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The Journal of Clinical Endocrinology & Metabolism Vol. 85, No. 12 4841-4850
Copyright © 2000 by The Endocrine Society

Original Studies

Tissue- and Site-Specific Gene Expression of Type 2 17ß-Hydroxysteroid Dehydrogenase: In Situ Hybridization and Specific Enzymatic Activity Studies in Human Placental Endothelial Cells of the Arterial System1

Martin Bonenfant, Charles H. Blomquist, Pierre R. Provost, Renée Drolet, Peter D’Ascoli and Yves Tremblay2

Laboratory of Ontogeny and Reproduction, Laval University Medical Center, CHUQ (M.B., P.E.P., R.D., Y.T.); Department of Obstetrics and Gynecology, Faculty of Medicine and CRBR, Laval University (Y.T.), Québec, Canada G1V 4G2; and Department of Obstetrics and Gynecology, HealthPartners Regions Hospital (C.H.B., P.D.), St. Paul, Minnesota 55101

Address all correspondence and requests for reprints to: Dr. Yves Tremblay, Laboratory of Ontogeny and Reproduction, Laval University Medical Center, CHUQ, Québec, Canada G1V 4G2. E-mail: Yves.Tremblay{at}crchul.ulaval.ca


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Progesterone and estradiol are the most potent human sex steroid hormones of placental origin and are essential to the maintenance of pregnancy, the timing of parturition, the maturation of many fetal organs, and the preparation of the maternal reproductive system. Naturally, regulatory mechanisms must be in place to coordinate the synthesis and inactivation of these two hormones. We have previously shown that the highest levels of type 1 and type 2 17ß-hydroxysteroid dehydrogenase (17ßHSD) messenger ribonucleic acids (mRNAs) occur in the placenta, particularly in the villi. However, in contrast to type 1 17ßHSD mRNA, type 2 17ßHSD mRNA was not detectable in cell cultures of human cytotrophoblasts or syncytiotrophoblasts. Using in situ hybridization, we unequivocally identified endothelial cells as the only cell type expressing the type 2 17ßHSD gene in fetal villi. Moreover, type 2 17ßHSD mRNA was specifically detected in the endothelial cells of the arterial system, and at higher levels in the villi compared with endothelial cells of the cord arteries when the two tissue sections were cohybridized. In fact, both mRNA levels and enzymatic activity are at their highest levels in arterial endothelial cells. In conclusion, the endothelial cells of the villous arterioles are the primary site of type 2 17ßHSD gene expression. This suggests a regulatory role for these cells in the control of progestin, androgen, and estrogen levels during pregnancy, thus opening a whole new way of viewing regionalization and localization of steroidogenesis in the human villi.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
AS THE DEVELOPING placenta differentiates during pregnancy (1), steroidogenesis increases, thus stimulating the placenta to secrete high levels of progesterone and estrogens [estrone (E1) and estradiol (E2)] (2). Consequently, concentrations of circulating maternal progesterone and estrogens rise progressively (3, 4, 5, 6). However, from the 20th week through until the end of gestation, the ratio between maternal plasma progesterone and E2 concentrations remains relatively constant (5, 7), suggesting that their levels are regulated. Such control of progesterone and E2 levels is important because these steroids exert opposing effects on myometrium contractile activity and also because E2 induces the timely vital maturation of fetal organ systems (8, 9). Therefore, control between the synthesis and the inactivation of the biologically active progesterone and E2 is important for a normal pregnancy in terms of fetal development and delivery time.

The production of placental progesterone depends on maternal lipoprotein-cholesterol delivery, whereas E2 depends on dehydroepiandrosterone produced by the fetal adrenals (2, 9, 10, 11, 12). Several steroidogenic enzymes have a role to play in these syntheses: cholesterol side-chain cleavage cytochrome P450 (P450scc), type 1 3ß-hydroxysteroid dehydrogenase (3ßHSD), aromatase cytochrome P450 (aromatase), and 17ßHSDs. Among 17ßHSDs, type 1 and type 2 isoforms, characterized by specific substrates and activities, control the amounts of biologically active 17-hydroxysteroids and inactive 17-ketosteroids produced by the placenta. Therefore, these two enzymes could be implicated in such a regulatory mechanism (13, 14, 15, 16). Type 1 17ßHSD exclusively reduces E1 into E2 (17, 18), whereas type 2, a dehydrogenase, oxidizes E2, testosterone, and 5{alpha}-dihydrotestosterone with similar reactivity in addition to exerting a 20{alpha}-dehydrogenase activity on 20{alpha}-dihydroprogesterone (20{alpha}DHP) (19, 20).

