| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Canadian Institutes for Health Research (CIHR), Institute of Human Development, Child and Youth Health, Department of Obstetrics and Gynecology, and Department of Physiology (N.A., W.L., T.M., J.C.), University of Toronto, Toronto, Ontario, Canada M5S 1A8; and CIHR Group in Fetal and Neonatal Health and Development, Department of Obstetrics and Gynecology (K.Y., J.C.), University of Western Ontario, London, Ontario, Canada N6A 5B8
Address all correspondence and requests for reprints to: Dr. Nadia Alfaidy, University of Toronto, Department of Physiology, Medical Sciences Building (MSB) Room 3344, 1 King’s College Circle, Toronto, M5S 1A8, Ontario, Canada. E-mail: n.alfaidy{at}utoronto.ca.
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
, hormones that are produced in the fetal membranes (FM) at term. In the present study, we hypothesized that 11ß-HSD 1 expression would increase in FM during pregnancy and at labor, creating the potential for local increase in F production at term. We examined 11ß-HSD 1 expression in placenta and FM obtained during normal pregnancy from nonlaboring women [26–28 wk (n = 3); 29–30 wk (n = 3); 32–33 wk (n = 3); 35–36 wk (n = 3)] and from uncomplicated term pregnancies after elective cesarean section (n = 6). 11ß-HSD 1 expression was also examined in amnion and chorionic tissues in relation to term labor (n = 12). Immunohistochemistry and Western blot analysis were used to examine 11ß-HSD 1 localization and expression. 11ß-HSD 1 activity was also measured in microsomal fractions prepared from whole fetal membranes. At term, immunoreactive 11ß-HSD 1 expression was localized predominantly to the chorion trophoblast cells, attached decidua, and amnion epithelial cells. 11ß-HSD 1 expression in FM increased with gestational age and reflected increased enzyme reductase activity. No change in 11ß-HSD 1 expression was found in placental tissue from the same patients. There was a significant increase in 11ß-HSD 1 expression in amnion but not in chorion with the onset of labor. We suggest that increases in 11ß-HSD 1 expression/activity by intrauterine membranes during late gestation may result in increased potential for a local increase in F production and that FM should be considered as an extraadrenal source of F during late gestation. This local F production may be involved in different pathways contributing to the regulation of parturition. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
and PGE2 stimulation (16). Activation of 11ß-HSD 1 may contribute to local F production within the fetal membranes, which in turn increases PG production by activating the PG synthesizing enzymes and inhibiting the PG metabolism (17, 18). These findings led us to hypothesize that there might be an increase during late gestation and labor in 11ß-HSD 1 expression within the fetal membranes. Therefore, in the present study, we examined expression and reductase activity of 11ß-HSD 1 in the placenta, chorion, and amnion during late human gestation and with respect to the labor process. |
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Patient consent and ethical approval were obtained before the onset of the study and tissue collection, according to the guidelines of Mount Sinai Hospital (Toronto, Canada) and the University of Toronto. Placentae (n = 30) were collected at preterm no labor (26–36 wk), term no labor (38–41 wk), and term with labor. The indications for the preterm nonlaboring patients in this study included antepartum hemorrhage, cervical cancer, and multiple pregnancy. There were no clinical or pathological signs of preeclampsia or other placental disease. Term human placentae were obtained from uncomplicated pregnancies after elective cesarean section and after spontaneous labor.
