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Endocrinology Vol. 142, No. 10 4496-4503
Copyright © 2001 by The Endocrine Society


ARTICLES

Estrogen Regulates 11ß-Hydroxysteroid Dehydrogenase-1 and -2 Localization in Placental Syncytiotrophoblast in the Second Half of Primate Pregnancy

Gerald J. Pepe, Marcia G. Burch and Eugene D. Albrecht

Department of Physiological Sciences, Eastern Virginia Medical School (G.J.P., M.G.B.), Norfolk, Virginia 23507; and Departments of Obstetrics/Gynecology/Reproductive Sciences and Physiology, Center for Studies in Reproduction, University of Maryland School of Medicine (E.D.A.), Baltimore, Maryland 21201

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


    Abstract
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We recently demonstrated that the 11ß-hydroxysteroid dehydrogenase enzymes catalyzing cortisol-cortisone reduction (11ß-hydroxysteroid dehydrogenase-1) and oxidation (11ß-hydroxysteroid dehydrogenase-2) are located in different regions of the baboon and human placental syncytiotrophoblast. Moreover, there was a 2-fold increase in the ratio of 11ß-hydroxysteroid dehydrogenase-2 to 11ß-hydroxysteroid dehydrogenase-1 in syncytiotrophoblast membranes contiguous with the basal membrane (BMm) between mid and late baboon gestation. Our laboratories have also shown that estrogen regulates syncytiotrophoblast functional differentiation. Therefore, the current study determined whether the change in the ratio of 11ß-hydroxysteroid dehydrogenase-2 to 11ß-hydroxysteroid dehydrogenase-1 in the BMm was regulated by estrogen. Placentas were obtained on d 165 of gestation (term = d 184) from baboons that were untreated or were treated daily beginning on d 100 with the aromatase inhibitor CGS 20267, which reduced uterine and maternal serum E2 by more than 95% or with CGS 20267 plus E2 benzoate. Western blot analyses and immunofluorescence confirmed that in untreated controls the expression of 11ß-hydroxysteroid dehydrogenase-1 was abundant in the microvillus membranes and considerably less in the BMm. In contrast, expression of 11ß-hydroxysteroid dehydrogenase-2 was abundant in more internal regions of the syncytiotrophoblast, including the BMm, but was not detected in the microvillus membranes. The 11ß-hydroxysteroid dehydrogenase-2 protein level was significantly decreased in the BMm of placentas from estrogen-suppressed baboons, resulting in a 2-fold decrease in the ratio of these enzymes in membranes juxta the fetal blood, and these changes were partially restored by CGS 20267 and E2. In contrast, estrogen had no effect on the ratio of 11ß-hydroxysteroid dehydrogenase-2 to 11ß-hydroxysteroid dehydrogenase-1 in whole villous homogenate or the micro-villus membranes. Collectively, these results indicate that estrogen regulates the developmental increase in the ratio of 11ßhydroxysteroid dehydrogenase-2 to 11ß-hydroxysteroid dehydrogenase-1 in syncytiotrophoblast membranes juxta fetal blood, providing the subcellular architectural mechanism responsible for the previously demonstrated estrogen-dependent switch in transplacental glucocorticoid metabolism that regulates maturation of the primate fetal pituitary-adrenocortical axis.


    Introduction
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
WE RECENTLY DEMONSTRATED that the 11ß-hydroxysteroid dehydrogenase (11ßHSD) enzymes catalyzing the reduction of cortisone to cortisol (11ßHSD-1) and oxidation of cortisol to cortisone (11ßHSD-2) are located in different regions of the baboon and human placental villous syncytiotrophoblast (1). Thus, 11ßHSD-1 expression was abundant in the microvillus membrane (MVM) and less in more internal regions of the syncytiotrophoblast, including membranes contiguous with the basal membrane (BMm). In contrast, 11ßHSD-2 expression was limited in the MVM and was extensive throughout the remainder of the syncytiotrophoblast, including the BMm facing the fetal blood compartment. Our studies also showed that in the BMm, levels of 11ßHSD-1 declined and those of 11ßHSD-2 increased between mid and late baboon gestation in association with the increase in estrogen, resulting in a 2-fold increase in the ratio of 11ßHSD-2 to 11ßHSD-1 in syncytiotrophoblast membranes juxta the fetal blood compartment (1). It is well established that the pattern of glucocorticoid metabolism across the baboon placenta changes from reduction at midgestation to oxidation in late gestation (2). Because the baboon syncytiotrophoblast mRNA and protein levels for both 11ßHSD-1 and -2 increased with advancing gestation (3, 4), we (1) have proposed that the change in the ratio of these two enzymes in membranes contiguous with the fetal vasculature may be the subcellular mechanism responsible for the developmental switch in transplacental glucocorticoid metabolism that results in decreased secretion of maternal cortisol into the fetus and activation of the fetal pituitary-adrenocortical axis at term (5).

