in the fetal adrenal gland with advancing gestation (4, 10, 11). This regulatory process may involve the ACTH receptor, because although ACTH receptor messenger RNA (mRNA) expression was enhanced in the transitional/definitive zones in late gestation, ACTH receptor expression declined in the fetal zone between mid- and late gestation (10, 11).



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  		Figure 1. Proposed actions of placental estrogen (E2) during primate pregnancy in a) stimulating the expression of 11ßHSD-1/-2 and thus the change in transplacental cortisol (F) and cortisone (E) metabolism that results in fetal pituitary ACTH release and maturation of the fetal adrenal transitional zone (shaded area) for F synthesis, and b) inhibiting the responsivity of the fetal zone to ACTH with respect to the production of DHA.

 
It is proposed that the direct action of estrogen on the primate fetal adrenal is mediated by the estrogen receptor. Although estrogen receptor protein has been demonstrated by gradient shift receptor assay and immunocytochemistry in the adrenal gland of the rhesus monkey neonate and adult, only very weak estrogen receptor immunoreactivity was observed in the fetal adrenal (12). The mRNA for estrogen receptor ß has been demonstrated by RT-PCR in the whole human fetal adrenal gland at midgestation (13). However, the developmental expression and cortical zone-specific localization of estrogen receptor {alpha} and ß in the primate fetal adrenal gland have not been determined. Therefore, a developmental approach was used in the present study to determine the potential expression of mRNA and protein for the estrogen receptor by RT-PCR, in situ hybridization, and immunocytochemistry in the fetal and definitive/transitional zones of the baboon fetal adrenal gland at mid- and late gestation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Adrenal glands were obtained from baboon (Papio anubis) fetuses immediately after cesarean section under halothane-nitrous oxide anesthesia on days 98–104 (midgestation) and days 161–170 (late gestation) of gestation (term = 184 days). One of the adrenal glands was immediately frozen and stored in liquid nitrogen for mRNA analysis, and the other gland was fixed in 4% paraformaldehyde for immunocytochemistry. This experiment was approved by the institutional animal care and use committees of the University of Maryland School of Medicine and Eastern Virginia Medical School.

Competitive RT-PCR
RNA preparation. The mRNA levels for the estrogen receptor {alpha} were quantified by the competitive RT-PCR assay established by Riedy et al. (14) as modified in our laboratory (15). Total RNA was obtained from baboon fetal adrenals by 4 M guanidine isothiocyanate homogenization, chloroform-isoamyl alcohol extraction, and cesium chloride centrifugation.

Primer sequence. The estrogen receptor {alpha} oligonucleotide primers were synthesized by Life Technologies, Inc. (Grand Island, NY); they were selected from the human estrogen receptor {alpha} complementary DNA (cDNA) sequence (16) and flanked a portion of the sequence spanning exons 4–7 and overlapping introns D, E, and F. The primers were as follows: primer 1: downstream, 5'-TTC TCT TCC AGA GAC TTC AGG GTG CTC ATG CGG AAC CGA GAT GAT-3' (position 1642–1623 linked to 1544–1525); primer 2: upstream, 5'-AAT TTA ATA CGA CTC ACT ATA GGG AGA TCC TAC CAG ACC CTT CAG-3' (position T7 polymerase sequence; the underlined sequences are linked to 1226–1245); primer 3: downstream, 5'-TTC CAG AGA CTT CAG GGT GC-3' (position 1642–1623); and primer 4: upstream, 5'-GAT CCT ACC AGA CCC TTC AG-3' (position 1226–1245).