We have previously shown that type 1 17ßHSD and type 1 3ßHSD messenger ribonucleic acids (mRNAs), proteins, and enzymatic activity are present in syncytia formed in vitro, whereas the expression of the type 2 17ßHSD gene was not detectable in either freshly isolated cytotrophoblasts or the syncytium (17, 21). In fact, the latter is the major steroidogenic unit of the placenta and also expresses the P450scc and aromatase genes (22). However, high levels of type 2 17ßHSD activity and mRNA were detected in the term villi from which the trophoblasts were isolated (17). The disappearance of type 2 17ßHSD expression in cell cultures of cytotrophoblasts and the absence of reactivation of expression in syncytiotrophoblasts formed in vitro suggest either that trophoblast type 2 mRNA is stabilized in vivo by a regulatory factor(s) lost during the isolation procedure or that the type 2 17ßHSD gene is expressed not by trophoblasts but, rather, by other cell types present in villi.

In the present report we show that the pattern of type 2 17ßHSD gene expression in the human placenta is different from that of other expressed steroidogenic genes. We have, in fact, unequivocally identified the endothelial cells of the villous arterioles as the primary site of type 2 17ßHSD expression in the human term placenta. We also present strong evidence that the expression and activity of type 2 17ßHSD enzyme by the endothelial cells forming the wall of the arteries are higher than those of the veins. This suggests a role for these cells in the regulation of progestins, androgens, and estrogens, during pregnancy.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Human tissue preparation

Term placentas (38–42 weeks) with umbilical cords attached, membranes, and decidua were obtained after normal term spontaneous vaginal deliveries from 15 patients. Informed consent was obtained according to the policies of the human studies institutional review board of the Centre Hospitalier Universitaire de Québec and the HealthPartners Regions Hospital (St. Paul, MN). All tissues used were classified as normal placenta after routine pathological examination. Tissues were collected on ice and brought to the laboratory within 1 h after removal. To avoid contamination of the villi by maternal tissue or fetal membranes, a bed of 0.5 cm of tissue was removed on each side of the placenta, and then small pieces of villi (0.5 cm3) were cut, extensively rinsed in saline, embedded in Tissue-Tek OCT compound (Miles, Elkhart, IN), and kept at -70 C for immunohistochemistry (IHC) and in situ hybridization (ISH) as previously described (22). Figure 1Go illustrates the typical morphology of the term villi sections that we used in this study. These are fetal villi sections and were not contaminated by maternal septa or the basal plate. Each square indicates the precise area that was chosen for observation.



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  		Figure 1. Lower magnification of the tissue sections presented in Figs. 3–5GoGoGo. The inset in A corresponds to Fig. 3Go, E–H. The inset in B corresponds to Fig. 4Go, A–F and H. The inset in C was used for Fig. 4GGo. The inset in D was used for Fig. 5Go, A, C, and E. The inset in E was used for Fig. 5Go, B, D, and F. The inset in F was used for Fig. 5Go, G and H. Scale bar in A, 250 µm (A–H).

 
Northern blot preparation and hybridization

One hundred milligrams of tissue were homogenized in 1 mL Tri-Reagent, a mixture of phenol and guanidine thiocyanate in a monophasic solution (Molecular Research Center, Cincinnati, OH). Total RNA was extracted by adding 0.2 mL chloroform and was recovered from the upper phase by isopropanol precipitation. RNA samples were glyoxalized and electrophoresed as previously described (23, 24). Membranes were hybridized and washed under high stringency conditions (18). The following human complementary DNA (cDNA) probes were used: type 2 17ßHSD full-length fragment (17), type 1 17ßHSD EcoRI/SacI 964-bp segment (25), type 1 3ßHSD EcoRI/PvuII 1038-bp segment (25), and {gamma}-actin full-length fragments. Probes were labeled with [{alpha}-32P]deoxy-CTP to 2 x 106 dpm/ng with random primers (26).

Complementary RNA probes

RNA probes were synthesized from the 964-bp EcoRI/SacI fragment of the type 1 17ßHSD (18) and the 303-bp EcoRV fragment of the type 2 17ßHSD (17) inserted into pSV-SPORT-1 (23). Antisense and sense type 1 and type 2 17ßHSD RNA probes were synthesized after linearization of the plasmid DNAs with EcoRI and SacI for the type 1 and with BamHI and XhoI for the type 2, respectively, using [35S]UTP (NEN Life Science Products, Boston, MA). Probes were synthesized using the Riboprobe Combination System kit (Promega Corp., Madison, WI), and riboprobes with less than 1.4 x 109 dpm/µg DNA matrix were discarded.