11ß-HSD 1 expression
Immunohistochemical analysis (IHC). Fetal membranes: Rolls of fetal membranes were embedded in paraffin, sectioned at 5 µm on a microtome (Histocut, Reichert-Jung, Cambridge Instruments, Heidelberg, West Germany), placed on Superfrost Plus slides (Fisher Scientific, Fair Lawn, NJ), and processed for immunohistochemistry as described (16). Briefly, slides were incubated in xylene substitute (BD Diagnostic Systems, Franklin Lakes, NJ) to remove the paraffin and then rehydrated in a graded series of ethanol dilutions and a final 0.01 M PBS [pH 7.4; 150 mM NaCl, 19 mM Na2HPO4, 1.5 mM NaH2PO4] wash. Slides were then incubated with rabbit antisheep 11ß-HSD 1 antibody (1:500 dilution in PBS) (19). The avidin-biotin-peroxidase technique (Vectastain ABC Kit; Vector Laboratories, Burlingame, CA) for immunostaining was used with diaminobenzidine (Sigma, St. Louis, MO) as the substrate. Slides were counterstained with Carazzi’s hemotoxylin, followed by dehydration in a graded series of ethanols, cleared in xylene substitute, and mounted with permount (Fisher Scientific). To test the specificity of the antibody, fetal membranes were also stained with primary antibody preabsorbed with excess immunizing peptide (19).
IHC. Placenta: Staining was performed according to the same protocol described above, except that an antigen retrieval technique was applied to enhance detection of 11ß-HSD 1 protein (20). Before adding the primary antibody, placental sections were placed in Coplin jars containing 10 mM citrate buffer (pH 6.0; Sigma) and placed in a microwave oven at 96 C for 20 min. After cooling for 40 min, sections were washed in PBS, preincubated for 30 min in 5% normal goat serum, and then incubated overnight at 4 C with primary antibody (1:250 dilution).
Western blot analysis. Frozen fetal membrane samples were homogenized on ice for 1 min in RIPA lysis buffer [50 nM Tris HCl (pH 7.5), 150 mM NaCl, 1% (wt/vol) sodium deoxycholate, 0.1% sodium dodecyl sulfate, 100 mM sodium orthovanadate (Sigma), 1% (vol/vol) Triton X-100 (Fisher Chemicals), and Complete MiniEDTA-free protease inhibitors (Roche Molecular Biochemicals, Dorval, Canada)]. Homogenates were centrifuged (4 C at 15,000 x g) for 15 min, and supernatants were collected. Protein concentrations were determined by the Bradford assay (21). Polyacrylamide gels were prepared (stacking gel, 4%; separating gel, 12% for 11ß-HSD 1). Proteins (50 µg/well) were separated by PAGE and then transferred electrophoretically to a 0.45-µm pore nitrocellulose membrane (Bio-Rad Laboratories, Inc., Hercules, CA). Transfer was confirmed by protein visualization with Ponceau S (Sigma). Blots were washed with PBS-T [150 mM NaCl, 10 mM Na2HPO4, 1.5 mM NaH2PO4, and 0.1% Tween-20 (Sigma); pH 7.5] and incubated overnight with blocking solution (5% skim milk powder in PBS-T). Subsequently, blots were incubated with primary antibody for rabbit antisheep 11ß-HSD 1 (1:1000 dilution in blocking solution) for 1 h. Blots were then rinsed six times for 5 min each with PBS-T and incubated with secondary rabbit antiserum conjugated with horseradish peroxidase (1:3000 dilution in blocking solution; Amersham Pharmacia Biotech, Piscataway, NJ) for 1 h. Blots were washed six times, 5 min each, and the antibody-antigen complex was detected using the Amersham Pharmacia Biotech ECL detection system (Amersham Pharmacia Biotech). The membranes were then exposed to X-OMAT blue film (Kodak Scientific Imaging Products, Rochester, NY). A major band of 34 kDa, corresponding to the known molecular mass of 11ß-HSD 1, was clearly visible in all specimens tested. A second band of 32 kDa, corresponding to the nonglycosylated form of 11ß-HSD 1, was also visible in some specimens. The intensities of immunoreactive bands were measured by scanning (6200C scanner, Hewlett Packard (Canada) Ltd., Mississauga, Ontario, Canada) and analyzing the image on a desktop computer using Scion Image software (v.4.0.2; Scion Corporation, Frederick, MD). The mean pixel density for each band was analyzed to obtain relative OD units for 11ß-HSD 1 protein. To standardize for sample loading, the relative OD units of 11ß-HSD 1 for each band were standardized to the relative OD units of the total protein in the band as analyzed by Ponceau S (Bio-Rad Laboratories, Inc.). To compare measurements between blots prepared at different times, a reference sample from human placenta was included in each gel.