Construction of internal standard RNA. The estrogen receptor {alpha} competitive reference standard (CRS) was prepared by the method of Riedy et al. (14), using RT-PCR to generate the cDNA template and transcription with T7 polymerase. Total RNA (2 µg) from baboon fetal adrenals was reversed transcribed at 42 C for 60 min in a reaction mixture (20 µl) containing 1 mM each of deoxy (d)-ATP, dCTP, dGTP, and dTTP (Promega Corp., Madison, WI); 1 mM dithiothreitol; 200 U SUPERSCRIPT ribonuclease (RNase) H-RT (Life Technologies, Inc.); 40 U RNAguard (Pharmacia Biotech, Piscataway, NJ); 50 mM Tris-HCl; 75 mM KCl; 3.0 mM MgCl2; and 250 ng random primers (Life Technologies, Inc.). After 60 min, the RT mixture was incubated at 70 C for 15 min and cooled to 4 C, and 5 µl were added to a PCR mixture (45 µl) containing 0.2 mM each of dATP, dCTP, dGTP, and dTTP; 10 mM Tris-HCl; 1.5 mM MgCl2; 50 mM KCl; 1.25 U cloned thermus aquaticus DNA polymerase (Amplitag, Perkin Elmer Corp./Cetus, Norwalk, CT); and 20 pmol each of primers 1 and 2. PCR was performed in a programmable thermal cycler (MJ Research, Inc., Cambridge, MA), and the sample was amplified in 28 or 34 sequential cycles at 94 C for 1 min, 60 C for 1 min, and 72 C for 2 min. After the last cycle, the sample was incubated for an additional 5 min at 72 C. An aliquot of the PCR reaction was fractionated by electrophoresis in a 2% agarose gel and visualized in ethidium bromide. The 417-bp amplified target (i.e. wild-type) mRNA strand and 339-bp CRS synthesized with the MEGAscript T7 in vitro transcription kit (Ambion, Inc., Austin, TX) were gel purified using the QIAEX II gel extraction kit (QIAGEN, Valencia, CA).

Competitive RT-PCR. A constant amount of fetal adrenal total RNA (3.0 µg) was added to the RT mixture containing 3-fold serial dilutions of the estrogen receptor {alpha}-CRS (30–2438 attomoles). After completion of the RT, 20 pmol each of primers 3 and 4 were added for the PCR. Negative controls in which either RNA or RT was omitted from the reaction were also performed. The PCR products were fractionated by electrophoresis, visualized with a UV transilluminator, and photographed using type 665 positive/negative film (Polaroid Corp., Cambridge, MA).

Quantification of estrogen receptor {alpha}. Photographs (negative image), representing the amplified products, were analyzed by audioradiography using a model 620 video densitometer (Bio-Rad Laboratories, Inc., Richmond, CA). The intensity of the amplified products was represented as the relative area under each sample band. A correction factor (17) was used to account for the differences in size of the target and CRS cDNAs. The logarithm (log) of the ratio of CRS to target area was plotted as a function of the concentration of estrogen receptor {alpha}-CRS added to each PCR reaction. The concentration of estrogen receptor {alpha} mRNA was determined where the ratio of CRS/target area was equal to 1 (i.e. equivalence point).

In situ hybridization
The methods for in situ hybridization previously employed in our laboratories (18) were modified essentially as described by Liuzzi et al. (19) for localization and zone-specific expression of estrogen receptor {alpha} and ß mRNA in the baboon fetal adrenal gland at mid- and late gestation. Sense and antisense estrogen receptor {alpha} and ß riboprobes were prepared from the SacII-SmaI fragment (bases 222–478) of the human estrogen receptor {alpha} (IGBMC, Strassburg, France) and the SacII-PstI fragment (bases 1–308) of the human estrogen receptor ß (provided by P. Webb, University of California, San Francisco, CA) cDNAs, which were subcloned into pBluescript SK (Stratagene, La Jolla, CA), and orientations were verified by DNA sequencing (20). Probes were radiolabeled with [33P]UTP using a Riboprobe In Vitro Transcription Kit (Promega Corp.), and unincorporated isotope was removed by filtration through a TE Midi Select Column (5 Prime-3 Prime, Boulder, CO).