Our laboratories have demonstrated that estrogen has a central role in regulating syncytiotrophoblast functional differentiation during primate pregnancy (6). Thus, estrogen increases placental syncytiotrophoblast mRNA and protein levels for 11ßHSD-1 and -2 (7, 8) and components of the progesterone biosynthetic pathway (9, 10, 11). It is possible, therefore, that the developmental change in the 11ßHSD-1/11ßHSD-2 ratio in syncytiotrophoblast membranes juxta fetal blood is also regulated by estrogen. To determine this possibility, we compared the expression of 11ßHSD-1 and 11ßHSD-2 proteins in enriched fractions of syncytiotrophoblast membranes prepared from baboon placentas obtained in late gestation from untreated animals with that in placentas from baboons in which placental estrogen production was suppressed by administration of an aromatase inhibitor throughout the second half of pregnancy.


    Materials and Methods
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Animals
Sixteen female baboons (Papio anubis/cynocephalus), weighing 10–15 kg, were mated and maintained as described previously (12). Six of the animals were treated daily beginning on d 100 of gestation (term = d 184) with the aromatase inhibitor CGS 20267 (4,4-[1,2,4,-triazol-1yl-methylene]bis-benzonitrite, Letrozol, Novartis Pharma AG, Basel, Switzerland) administered to the mother sc (2 mg/d·ml sesame oil) as described previously (13). Six additional animals were injected with CGS 20267 (2 mg/d) and E2 benzoate in increasing doses (1.0–2.0 mg/d·ml sesame oil) designed to replicate the normal pattern of serum E2 also beginning on d 100 of gestation. Four baboons served as untreated controls. Blood samples (3–4 ml) were obtained at 1- to 4-d intervals between d 85 and 165 of gestation via a maternal saphenous vein after sedation with an im injection of ketamine-HCl (10 mg/kg BW; Parke-Davis, Detroit, MI). On d 161–165 of gestation, baboons were sedated with ketamine and anesthetized with isoflurane/nitrous oxide, blood samples were obtained from the maternal saphenous and right and left uterine veins, and fetus and placenta were delivered. Serum samples were stored at -20 C and assayed for E2 as described previously (13). Animals were cared for and used strictly in accordance with USDA regulations and the NIH Guide for the Care and Use of Laboratory Animals (HHS, NIH Publication 86-23, 1985). The experimental protocol used in this study was approved by the institutional animal care and use committees of the Eastern Virginia Medical School and the University of Maryland School of Medicine.