11ß-HSD 1 activity
Reagents and supplies. [1,2,6,7-3H(N)]F (80 Ci/mmol) was purchased from DuPont Canada Inc. (Mississauga, Ontario, Canada). [1,2,6,7-3H(N)]E was prepared from [1,2,6,7-3H(N)]F in our laboratory as described previously (16). Nonradioactive steroids were obtained from Steraloids, Inc. (Wilton, NH). The cofactor H+ nicotinamide adenosine dinucleotide phosphate (NADPH) was purchased from Sigma. Polyester-backed thin-layer chromatography plates were obtained from Fisher Scientific (Unionville, Ontario, Canada). All solvents used were OmniSolv grade from BDH, Inc. (Toronto, Ontario, Canada).
Microsomal preparation. Fetal membrane tissues were homogenized in [10% glycerol, 300 mM NaCl, 1 mM EDTA, and 0.02 M Tris HCl (pH 7.4)]. Tissue homogenate from each sample was centrifuged at 4 C at 790 x g for 10 min, and the supernatant was then centrifuged at 4 C at 25,000 x g for 40 min. This supernatant was centrifuged at 4 C at 110,000 x g for 60 min, and the pellet containing the microsomal fraction was resuspended in 200–300 µl 11ß-HSD1 homogenization buffer. The protein content was measured by the Bradford method using a protein assay kit (Bio-Rad) with BSA as standard (21).
Assay of 11ß-HSD 1 reductase. 11ß-HSD1 reductase activity was assessed by measuring the rate of conversion of [3H]E to [3H]F. Microsomal fractions from fetal membranes (20 µg of protein) were incubated in 250 µl buffer containing E (10-6 M) and [3H]E as tracer, in the presence of NADPH (10-3 M) at 37 C for 10 min. The reaction was stopped by immediate transfer of the tubes to ice and addition of ethyl acetate (750 µl in a final volume of 1 ml; Sigma). The steroids were extracted using ethyl acetate and separated on silicon-coated thin-layer chromatography (TLC) plates (Fisher Scientific) using the solvent system chloroform/ethanol (95:5, vol/vol). Radioactivity was counted using a ß-counter. 11ß-HSD1 activity was expressed as picomoles of F formed per milligram of microsomal protein per minute. The percentage conversion of E to F was always less than 15%. Under these conditions the enzyme activity was not limited by substrate availability.
Statistical analysis
Results are expressed as the mean ± SEM. Statistical comparisons were made using one-way ANOVA test ( = 0.05) (SigmaStat, Jandel Scientific Software, San Rafael, CA).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Sections of normal human fetal membranes at 26 wk gestation stained with anti-11ß-HSD 1 antibody revealed specific staining in the chorion trophoblast layer and amnion epithelial cells. The intensity of the staining in chorion increased during pregnancy and reached a maximum at term (Fig. 1A
). Western blotting analysis of proteins from human fetal membranes homogenate confirmed the presence of 11ß-HSD 1, which was detected as a major band at 34 kDa. The amount of 11ß-HSD 1 protein in the homogenates increased with advancing gestation and was significantly greater (P < 0.05) at term than at any other gestational age examined (Fig. 1B).
|
11ß-HSD 1 reductase activity was measured in the microsomal fraction of fetal membranes from 18 patients at gestational ages ranging from 26 wk gestation to term. Protein (20 µg) was incubated for 10 min with E (1 µM) in the presence of 1 mM NADPH. These conditions were chosen so that the 11ß-HSD 1 reductase activity was measured in the linear range of the initial velocity and a maximum of 15% substrate was transformed at the end of the reaction. The 11ß-HSD 1 activity increased gradually from the first age bracket (26–30 wk) to term; however, the increase was only significant at term compared with each of the other age groups (Fig. 2). There was more than a 2-fold increase in 11ß-HSD 1 reductase activity, confirming our finding of increased 11ß-HSD 1 protein expression.