At the time of hybridization, paraffin-embedded fetal adrenal sections (4 µm) were incubated (10 min) with 0.02 M HCl, washed, incubated (70 C, 30 min) with 2 x SSC (1 x SSC = 0.15 M NaCl and 0.015 M sodium citrate buffer, pH 7.2), and then treated (15 min; 37 C) with 5 µg/ml proteinase K (Sigma Chemical Co., St. Louis, MO). After incubation (10 min) with 10% buffered formalin, sections were rinsed in PBS and treated with 0.25% acetic anhydride in 0.1 M triethanolamine hydrochloride-0.9% NaCl. After dehydration in ethanol, delipidation in chloroform, and rehydration, sections were hybridized overnight at 55 C with labeled sense or antisense estrogen receptor {alpha} or ß 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 transfer RNA (Sigma Chemical Co.), 400 µl 50 x Denhardt’s solution (Sigma Chemical Co.), 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 4 x SSC, serially dehydrated, and washed in 50% formamide, 300 nM NaCl, 25 mM Tris, and 1 mM EDTA. After a 10-min rinse in 2 x SSC, nonspecific hybridized probe was removed by incubation (10 min; 37 C) with RNase A (26 µg/ml; Promega Corp.) and RNase T1 (2000 U; Promega Corp.) in RNase buffer consisting of 500 mM NaCl, 10 mM Tris, and 1 mM EDTA (pH 8.0). Subsequently, slides were washed twice in RNase buffer at 50 C for 15 min each time and then four times in 2 x SSC for 15 min each time at 50 C. Slides were then washed in 0.2 x SSC at 60 C for 2 h before serial dehydration and vacuum drying. Slides were coated with Kodak NTB2 nuclear emulsion (Eastman Kodak Co., Rochester, NY) diluted 1:1 with distilled water, and after 21 days were developed and lightly counterstained with hematoxylin.

Specific zonal localization of estrogen receptor expression in the fetal adrenal was confirmed by both light microscopy and darkfield optics. Quantification of estrogen receptor {alpha} mRNA expression was performed by counting the specific number of silver grains per 0.025 mm2 using an Optiphot 2 microscope attached to a video-based Image-1 analysis system (Universal Imaging Corp., West Chester, PA). Spatial calibration of the imaging system using a x40 objective was 512 pixels along the horizontal axis and 480 pixels along the vertical axis. An average of 10 different 145 x 180-µm areas of the fetal zone and the definitive/transitional zones were examined. After establishing a gray area, cells with a grain density 10-fold greater than background were considered estrogen receptor positive. All silver grain counts (number per 0.025 mm2) were averaged, and a single value for each animal was determined and used to calculate the overall group mean value.

Laser capture microdissection
Zone-specific cells of fetal adrenal glands obtained in late gestation were isolated by laser capture microdissection using an Arcturus Pix Cell LCM System (Arcturus, Los Angeles. CA) and methods described by the manufacturer and described originally by Emmert-Buck et al. (21). Briefly, frozen adrenal tissue was sectioned (4 µm) onto nontreated glass microscope slides, and the definitive/transitional zone was demarcated from the fetal zone by previously determined immunocytochemical expression/image analysis of definitive/transitional zone-specific 3ßHSD. Approximately 200 cells from each zone were microdissected and transferred to microfuge tubes, RNA was extracted using the RNeasy kit (QIAGEN), and estrogen-receptor {alpha} mRNA was determined by RT-PCR as described above. As a control, endometrium was obtained by laser capture from a section of frozen baboon uterus from the late follicular phase of the menstrual cycle, and estrogen receptor {alpha} mRNA was determined by RT-PCR concomitantly with cells from the fetal adrenal.