Tissue preparation
Placentas were immediately rinsed in ice-cold 0.9% saline, and after removal of membranes, decidua basalis, and chorionic plate, sections of villous tissue were placed in 10% buffered formalin for subsequent immunofluorescence or were stored in liquid nitrogen. A small portion was homogenized in Tris-sucrose (homogenate) and stored at -80 C. Approximately 60–70 g whole villous tissue were then rinsed in saline, and used for preparation of syncytiotrophoblast MVM and BMm by methods recently developed in our laboratories (1). Briefly, villous tissue was minced, stirred with a magnetic stir bar for 60 min (4 C) in PBS (pH 7.4; Amresco, Solon, OH), and filtered through nylon mesh (Sefar America, Kansas City, MO) to obtain filtrate and particulate fractions. The filtrate fraction was centrifuged at 800 x g (to pellet debris), 10,000 x g for 10 min (to pellet intracellular organelles including nuclei), and then at 150,000 x g for 25 min, after which the membrane pellet was homogenized in Tris-mannitol buffer to which 10 mM MgCl2 were added. After incubation on ice for 10 min, the sample was centrifuged at 2,200 x g for 12 min and then at 150,000 x g for 25 min to obtain MVM. The particulate fraction was washed with PBS and 50 mM Tris, then placed in an ice-ethanol bath and sonicated (15 g/100 ml 50 mM Tris) for 10 sec using a Biosonik IV sonicator (VWR Scientific, Baltimore, MD) to release the BMm, and then sonicated again in EDTA to release the BM which was obtained by centrifugation. All fractions collected were aliquoted and stored at -80 C until analyzed for 11ßHSD-1 and -2 by Western blot, for alkaline phosphatase activity (Sigma) and protein concentration using the bicinchoninic acid procedure (Sigma, St. Louis, MO), and for dihydroalprenolol binding essentially as described by Kelley et al. (14). Briefly, fractions were incubated (37 C) for 30 min with 10 nM [3H]dihydroalprenolol (40 Ci/mmol; NEN Life Science Products, Boston, MA) in the presence (nonspecific binding) or absence of 30 µM d,l-propanolol (Sigma), followed by filtration onto glass-fiber filters (GFC, Whatman, Clifton, NJ). To confirm the site of localization of syncytiotrophoblast nuclei, aliquots (10 µg protein/20 µl) of the various fractions were added to 20 µl Vectashield mounting medium (Vector Laboratories, Inc., Burlingame, CA) containing 1 µg/ml 4',6-diamidino-2-phenylindole dihydrochloride hydrate (DAPI; Sigma), placed onto microscope slides, coverslipped, and examined for immunofluorescence as described previously (1).

Western blot analyses
Western blot analysis of 11ßHSD-1 and -2 was performed essentially as described previously (1, 4). Briefly, after determination of protein concentration using the bicinchoninic procedure (Sigma) and addition of Laemmli buffer (15), samples were heated (100 C for 5 min), cooled, and then loaded (25 µg protein/lane) onto discontinuous SDS-polyacrylamide minigels (12% for 11ßHSD-1; 10% for 11ßHSD-2) in electrophoresis chambers containing chilled 0.025 M Tris, 0.192 M glycine (Bio-Rad Laboratories, Inc., Richmond, CA), and 0.1% SDS buffer (pH 8.3). Samples were electrophoresed and wet-transferred to Immobilon P (Millipore Corp., Bedford, MA), and membranes were blocked and then incubated with polyclonal antibodies to 11ßHSD-1 and -2 diluted to 10 µg/ml in buffer I [50 mM Tris (pH 7.5), 150 mM NaCl, 0.05% Tween-20, and 0.05% IGEPAL CA-630] containing 1.5% BSA. Membranes were washed and then incubated with donkey antirabbit IgG horseradish peroxidase-conjugated second antibody (Amersham Pharmacia Biotech, Arlington Heights, IL) in buffer I containing 1.5% BSA. After washing and application of enhanced chemiluminescent reagent (ECL, Amersham Pharmacia Biotech), membranes were placed in x-ray film cassettes containing Fugi medical x-ray film (Fugi Medical Systems, Stamford, CT) and exposed in a dark room for 15–180 sec. The second antibody contributed no nonspecific bands at the concentrations employed. The specificity of the primary antibodies prepared (Cocalico Biologicals, Reamstown, PA) from rabbits injected with multiple antigenic peptides to which amino acids 22–36 of human 11ßHSD-1 and amino acids 332–345 of human 11ßHSD-2 were coupled (4) was confirmed in previous experiments (1, 4) demonstrating elimination of signal in syncytiotrophoblast samples probed with primary antibodies preabsorbed with excess immunizing peptide. To compare the levels of 11ßHSD-1 and -2 in the MVM or the BMm between treatment groups, Western blots were developed in the same film cassette for both 11ßHSD-1 and -2. After film development, samples were quantified by one-dimensional densitometry using an Image 1 analysis system (Universal Imaging Corp., Reamstown, PA), and results were expressed as arbitrary densitometric units per µg protein (1).