IHC and ISH

Embedded samples were thawed and washed in 22 mmol/L K2HPO4, 3 mmol/L KH2PO4, and 140 mmol/L NaCl. Human placental lactogen (hPL) or vimentin antisera were diluted 1:5000 in 22 mmol/L K2HPO4, 3 mmol/L KH2PO4, and 140 mmol/L NaCl containing 0.4% Triton X-100 and 2.5 mg/mL heparin as blocking agent (176 USP units/mg; Sigma, St. Louis, MO), applied to slides (100 µL/slide), and left at 4 C overnight. All slides were treated as described above, except for negative controls, where antiserum was omitted. Biotinylated antibodies (antirabbit IgG for hPL and antimouse IgG for vimentin; Dimension Laboratories, Inc., Mississauga, Canada) were added for 90 min at room temperature. Immunostaining was revealed with an avidin-biotin peroxidase reaction method (27) using the ABC Vectastain kit (Vector Laboratories, Inc., Burlingame, CA) with diaminobenzidine (60 µg/mL; Sigma) as the chromagen. After IHC, slides were immediately processed for ISH. Tissues were fixed in 4% paraformaldehyde (Sigma) for 20 min, treated with proteinase K (10 µg/mL; Sigma) for 25 min at 37 C, acetylated in 37.5 mmol/L triethanolamine solution (Sigma) containing 0.25% (vol/vol) anhydric acid (Sigma) for 10 min, dehydrated in graded alcohol, and air-dried. Hybridization (2 x 106 dpm/100 µL/slide) was performed overnight at 60 C in 50% formamide, 0.3 mol/L NaCl, 10 mmol/L Tris (pH 8.0), 1 mmol/L ethylenediamine tetraacetate, 1 x Denhardt’s (100x = 2% BSA, 2% polyvinylpyrrolidone, and 2% Ficoll), 1% dextran sulfate, 10 mmol/L dithiothreitol, and 500 µg/mL transfer RNA. The slides were then treated with ribonuclease A for 30 min and washed in high stringency in 0.1 x SSC (standard saline citrate) and 1 mmol/L dithiothreitol at 60 C for 30 min. After defatting, tissues were coated with NTB-2 emulsion (Eastman Kodak Co., Rochester, NY) and kept at 4 C for 10 days. Slides were developed with D-19 solution (Eastman Kodak Co., Rochester, NY), counterstained with 0.25% (wt/vol) thionine (Sigma), dehydrated, and mounted with DPX (Electron Microscopy Sciences, Fort Washington, PA).

Isolation of umbilical cord blood vessels

Vein and arteries were mechanically separated, and about 5 cm of these were dissected free of connective tissue. Tissues were washed in ice-cold Dulbecco’s PBS, minced, homogenized, and fractionated into cytosol and microsomes.

17ßHSD activity in cytosol and microsomes

Samples were homogenized in 0.04 mol/L potassium phosphate, pH 7.0, containing 1 mmol/L ethylenediamine tetraacetate and 20% (vol/vol) glycerol, and centrifuged at 1000x g to remove cellular debris, then again at 105,000 x g for 1 h. Supernatants were saved as cytosol, and the pellets as microsomes. The microsomal and cytosolic17ßHSD specific activities were immediately assayed as described previously (28). Types 1 and 2 17ßHSD specific activities were obtained by calculating the conversion of E2 to E1 and the conversion of testosterone to androstenedione, respectively. Product yields were expressed as a percentage of total radioactivity recovered. The resulting percentage values were converted to nanomoles of steroid produced per mg protein/30 min (specific activity). High pressure liquid chromatography-purified labeled steroids were used as standards.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Progesterone and E2 synthesis in the placenta has been attributed to the villous trophoblasts. In a previous study we found that type 2 17ßHSD mRNA and activity were barely detectable in freshly isolated cytotrophoblasts and undetectable in syncytiotrophoblasts formed in vitro, whereas high levels of type 2 17ßHSD activity and mRNA were detected in the term villi (17). In the present study we first confirmed that the whole term villi (TV) expresses the type 2 17ßHSD gene (Fig. 2Go, lane TV). Then, TV was trypsinized; after removal of cytotrophoblasts, the resulting material, called trypsinized term villi (TTV), composed mainly of blood vessels and connective tissues, contained type 2 17ßHSD mRNA (Fig. 2Go, lane TTV). To confirm that the gene is not expressed by trophoblasts but, rather, by a different cell type remaining in TTV, Northern blots were probed with two specific markers of trophoblasts (type 1 17ßHSD and type 1 3ßHSD). TV was positive and TTV RNA was negative for both, showing the TTV sample was devoid of trophoblasts.



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  		Figure 2. Expression of type 2 17ßHSD, type 1 17ßHSD, and type 1 3ßHSD genes in TV and TTV. TTV results from trypsinization of TV according to the cytotrophoblast isolation procedure previously described (17 21 45 ) and corresponds to the material remaining after cytotrophoblast isolation. Total RNA samples (20 µg) were subjected to Northern blot analysis. The membrane was successively hybridized with the indicated specific cDNA probes. The autoradiograph was exposed 24 h. Similar results were obtained with RNA preparations from tissues of three other patients (data not shown).