Immunocytochemistry
Estrogen receptor {alpha} and ß protein expression in the baboon fetal adrenal was determined by immunocytochemistry using methods previously established in our laboratory (22). Fetal adrenals were sectioned at 4 µm, and tissue was mounted on slides, heat fixed, placed in a microwave for 20 min, and endogenous peroxidase inhibited with 0.4% H2O2 in methanol. Tissue was then incubated overnight at 4 C with polyclonal NCL-ER6 F11 antibody to the rat estrogen receptor {alpha} (Vector Laboratories, Inc., Burlingame, CA) diluted 1:400 in 5% goat serum or polyclonal PA1–313 antibody to the C-terminal amino acid 467–485 of the human estrogen receptor ß (Affinity BioReagents, Inc., Golden, CO) diluted in PBS (2 µg/ml). The expression of 3ßHSD was determined by incubation of nonmicrowaved adrenal sections overnight at 4 C with polyclonal antibody to rabbit antihuman 3ßHSD (supplied by Dr. Ian Mason, University of Edinburgh, Edinburgh, Scotland) diluted 1:2500 in 5% goat serum. Sections were then washed and incubated with biotinylated goat antimouse IgG (estrogen receptor {alpha}) or antirabbit IgG (estrogen receptor ß and 3ßHSD; Vector Laboratories, Inc.) and then with Vectastain Elite Kit (Vector Laboratories, Inc.). After rinsing in PBS, sections were stained with diaminobenzidene (DAB)-imidazole-H2O2 (estrogen receptor ß and 3ßHSD) or DAB-nickel sulfate (estrogen receptor {alpha}, 0.250 g/10 ml, in 0.05 M Tris buffer, pH 7.2; Sigma Chemical Co.) as described by Berghorn et al. (23). Tissue sections were then counterstained with Gill’s hematoxylin and mounted in Biomount.

Statistics
mRNA levels were analyzed by one-way ANOVA and Newman-Keuls multiple comparison test or by Student’s t test for independent observations to identify significant differences between individual group means.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Estrogen receptor {alpha} mRNA levels by competitive RT-PCR
Figure 2Go shows a representative quantitative analysis of estrogen receptor {alpha} mRNA levels by competitive RT-PCR in whole baboon fetal adrenal tissue obtained at mid- and late gestation. The expected 417-bp estrogen receptor target product and the 339-bp estrogen receptor CRS product generated by PCR are shown in Fig. 2AGo. In contrast, there was no PCR product when either RNA or RT was omitted from the reaction (data not shown). The slopes of the log of the CRS to target areas plotted as a function of increasing amounts of CRS were similar for RNA obtained from the fetal adrenal at mid- and late gestation (Fig. 2BGo), indicating no difference in amplification efficiency. However, analysis of the equivalence points from each plot indicated that fetal adrenal estrogen receptor {alpha} mRNA levels were different at mid- and late gestation. Thus, overall mean (±SE) mRNA levels for estrogen receptor {alpha} were approximately 7-fold greater (P < 0.02) in the fetal adrenal of late (187.8 ± 40.3 attomoles/µg RNA) compared with mid (27.4 ± 5.4 attomoles/µg RNA) gestation (Fig. 2CGo).



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  		Figure 2. Representative competitive RT-PCR of estrogen receptor {alpha} in the baboon fetal adrenal. Total RNA (3.0 µg) from fetal adrenals obtained at mid- and late gestation were mixed with 3-fold serial dilutions of estrogen receptor {alpha} CRS. Samples were reversed transcribed and amplified for 28 cycles in the presence of specific primers. A, The 417-bp target product from total RNA and the 339-bp product from the CRS separated on 2% agarose gels and stained with ethidium bromide. B, The intensities of the amplified products in A were analyzed by densitometry, and the log of the ratios of estrogen receptor {alpha}-CRS and target areas in tissue of mid ({blacktriangleup}) and late (•) gestation was plotted as a function of CRS added to each PCR reaction. Lines were constructed by linear regression analysis, and estrogen receptor {alpha} mRNA levels were determined from the equivalence points (i.e. intersection of vertical with regression lines). C, Mean (±SE) estrogen receptor {alpha} mRNA levels determined by RT-PCR in adrenal tissue of baboons at mid (days 98–104, RNA from 9 baboons used to yield four samples) and late (days 161–170, RNA from six baboons used to yield four samples) gestation (term = 184 days). *, P < 0.02 late vs. midgestation, determined by t test for independent observations.