Immunofluorescence
Localization of 11ßHSD-1 and -2 proteins in baboon syncytiotrophoblast was determined by immunofluorescence essentially as described previously (1). Briefly, peroxidase-blocked sections (4 µm) were microwaved for 20 min in sodium citrate buffer (pH 6.0; Sigma), cooled (30 min), washed in PBS, blocked (30 min) in 5% normal goat serum (NGS; Vector Laboratories, Inc.), and incubated overnight at 4 C with rabbit primary antibodies to 11ßHSD-1 or 11ßHSD-2 diluted 1:2000 in 5% NGS. After washing, sections were incubated for 2 h at room temperature with biotinylated antirabbit IgG (Zymed Laboratories, Inc., South San Francisco, CA). Biotin was detected after treatment (30 min) in the dark with streptavidin conjugated with a Cy3 fluorotag (Zymed Laboratories, Inc.) diluted 1:50 in 5% NGS (1). Autofluorescence was quenched by incubation with Sudan Black (Sigma; 1% in 70% methanol), slides were rinsed, and Vectashield mounting medium (Vector Laboratories, Inc.) was applied with/without 1 µg/ml DAPI (Sigma). Slides were then coverslipped and stored at 4 C in the dark until photographed with Elite Chrome 400 color film (Eastman Kodak Co., Rochester, NY) using a Nikon FX-35 WA camera and Microphot-FX microscope and rhodamine or DAPI filters (Nikon, Melville, NY).

Statistical analysis
Data are expressed as the mean ± SE. Statistical differences between groups were determined by ANOVA, with post-hoc comparisons of the means by Newman-Keuls multiple comparison test.


    Results
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Serum E2
Serum E2 in untreated baboons increased from approximately 1 ng/ml on d 85–120 of gestation to 2.5–3.5 ng/ml by d 165 (Fig. 1AGo). Within 1–3 d after the onset of CGS 20267 treatment on d 100 of gestation, maternal serum E2 decreased to and remained at levels less than 0.1 ng/ml. The qualitative pattern of maternal serum E2 after treatment with CGS 20267 plus E2 benzoate approximated that in the control. Mean (±SE) E2 levels in uterine vein serum on the day of delivery in untreated animals (5.74 ± 1.47 ng/ml) were decreased (P < 0.05) by CGS 20267 (0.34 ± 0.09 ng/ml) and restored (P < 0.05) by CGS 20267 plus E2 benzoate (4.98 ± 0.97 ng/ml; Fig. 1BGo).



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Figure 1. A, Mean maternal peripheral serum E2 levels between d 85 and 165 of gestation in baboons that were untreated or treated with CGS 20267 (2 mg/d, sc) or CGS 20267 and E2 benzoate (1–2 mg/d; sc) beginning on d 100 of gestation (term = d 184). B, Mean (±SE) uterine vein serum E2 levels on d 165 of gestation in baboons that were untreated (n = 4) or treated with CGS 20267 (n = 6) or CGS 20267 and E2 benzoate (n = 6). Values with different letter superscripts differ from each other at P < 0.05 (by ANOVA and multiple comparison of the means using the Student-Newman-Keuls statistic).

 
Immunofluorescence of 11ßHSD-1 and -2
11ßHSD-1 was detected by immunofluorescence in syncytiotrophoblast MVM and to a limited extent in regions of the syncytiotrophoblast in proximity to the basal membrane (i.e. the BMm; Fig. 2AGo). In estrogen-deprived baboons (Fig. 2B), 11ßHSD-1 was still detected in the MVM, but was also detected in the BMm in the majority, but not all, of the placental sections examined. In baboons treated with CGS 20267 and estrogen, 11ßHSD-1 syncytiotrophoblast localization was more like that in the untreated animals and thus was not detected in the BMm in the majority of placental sections examined (Fig. 2CGo). Although 11ßHSD-1 was occasionally detected in fetal vascular cells, 11ßHSD-1 was not detected in syncytiotrophoblast nuclei, which bound the blue fluorescent dye DAPI, regardless of treatment in vivo (Fig. 2DGo). In contrast to 11ßHSD-1, 11ßHSD-2 was detected by immunofluorescence in regions of the syncytiotrophoblast including nuclei, identified by expression of DAPI (Fig. 2HGo), close to the inner villous core, but was not detected in the MVM in all three groups (Fig. 2Go, E–G).