|
The effect of labor on 11ß-HSD 1 expression in chorion and amnion tissue was examined from six nonlaboring patients and six patients in labor, using immunohistochemistry and Western blot analysis. Immunoreactive 11ß-HSD 1 was localized to amnion, chorion, and decidua. The intensity of the staining in both chorion and amnion was higher in the laboring group compared with the nonlabor group (Fig. 3A). 11ß-HSD1 protein levels increased in both chorion (P = 0.06) and amnion (P < 0.05) with labor (Fig. 3B).
|
11ß-HSD 1 is also present in the human placenta. Previously, we found that the enzyme was localized mainly to intermediate trophoblast and blood vessels (15). This expression at both protein and mRNA levels was significantly lower than the expression of the type 2 11ß-HSD (15). Using the antigen retrieval technique to enhance 11ß-HSD 1 detection, we found that 11ß-HSD 1 was not only expressed in the intermediate trophoblast and endothelial cells but was also present in the syncytiotrophoblast (Fig. 4A). However, the expression of 11ß-HSD 1 was significantly lower in placenta compared with the amnion and chorion (Fig. 4B). There was no gestational age-related change in placental 11ß-HSD 1 protein, in contrast to the increase in the 11ß-HSD 1 expression observed in the fetal membranes (Fig. 5).
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
(16). Production of these eicosanoids is well recognized as a critical step in the processes leading to parturition. Their synthesis is stimulated by glucocorticoids, and their interaction with 11ß-HSD 1 creates a short loop feed-forward loop within chorion (16). In the present study, we demonstrated that both expression and activity of the enzyme 11ß-HSD 1 increases in human fetal membranes during late gestation, with the most significant changes occurring at term. These data are consistent with previous reports describing the chorionic membrane as an extraadrenal source of fetal F in human amniotic fluid (9) and suggest that 11ß-HSD 1 is likely the key enzyme responsible for this extraadrenal source. Our results also show that 11ß-HSD 1 expression increases in amnion and chorion tissues with labor. However, this increase was only significant in amniotic tissue. This difference might be explained by variation in the local stimulation of 11ß-HSD1 by PGs in the fetal membranes (16). The chorion is a major site of PG inactivation due to the presence of the enzyme PG dehydrogenase (PGDH), whereas amnion is essentially the site of increased PG synthesis due to the expression of PG synthesizing enzymes without PGDH. Hence, elevations in PG output may be greater in amnion than chorion, and these tissues may constitute two different environments for 11ß-HSD 1 regulation by PGs. The present study suggests that in the fetal membranes 11ß-HSD1 may be facilitatory to the integrated cellular regulation between glucocorticoids and PGs. 11ß-HSD 1 is discretely colocalized in chorion trophoblast cells with PGH synthase 2 and PGDH, consistent with paracrine/autocrine regulation between glucocorticoids and PGs. We speculate that the increase of 11ß-HSD 1 in late gestation, and with the onset of labor in human fetal membranes to generate active F, could have more general applicability in these tissues. For example, glucocorticoids stimulate CRH output in human chorion and placental trophoblast (29). CRH is discretely elevated in patients presenting in preterm labor at 28–32 wk who go on to deliver prematurely (30). Feed forward effect of PG to increase 11ß-HSD 1 in chorion (16) or to decrease 11ß-HSD 2 in placenta (31) would create an environment of increased local F with potential for enhanced stimulation of CRH output. We speculate that these relationships, combined with a reduction of placental 11ß-HSD 2 expression with lowered oxygenation (32), may contribute to the associations between elevated cord blood CRH in intrauterine growth restricted pregnancies, the inhibition effects of F of fetal growth, and the higher predisposition of these pregnancies to preterm delivery (33)
Many groups have reported that the F:E ratio in amniotic fluid increases significantly throughout gestation, with the greatest change after 30 wk of pregnancy. At term, the amniotic fluid F:E ratio is higher than that of the cord serum (34). In late gestation, F present in the amniotic fluid comes from at least two sources: the fetal adrenals, which generate largely conjugated F from the transitional cortex, and the fetal membranes, which produced unconjugated F (35). Most authors have found significant correlations between total F in amniotic fluid and parameters of lung maturation (36). The local increase in expression and activity of the 11ß-HSD 1 enzyme, potentially increasing local F, emphasizes the importance of the physiological role that this enzyme might play during late gestation. Although we have focused on the potential intrauterine actions of the locally produced F, it remains possible that this steroid also reaches the fetus through the chorionic vasculature and amniotic fluid.
