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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
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Abstract |
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Introduction |
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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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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.
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Results |
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). 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. 1B).
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). 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. 2C). 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. 2D). In contrast to 11ßHSD-1, 11ßHSD-2 was detected
by immunofluorescence in regions of the syncytiotrophoblast including
nuclei, identified by expression of DAPI (Fig. 2H), close to the inner
villous core, but was not detected in the MVM in all three groups (Fig. 2, E–G).
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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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), 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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). 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. 4C) 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. 4D, 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 1). 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. 5A), the height and perhaps the amount of
microvilli were markedly lower in placental syncytiotrophoblast of
estrogen-deprived baboons (Fig. 5B), a structural change that appeared
to be prevented by restoration of estrogen (Fig. 5C).
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), 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. 5, 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. 5B), a structural change that appeared to be prevented in
part in syncytiotrophoblast of baboons treated with CGS 20267 plus
estrogen (Fig. 5C).
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Discussion |
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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.
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Acknowledgments |
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Footnotes |
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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.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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5-3ß-hydroxysteroid dehydrogenase
and 17
-hydroxylase-17,20-lyase activity. Endocrinology 128:2395–2401[Abstract]
-ergocryptine, estradiol, and thyrotropin-releasing hormone in
rat anterior pituitary cells in culture. J Cell Biol 86:377–387This article has been cited by other articles:
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R. B. Billiar, N. C. Zachos, M. G. Burch, E. D. Albrecht, and G. J. Pepe Up-Regulation of {alpha}-Inhibin Expression in the Fetal Ovary of Estrogen-Suppressed Baboons Is Associated with Impaired Fetal Ovarian Folliculogenesis Biol Reprod, June 1, 2003; 68(6): 1989 - 1996. [Abstract] [Full Text] [PDF]
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N. C. Zachos, R. B. Billiar, E. D. Albrecht, and G. J. Pepe Developmental Regulation of Baboon Fetal Ovarian Maturation by Estrogen Biol Reprod, October 1, 2002; 67(4): 1148 - 1156. [Abstract] [Full Text] [PDF]
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