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Figure 2. Representative photomicrographs of 11ßHSD-1 (A–D) and 11ßHSD-2 (E–H) immunofluorescence in the baboon placenta on d 165 of gestation in animals that were untreated (A and E) or treated during the second half of gestation with CGS 20267 (B and F) or CGS 20267 plus E2 benzoate (C and G) as described in Fig. 1Go. Sections were incubated with primary antibody to 11ßHSD-1/-2, and proteins were detected with streptavidin conjugated with a Cy3 fluorotag and photographed with Elite Chrome 400 color film. Sections D and H are the same as those in C and G, but were coated with DAPI before photography to confirm localization of syncytiotrophoblast nuclei. Original magnification, x400. IVC, Inner villous core.

 
Western blot of 11ßHSD-1/-2
Figure 3Go depicts representative Western blots for 11ßHSD-1 and -2 expression in the MVM, the 10,000 x g fraction (i.e. intracellular organelles and 10K fraction), and the BMm of the baboon placental syncytiotrophoblast. 11ßHSD-1 was expressed as a 32-kDa protein, and 11ßHSD-2 was expressed as a doublet of approximately 40 and 42 kDa as previously shown (1, 4). In all groups 11ßHSD-1 expression was most extensive in the MVM, was relatively low in the 10K fraction, and was lower, but still detectable, in the BMm. In contrast, 11ßHSD-2 was expressed in low abundance in MVM and was present in relatively high levels in the 10K fraction and the BMm. Finally, as previously demonstrated (1), based on location in the gel it would appear that the sonication procedure used to isolate the BMm modified the 11ßHSD-2 protein.



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Figure 3. Representative Western immunoblot of 11ßHSD-1 and -2 proteins in fractions of placenta from baboons that were untreated or treated with CGS 20267 or CGS 20267 plus E2 benzoate. Syncytiotrophoblast MVM and intracellular organelles (10,000 x g; 10K) were isolated by mechanical agitation and differential centrifugation, and BMm were released from the BM by sonication. Membrane-associated proteins were solubilized and loaded (25 µg/lane) onto discontinuous SDS-polyacrylamide gels. Electrophoresed samples were transferred to Immobilon P and incubated with antibodies to 11ßHSD-1 or -2.

 
In homogenate of whole villous placenta (Fig. 4AGo), the mean SE) 11ßHSD-2 concentration (67.6 ± 1.4 arbitrary densitometric units x 10-3/µg protein) was decreased (P < 0.05) in baboons in which estrogen production was suppressed by treatment with CGS 20267 (49.3 ± 2.6) and was restored (P < 0.05) to normal by CGS 20267 and E2 benzoate (63.9 ± 1.7). In contrast, 11ßHSD-1 levels were similar in whole villous homogenate of placentas from baboons that were untreated (34.3 ± 4.9) or treated with CGS 20267 (29.9 ± 1.4) or CGS 20267 plus estrogen (32.2 ± 1.8). Moreover, the ratio of 11ßHSD-2 to 11ßHSD-1 in whole villous placenta was similar in untreated (2.1 ± 0.4), CGS 20267-treated (1.7 ± 0.1), and CGS 20267- plus E2 benzoate-treated (2.0 ± 0.1) baboons.



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Figure 4. Mean (±SE) levels of 11ßHSD-1 and -2 in whole villous homogenate (A), MVM (B), and BMm (C) of placentas obtained on d 165 of gestation from baboons that were untreated (n = 4) or treated with CGS 20267 (n = 6) or CGS 20267 and E2 benzoate (n = 6) as described in Fig. 1Go. Membranes were prepared, and 11ßHSD-1 and -2 proteins were detected by Western blot as described in Fig. 3Go. D, Ratio (mean ± SE) of the 11ßHSD-2 to 11ßHSD-1 protein concentrations determined by Western blot in BMm of samples from C. Values with different letter superscripts differ from each other at P < 0.05 (by ANOVA and Student-Newman-Keuls statistic).