In the fetal membranes, 11ß-HSD 1 acts mainly as a reductase. Dehydrogenase activity can also be detected, but only if some adherent decidual tissue is present in the sample, because decidual 11ß-HSD 1 exhibits mainly dehydrogenase activity (15). Therefore, decidual tissue was removed as completely as possible at the time of tissue collection from all samples used in the present study. Substantial 11ß-HSD 1 was reported to be present in human placenta. Here, the enzyme might act as a dehydrogenase along with the type 2 11ß-HSD (37), which is the predominant enzyme type in the syncytiotrophoblast (15, 28). Previously, we (15) and others (14) reported only low 11ß-HSD 1 immunoreactivity by IHC, although Pepe and Albrecht (38) had clearly shown that the enzyme was present. This apparent discrepancy was reconciled by use of antigen retrieval procedures, allowing us to demonstrate clearly the presence of 11ß-HSD 1 protein in the syncytiotrophoblast and confirming the original observations of Pepe and Albrecht (38). However, Western blotting revealed that the level of 11ß-HSD 1 protein in the placenta was much less than that in the chorion and amnion and did not change with gestation. It remains possible that altered subcellular localization of this enzyme in placental tissue affects its biological activity (37), but we have not examined this option further. Clearly, the increase in 11ß-HSD 1 protein in late pregnancy and at term is peculiar to the membranes, consistent with a physiological role at that locale.
In summary, the results of the present study show that 11ß-HSD 1 is present and active in human fetal membranes from 26 wk gestation to term and that both activity and expression increase gradually in late gestation and further at the time of labor. Consequently, the final regulation and local availability of glucocorticoid in the membranes and amniotic fluid may depend upon normal expression of 11ß-HSD1 and its local regulation.
|
Footnotes |
|---|
Abbreviations: E, Inactive cortisone; F, cortisol; 11ß-HSD, 11ß-hydroxysteroid dehydrogenase; IHC, immunohistochemical analysis; NADPH, nicotinamide adenosine dinucleotide phosphate; PG, prostaglandin; PGDH, PG dehydrogenase.
Received December 5, 2002.