 
Estrogen receptor {alpha} and ß mRNA expression by in situ hybridization
Representative darkfield photomicrographs illustrating estrogen receptor {alpha} mRNA expression are shown for sections of baboon fetal adrenals of mid (Fig. 3Go, A and C) and late (Fig. 3Go, B, D, and F) gestation incubated with 33P-labeled antisense (Fig. 3Go, A, B, and F) or sense (Fig. 3Go, C and D) probes. The specificity of estrogen receptor labeling was evident by the relative absence of silver grains over cells with the sense riboprobe and the selectively greater distribution of silver grains over cells of the outer definitive/transitional zones (Fig. 3BGo, DZ) compared with the fetal zone (Fig. 3FGo, FZ) in fetal adrenals of late gestation. Estrogen receptor {alpha} mRNA was expressed in the fetal adrenal at midgestation (Fig. 3AGo), a time when the gland is comprised almost exclusively of the fetal zone. In late gestation there was extensive expression of estrogen receptor {alpha} mRNA in the outer definitive/transitional zones (Fig. 3BGo), which also stained selectively and intensely for 3ßHSD (not shown). Estrogen receptor {alpha} was still expressed in the fetal zone late in gestation (Fig. 3FGo), although at an apparently lower level than in the definitive/transitional zones. Localization of the estrogen receptor {alpha} in the endometrium of the baboon uterus, a well established site of this steroid receptor, is shown in Fig. 3EGo. When quantified by image analysis, estrogen receptor {alpha} mRNA levels (silver grains per 0.025 mm2) in the fetal adrenal appeared to decline, although not significantly, in the fetal zone between mid- and late gestation (Fig. 4Go) and were approximately 2.5-fold greater (P < 0.05) in the definitive/transitional zones (21.6 ± 0.5) than in the fetal zone (8.3 ± 1.5) in late gestation.



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  		Figure 3. Representative darkfield photomicrographs of 4-µm sections of baboon fetal adrenal glands after in situ hybridization with 33P-labeled estrogen receptor {alpha} antisense (A, B, and F) or sense (C and D) riboprobes. Fetal adrenals were obtained on day 100 (mid; A and C) and day 170 (late; B, D, and F) of gestation. FZ, Fetal zone; DZ, definitive/transitional zones; cap, capsularis. Magnification is approximately x400. E, Localization of estrogen receptor {alpha} mRNA in the baboon endometrium on day 12 of the menstrual cycle.

 


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  		Figure 4. Estrogen receptor {alpha} mRNA levels (silver grains per 0.025 mm2) determined by quantitative in situ hybridization in the fetal and definitive/transitional zones of adrenals obtained from baboon fetuses at mid (day 100; n = 4) and late (day 170; n = 4) gestation. *, Significantly different (P < 0.05) from fetal zone of late gestation (by ANOVA and Newman-Keuls multiple comparison test).

 
Expression of estrogen receptor {alpha} mRNA observed by in situ hybridization in the baboon fetal adrenal was confirmed in tissue isolated by laser capture microdissection and analyzed by RT-PCR (Fig. 5Go). Thus, estrogen receptor {alpha} mRNA was detected in both the fetal (lane 2) and definitive/transitional (lanes 3 and 4) zones of the baboon fetal adrenal obtained in late gestation.



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  		Figure 5. Estrogen receptor {alpha} mRNA determined by RT-PCR (34 cycles at 60 C) in tissue (~200 cells) obtained by laser capture microdissection from the fetal (lane 2) and definitive/transitional (lanes 3 and 4) zones of the baboon fetal adrenal of late gestation. Lane 5, Baboon endometrium; lane 1, ladder.

 
The mRNA for estrogen receptor ß was also demonstrable by in situ hybridization in the fetal zone of the baboon fetal adrenal gland at midgestation (Fig. 6AGo) and the fetal and definitive/transitional zones in late gestation (Fig. 6BGo). The specificity for the ß isoform was evident by the relative lack of silver grains over the adrenal cortex in sections incubated with a sense riboprobe (Fig. 6Go, C and D).