 
In the MVM, 11ßHSD-2 protein was considerably lower than that of 11ßHSD-1 (Fig. 4BGo). Moreover, levels of both proteins in the MVM and their ratios were not affected by treatment with CGS 20267 and/or E2.

In contrast to the MVM, 11ßHSD-2 protein concentrations in the BMm (Fig. 4CGo) were lower (P < 0.05) in baboons in which estrogen production was decreased by CGS 20267 (3.8 ± 0.6) than in untreated baboons (8.2 ± 0.8) and were increased (P < 0.05) to an intermediate value (5.9 ± 0.7) in animals in which estrogen was restored by CGS 20267 and E2 benzoate treatment. However, the level of 11ßHSD-1 in the BMm of placentas from untreated baboons (3.4 ± 0.5) was slightly, but not significantly, higher in BMm of animals treated with CGS 20267 (4.1 ± 1.0). Thus, as shown in Fig. 4DGo, the ratio of 11ßHSD-2 to 11ßHSD-1 in the BMm of untreated baboons (2.6 ± 0.4) was decreased (P < 0.05) by approximately 50% by CGS 20267 (1.2 ± 0.3) and was partially restored by CGS 20267 plus E2 benzoate (1.7 ± 0.2).

Alkaline phosphatase activity and protein concentrations in syncytiotrophoblast membranes
Alkaline phosphatase activity was minimal in the BMm and was enriched approximately 13- to 20-fold in the MVM in all treatment groups (Table 1Go). Alkaline phosphatase was also enriched approximately 4-fold in the 10K fraction, indicating contamination of this fraction with MVM. In whole villous homogenate and MVM, alkaline phosphatase was reduced, although not significantly, by approximately 40% and 25%, respectively, by CGS 20267 and was restored to normal by CGS 20267 plus E2. Protein concentrations were greatest in the BMm and were not significantly altered by CGS 20267. However, compared with those in untreated baboons (Fig. 5AGo), the height and perhaps the amount of microvilli were markedly lower in placental syncytiotrophoblast of estrogen-deprived baboons (Fig. 5BGo), a structural change that appeared to be prevented by restoration of estrogen (Fig. 5CGo).


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Table 1. Mean (±SE) alkaline phosphatase activity and soluble protein concentration in baboon placental membrane preparations in late gestation

 


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Figure 5. Representative histology of sections of villous placenta obtained on d 165 of gestation from baboons that were untreated (A) or treated with CGS 20267 (B) or CGS 20267 plus E2 benzoate (C; see Fig. 1Go). Paraffin sections (4 µm) were rehydrated in graded alcohols and stained with hematoxylin. Magnification, x1,200. IVC, Inner villous core.

 
Dihydroalprenolol binding and detection of nuclei in syncytiotrophoblast membrane fractions
Binding of dihydroalprenolol, a marker of the basal membrane (BM) (14), was enriched approximately 16-fold in the BM, but not in the BMm or the 10K fraction (Table 2Go), compared with that in the whole villous homogenate. The MVM, however, did exhibit dihydroalprenolol binding, which appeared to be approximately 7-fold greater than that in the homogenate. It is also apparent that syncytiotrophoblast nuclei released after mechanical disruption of the baboon villous placenta pelleted and thus were detected primarily in the 10K fraction. In contrast, very few nuclei were detected in the BMm, and none was found in the BM or MVM. Finally, as shown in Fig. 5Go, in untreated baboons the syncytiotrophoblast nuclei were uniformly aligned and in close proximity to the BM. In contrast, in estrogen-deprived animals, the nuclei appeared to be less uniformly distributed and more distant from the BM (Fig. 5BGo), a structural change that appeared to be prevented in part in syncytiotrophoblast of baboons treated with CGS 20267 plus estrogen (Fig. 5CGo).