Accepted July 17, 2003.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
potentiates cortisol production by stimulating 11ß-hydroxysteroid dehydrogenase 1: a novel feedback loop that may contribute to human labor. J Clin Endocrinol Metab 86:5585–5592This article has been cited by other articles:
 |
|
 |
|
  A. E. Michael and A. T. Papageorghiou Potential significance of physiological and pharmacological glucocorticoids in early pregnancy Hum. Reprod. Update, June 13, 2008; (2008) dmn021v1. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Z. Yang, C. Guo, P. Zhu, W. Li, L. Myatt, and K. Sun Role of glucocorticoid receptor and CCAAT/enhancer-binding protein {alpha} in the feed-forward induction of 11{beta}-hydroxysteroid dehydrogenase type 1 expression by cortisol in human amnion fibroblasts J. Endocrinol., November 1, 2007; 195(2): 241 - 253. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Chan, E. H Rabbitt, B. A Innes, J. N Bulmer, P. M Stewart, M. D Kilby, and M. Hewison Glucocorticoid-induced apoptosis in human decidua: a novel role for 11{beta}-hydroxysteroid dehydrogenase in late gestation J. Endocrinol., October 1, 2007; 195(1): 7 - 15. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Takano, Z. Lu, T. Goto, L. Fusi, J. Higham, J. Francis, A. Withey, J. Hardt, B. Cloke, A. V. Stavropoulou, et al. Transcriptional Cross Talk between the Forkhead Transcription Factor Forkhead Box O1A and the Progesterone Receptor Coordinates Cell Cycle Regulation and Differentiation in Human Endometrial Stromal Cells Mol. Endocrinol., October 1, 2007; 21(10): 2334 - 2349. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Sun, D. Brockman, B. Campos, B. Pitzer, and L. Myatt Induction of Surfactant Protein A Expression by Cortisol Facilitates Prostaglandin Synthesis in Human Chorionic Trophoblasts J. Clin. Endocrinol. Metab., December 1, 2006; 91(12): 4988 - 4994. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. C. Jonas, C. Chandras, D. R. E. Abayasekara, and A. E. Michael Role for Prostaglandins in the Regulation of Type 1 11{beta}-Hydroxysteroid Dehydrogenase in Human Granulosa-Lutein Cells Endocrinology, December 1, 2006; 147(12): 5865 - 5872. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Ball, J. W. Wang, S. Wong, B. Zielnik, J. Mitchell, N. Wang, M. B. Stemerman, and B. F. Mitchell Phorbol ester treatment of human myometrial cells suppresses expression of oxytocin receptor through a mechanism that does not involve activator protein-1 Am J Physiol Endocrinol Metab, November 1, 2006; 291(5): E922 - E928. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A Luczay, D Torok, A Ferenczi, J Majnik, J Solyom, and G. Fekete Potential advantage of N363S glucocorticoid receptor polymorphism in 21-hydroxylase deficiency. Eur. J. Endocrinol., June 1, 2006; 154(6): 859 - 864. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. Li, L. Gao, Y. Wang, T. Duan, L. Myatt, and K. Sun Enhancement of Cortisol-Induced 11{beta}-Hydroxysteroid dehydrogenase Type 1 Expression by Interleukin 1{beta} in Cultured Human Chorionic Trophoblast Cells Endocrinology, May 1, 2006; 147(5): 2490 - 2495. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. E. Murphy, R. Smith, W. B. Giles, and V. L. Clifton Endocrine Regulation of Human Fetal Growth: The Role of the Mother, Placenta, and Fetus Endocr. Rev., April 1, 2006; 27(2): 141 - 169. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Hoffmann, J.-J. Feige, and N. Alfaidy Expression and Oxygen Regulation of Endocrine Gland-Derived Vascular Endothelial Growth Factor/Prokineticin-1 and Its Receptors in Human Placenta during Early Pregnancy Endocrinology, April 1, 2006; 147(4): 1675 - 1684. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. D. Mitchell, M. C. Chang, T. Chaiworapongsa, H.-Y. Lan, R. J. A. Helliwell, R. Romero, and T. A. Sato Identification of 9{alpha},11{beta}-Prostaglandin F2 in Human Amniotic Fluid and Characterization of Its Production by Human Gestational Tissues J. Clin. Endocrinol. Metab., July 1, 2005; 90(7): 4244 - 4248. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. F. Johnstone, A. D. Bocking, E. Unlugedik, and J. R.G. Challis The Effects of Chorioamnionitis and Betamethasone on 11{beta}, Hydroxysteroid Dehydrogenase Types 1 and 2 and the Glucocorticoid Receptor in Preterm Human Placenta Reproductive Sciences, May 1, 2005; 12(4): 238 - 245. [Abstract] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