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  		Figure 6. Representative darkfield photomicrographs of 4-µm sections of baboon fetal adrenal glands after in situ hybridization with 33P-labeled estrogen receptor ß antisense (A and B) or sense (C and D) riboprobes. Fetal adrenals were obtained on day 100 (mid; A and C) and day 170 (late; B and D) of gestation. The arrow indicates the demarcation between the transitional zone and the fetal zone. cap, Capsularis. Magnification is approximately x400.

 
Estrogen receptor {alpha} and ß proteins by immunocytochemistry
A gradient of immunocytochemical localization of the estrogen receptor {alpha} protein using DAB-nickel sulfate (purple staining, Fig. 7AGo) and the estrogen receptor ß protein using DAB-imidazole (brown staining, Fig. 8BGo) was observed in the baboon fetal adrenal of late gestation. Estrogen receptor {alpha} and ß immunoreactivity was extensive in the definitive zone and somewhat less in the transitional zone. The definitive and transitional zones were identified by the specific staining for 3ßHSD in these two zones (Figs. 7BGo and 8CGo). Predominant nuclear localization of the estrogen receptor compared with cytoplasmic localization of the 3ßHSD was observed. Consistent with mRNA expression, there was a lower level of immunoreactivity for the estrogen receptor {alpha} and ß proteins in the fetal zone of the adrenal near term (lower halves of Figs. 7AGo and 8BGo). Specificity for estrogen receptor immunocytochemical staining was evident by the absence of reactivity when the primary antibody was omitted (Fig. 7CGo) and when the antibody was preabsorbed with estrogen receptor protein (data not shown).



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  		Figure 7. Immunocytochemistry of the estrogen receptor {alpha} (A) and 3ßHSD (B) in baboon fetal adrenal gland obtained on day 170 of gestation. cap, Capsularis; DZ, definitive zone; TZ, transitional zone; FZ, fetal zone. In C is a section of definitive zone in which the primary estrogen receptor {alpha} antibody was omitted. Magnification is approximately x100.

 


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  		Figure 8. Immunocytochemistry of the estrogen receptor ß in baboon fetal adrenal obtained on day 100 (A) and day 170 (B) of gestation. C shows immunocytochemical staining for 3ßHSD in the same adrenal as that shown in B. cap, Capsularis; DZ, definitive zone; TZ, transitional zone; FZ, fetal zone. Magnification is approximately x100 in A–C. The inset in the lower corner of B illustrates estrogen receptor ß staining in the fetal zone at a magnification of approximately x200.

 
Estrogen receptor ß immunoreactivity was also observed in the baboon fetal adrenal at midgestation (Fig. 8AGo). However, the estrogen receptor was again expressed to a greater extent in the outer region of the cortex.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study shows that the mRNAs for as well as the proteins of the estrogen receptor {alpha} and ß, as determined by RT-PCR, in situ hybridization, and immunocytochemistry, were expressed in the fetal zone and the definitive/transitional zones of the baboon fetal adrenal gland at mid- and late gestation. Expression of estrogen receptor {alpha} and ß in the fetal zone throughout the second half of baboon pregnancy would provide a mechanism for mediating the action of estrogen directly within this unique cortical zone of the developing fetal adrenal gland. We suggest, therefore, that the estrogen-dependent suppression of ACTH-regulated DHA formation by the baboon fetal adrenal gland (5, 6, 7) may be mediated in the classical manner by the estrogen receptor. A direct action of estrogen on the fetal zone would potentially provide a feedback system for modulating C19-steroid DHA/DHAS production by the fetal adrenal gland and thereby placental estrogen synthesis, as illustrated in Fig. 1Go.