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Table 2. Dihydroalprenolol binding and relative proportion of nuclei in baboon placental syncytiotrophoblast fractions in late gestation

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We previously demonstrated that the ratio of 11ßHSD-2 to 11ßHSD-1 in the BMm of the baboon placental syncytiotrophoblast in late gestation was approximately 2-fold greater than that at midgestation (1), and we proposed that the latter provided the biochemical-architectural basis for the estrogen-dependent change in transplacental metabolism of cortisol-cortisone from reduction at midgestation to oxidation by term (2). In the estrogen-deprived baboons of the current study, the ratio of 11ßHSD-2 to 11ßHSD-1 in the BMm was decreased to a level comparable to that previously observed at midgestation. Therefore, the results of the current study support the suggestion that the developmental increase in the 11ßHSD-2/11ßHSD-1 ratio in the BMm of the primate placenta is regulated by estrogen. Estrogen has been previously shown to regulate other critical aspects of primate syncytiotrophoblast functional maturation, including expression of the low density lipoprotein receptor and P-450 cholesterol side-chain cleavage enzyme (9, 10, 11). Collectively, therefore, the results of these studies lend further support to the concept put forth by our laboratories that estrogen plays a central role in regulating primate placental-fetal development (6).

We previously demonstrated that the estrogen-induced increase in transplacental oxidation of cortisol to cortisone during the second half of baboon gestation resulted in maturation of the fetal pituitary-adrenocortical axis (5, 16) and onset of de novo fetal adrenal cortisol production (16). Because cortisol is the dominant glucocorticoid formed within the baboon placenta and secreted into the fetus at midgestation, we suggest that in estrogen-suppressed baboons, cortisol would continue to be preferentially transported across the placenta and secreted into the fetus, potentially compromising fetal adrenal development. Although the latter remains to be determined, we (16) previously showed that 3ßHSD-isomerase activity in the fetal adrenal was 3-fold lower at term in baboons in which the action of estrogen was blocked during the second half of gestation by treatment of the mother with an ER antagonist that also prevented the increase in placental oxidation of cortisol to cortisone (17). Therefore, we further propose that the spatial separation of the 11ßHSD-1 and -2 enzymes and the estrogen-regulated developmental change in their relative ratio within the syncytiotrophoblast BMm juxta fetal blood are of utmost physiological importance to the maturation of the primate fetus. Indeed, exposure of the fetus to cortisol has been suggested to impact on function in adulthood (18, 19), and reductions in placental 11ßHSD-2 have been implicated in development of hypertension in adulthood (20). Whether physiological function has indeed been compromised in estrogen-depleted baboons remains to be determined. However, we suggest that the baboon provides a model to perform such critical studies.

The mechanism(s) by which estrogen regulates localization of the 11ßHSD enzymes in the syncytiotrophoblast remains to be determined. In our previous study (1) we showed that the developmental increase in the ratio of 11ßHSD-2 to 11ßHSD-1 in the BMm was due to a marked reduction in the concentration of 11ßHSD-1 and an increase in that of 11ßHSD-2. Consistent with the latter observations, in the current study there was a decrease in the ratio of 11ßHSD-2 to 11ßHSD-1 in the placental BMm from baboons deprived of estrogen, and this reflected a significant reduction, as determined by Western blot, in the level of 11ßHSD-2 and a concurrent, albeit slight, increase in 11ßHSD-1. The results of immunofluorescence indicated that expression of 11ßHSD-1 in the placental BMm of estrogen-suppressed baboons was more extensive than that in untreated baboons, although this increase was not uniform. It is possible, therefore, that regional differences in syncytiotrophoblast structure occur in estrogen-deprived animals, which might impact 11ßHSD-1 localization. In the human, and presumably the baboon, placenta, the trophoblast basement membrane is comprised of extracellular matrix components that are products of the villous trophoblast (21, 22), and changes in BM structure may reflect alterations in synthesis/secretion of its components. Although it remains to be shown whether estrogen plays a role in the latter process, experimental procedures that decrease the production of fetal adrenal androgens and thus placental estrogen synthesis in rhesus monkeys also cause a striking increase in the thickness of the trophoblast basement membrane of the retained placenta (23, 24).