Estrogen receptor {alpha} and ß mRNA and protein expression appeared to be much greater in the definitive/transitional zones than in the fetal zone of the baboon fetal adrenal late in gestation. Moreover, competitive RT-PCR analysis indicated that estrogen receptor {alpha} mRNA levels were much greater in the fetal adrenal of late than of midgestation, apparently reflecting the enhanced expression of this receptor in the definitive/transitional zones which develop in later stages of gestation. The relatively high expression of the estrogen receptor in the definitive/transitional zones was surprising, because our experimental findings suggest that estrogen only indirectly promotes maturation and function of the transitional zone (1, 3) while simultaneously directly suppressing function of the fetal zone (4). Further study is needed, therefore, to determine whether estrogen may have a potentially direct action on the definitive/transitional zones to modulate the development and/or function of the outer compartments of the maturing fetal adrenal cortex as well.

The presence of estrogen receptor ß in the baboon fetal adrenal gland at mid- and late gestation is consistent with the demonstration of estrogen receptor ß mRNA by RT-PCR in the human fetal adrenal (13). The molecular characterization, heterodimerization and interaction with the estrogen response element (24, 25), cellular distribution, and physiological roles of the various {alpha} and ß species are only beginning to be elucidated. Therefore, it remains to be determined whether the action of estrogen within the primate fetal adrenal involves interaction with either or both forms of the estrogen receptor.

Although the specific mechanisms by which the estrogen-estrogen receptor interaction inhibits DHA formation by the fetal zone remain to be determined, estrogen may act to suppress the expression of the ACTH receptor and thus cellular responsivity to ACTH. In support of this possibility, ACTH receptor mRNA levels become decreased in the fetal zone and elevated in the definitive/transitional zones of the baboon fetal adrenal between mid- and late gestation in association with the rise in estrogen in both the maternal and fetal circulations (10, 11). Consistent with these findings in the baboon, Jaffe and co-workers (26) have shown that ACTH receptor mRNA expression was greater in the 3ßHSD-positive definitive/transitional zones than in the fetal zone of the human fetal adrenal and have suggested that the cells in the outermost cortical zones are more sensitive to ACTH than are cells of the fetal zone. Estrogen, however, may also modify ACTH receptor-dependent secondary intracellular messengers within the fetal adrenal, e.g. protein kinase C, which decreases the activity of the P-450 17{alpha}-hydroxylase, 17–20-lyase enzyme that catalyzes the conversion of pregnenolone to DHA in the fetal adrenal (27). Indeed, estrogen has been shown to regulate this enzyme in the human adrenal (28) as well as in the rat ovary (29) and placenta (30).

In contrast to the observation of an inhibitory influence of estrogen on DHA output as studied in short term incubates of baboon fetal adrenal cells, in long term cultures of human fetal adrenal cells estrogen stimulated ACTH-induced DHAS production and inhibited cortisol synthesis by suppressing the 3ßHSD enzyme catalyzing the conversion of DHA to {Delta}4-steroids such as cortisol (31, 32, 33). These apparently disparate effects of estrogen may reflect a difference in the degree of fetal adrenal cellular differentiation associated with the different experimental conditions. For example, in tissue culture conditions, human fetal adrenal cells develop an ultrastructure of definitive or zona glomerulosa cells (34). Consequently, we suggest that the apparently different responses to estrogen reported in the literature for the human and baboon fetal adrenal do not reflect a species difference, but, rather, appear to be due to a difference in the qualitative nature of the cells resulting from the use of very different in vitro experimental approaches. Regardless of the nature of the response to estrogen, it is evident from the current study that the estrogen receptor is expressed within the primate fetal adrenal cortex, providing a means to mediate the action of estrogen.

In summary, the results of the present study show that estrogen receptor {alpha} and ß were expressed in the fetal and definitive/transitional zones of the baboon fetal adrenal cortex at mid- and late gestation. The presence of the estrogen receptor provides a mechanism for mediating the action of estrogen in modulating ACTH-dependent and cortical zone-specific development and function of the primate fetal adrenal gland.


   Acknowledgments
 
The secretarial assistance of Mrs. Wanda James is greatly appreciated. We gratefully acknowledge Dr. Reinhart Billiar for his assistance with the immunocytochemical analysis of estrogen receptor {alpha} and ß.


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

Received April 29, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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