Recently, we demonstrated that the activity of the baboon 11ßHSD-2 promoter was increased in JEG 3 cells cotransfected with ER and incubated with E2 (8) and that syncytiotrophoblast expression of 11ßHSD-2 increased with advancing gestation in association with the elevation in estrogen (3, 4). Moreover, placental NAD+-dependent 11ßHSD-2 activity was increased at midgestation in baboons treated with estrogen (25). That estrogen up-regulates expression of the 11ßHSD-2 enzyme is supported by observations in the current study demonstrating that the 11ßHSD-2 protein concentration in whole villous homogenate and that in the BMm were decreased in estrogen-deprived baboons and were restored by estrogen. Although the baboon 11ßHSD-1 promoter is also increased by E2 (8), and placental syncytiotrophoblast 11ßHSD-1 expression increases with advancing pregnancy (3, 4), the levels of 11ßHSD-1 protein in the whole villous tissue and BMm as well as the MVM, its major site of localization, were not decreased in syncytiotrophoblast from estrogen-depleted baboons. The reason(s) for this lack of change is unknown. However, because 11ßHSD-1 resides primarily in the MVM, and the amount of MVM, as determined by histology and alkaline phosphatase activity, was lower in syncytiotrophoblast of estrogen-depleted compared with untreated baboons, it may be difficult to measure changes specifically in 11ßHSD-1 in either whole villous homogenate or MVM relative to those in other protein components.

The results of the current study also suggest that syncytiotrophoblast nuclei, which pellet primarily in the 10K fraction, as has been shown for the human placenta (14), are a major site of 11ßHSD-2 expression. Moreover, the 11ßHSD-2 signal detected by Western blot in the 10K fraction exceeded that in the BMm. However, because the Western blots were normalized to a specific amount of protein, the total amount of protein in the BMm was more than 10-fold greater than that in the 10K fraction, and nuclei were not detected in the BMm, it appears that the BMm and not the nuclei is the major site of 11ßHSD-2 localization. Nevertheless, because the syncytiotrophoblast nuclei express 11ßHSD-2, but not 11ßHSD-1, it is possible that nuclear metabolism may contribute to the developmental increase in placental cortisol oxidation in late gestation. The results of the current study also showed that syncytiotrophoblast nuclei at term were localized in close proximity to the BM, a process that appears to have been disrupted in estrogen-deprived baboons. Therefore, we propose that estrogen not only regulates the levels and localization of the 11ßHSD-1 and -2 enzymes in the baboon placenta, but also structural maturation of the syncytiotrophoblast, including the BM and microvilli. Consistent with the latter, estrogen has been shown to increase the number and length of microvilli in rat pituitary cells (26) and in ER-positive MCF7 breast cancer cells (27). Moreover, in MCF7 cells estrogen increased the expression of EBP50/NHE-RF (28), a cytoskeletal PDZ domain-containing protein that is typically concentrated in microvilli, including those of the human (29) and baboon (30) term placenta. Whether estrogen regulates EBP50/NHE-RF expression in the MVM of the baboon placenta remains to be determined.

In summary, the results of the current study indicate that the localization of 11ßHSD-2 and the developmental increase in the ratio of 11ßHSD-2 to 11ßHSD-1 in baboon syncytiotrophoblast membranes juxta the fetal blood compartment are regulated by estrogen. We propose that this increase in the ratio of 11ßHSD-2 to 11ßHSD-1 in the BMm provides the subcellular architectural mechanism responsible for the previously demonstrated estrogen-dependent switch in transplacental glucocorticoid metabolism, which regulates maturation of the primate fetal pituitary-adrenocortical axis. Therefore, these studies further support the concept that estrogen plays a central role in regulating placental development essential to fetal maturation and neonatal adrenocortical self-sufficiency.


    Acknowledgments
 
CGS 20267 was provided by Novartis Pharma AG (Basel, Switzerland). The authors sincerely appreciate the secretarial assistance of Ms. Sandra Huband with the manuscript, Mr. Nicholas Zachos with the preparation of the photomicrographs, Ms. Pam Brien with the baboon husbandry, and R. B. Billiar, Ph.D., with advice and assistance with the Western blot analyses.


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

Abbreviations: BM, Basal membrane; BMm, membranes contiguous with the basal membrane; DAPI, 4',6-diamidino-2-phenylindole dihydrochloride hydrate; 11ßHSD, 11ß-hydroxysteroid dehydrogenase; MVM, microvillus membranes; NGS, normal goat serum.

Received January 18, 2001.

Accepted for publication June 22, 2001.


    References
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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