 
ISH was used to identify the cells expressing type 2 17ßHSD. Sections of term villi were first analyzed by IHC using antibodies against a specific marker of trophoblasts (hPL) and against vimentin, which is expressed by both fibroblasts and endothelial cells. Then, ISH for both type 1 and type 2 17ßHSD mRNAs was performed on the same tissue sections. IHC with anti-hPL IgG produces the dark yellow immunoreactive pattern typical of hPL staining of the syncytium layer (29, 30, 31, 32) (Fig. 3AGo), whereas antivimentin stained blood vessels (Fig. 3BGo). As expected, the distribution of type 1 17ßHSD mRNA was limited to the syncytiotrophoblast layer of the villi (Fig. 3CGo) and colocalized with hPL-positive cells (Fig. 3Go, A–D). In contrast, cells expressing the type 2 17ßHSD gene corresponded to cells that compose blood vessel walls (Fig. 3Go, E–H). These cells were clearly negative for hPL immunoreactivity. By examining a greater magnification of the term villous sections, we unambiguously identified the endothelial cells as the only cell type expressing the type 2 17ßHSD gene (Fig. 4EGo, greater magnification of an arteriole identified in A and C) because only the first layer of cells is positive. As expected, these cells were negative for hPL (Fig. 4Go, A, C, and E), but were positive for vimentin (Fig. 4GGo). Similar results were obtained with a cadherin-5 antiserum that only detects endothelial cells (data not shown).



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  		Figure 3. Expression of type 1 17ßHSD and type 2 17ßHSD genes in human term villi. ISH was performed on a serial of adjacent tissue sections using an antisense (A–C) or a sense (D) type 1 17ßHSD RNA probe and an antisense (E–G) or a sense (H) type 2 17ßHSD RNA probe. Before ISH, IHC was performed using an antiserum to hPL (A, E, and G) or an antiserum to vimentin (B, C, and F; yellow/brown color). Weak background staining with antisera was caused by endogenous peroxidase. Tissue sections were counterstained with thionine (blue) and visualized by brightfield (A, B, E, and F) and darkfield (C, D, G, and H) microscope images. Type 1 17ßHSD mRNA was detected only in syncytium forming the edge of each villous (indicated by arrows in C compared with negative control in D), whereas type 2 17ßHSD mRNA was detected exclusively on the wall of blood vessels (indicated by an arrow in G compared with the negative control in H). The bright and straight line in G over the syncytial layer is caused by the hPL antiserum, and no hybridization is associated with this structure. These results were reproduced with material obtained from four different placentas (data not shown). T, Trophoblasts; BV, blood vessels. Scale bar in A, 50 µm (A–H). Magnification, x50.

 


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  		Figure 4. Identification of the cell type expressing type 2 17ßHSD gene in human term villi. Double labeling of term villi tissue sections by IHC and ISH with an antisense or sense type 2 17ßHSD RNA probes. A and C, hPL immunostaining and antisense RNA probe; B, vimentin immunostaining and antisense RNA probe; D, sense RNA probe only on adjacent tissue section of A; E, magnification of the artery shown in A; F, sense RNA probe on tissue section adjacent to A; G, vimentin immunostaining and antisense RNA probe on a different artery and tissue section; H, antisense RNA probe hybridization of the vein shown in A. Type 2 17ßHSD hybridizing signals were only detected on endothelial cells composing the wall of blood vessels (first row of cells). Again, the bright and straight line in C over the syncytial layer is caused by the hPL antiserum, and no hybridization is associated with this structure. The arrow in C and the arrowheads in E and G point to endothelial cells. A, Artery; V, vein. Scale bar in A, 50 µm (A–D); in E, 10 µm (E–H). Magnification: A–D, x50; E–H, x250.

 
Placental blood vessels form an extensive arteriole-capillary-venule network within the fetal villi. Where the umbilical cord attaches to the placenta, arteries divide and enter the villi to form arterioles. Arterioles can be distinguished from venules by the thickness of smooth muscle cells surrounding endothelial cells, which is thin or nearly absent in the venules. Deoxygenated blood containing waste products exits the fetus through the arterioles of the placenta before attaining the maternal circulation. In contrast, the placental venule system carries nutrients and oxygen to the fetus. Sections presented in Fig. 4Go (A–H; G is a different area) contain two blood vessels: based on their morphology, one is an arteriole (identified by A), and the other is a venule (identified by V). Only the arterial endothelial cells express the type 2 17ßHSD gene (Fig. 4Go, A, C, and E are from the same tissue section). The endothelial cells of the vein are negative by in situ hybridization (Fig. 4HGo, magnification of the venule in A). Other examples presented in Fig. 5Go, A and C, show two arterioles (identified with A) positive with the type 2 17ßHSD probe, whereas the two venules (identified by V) are negative. Similar results were obtained for tissue sections presented in Fig. 5Go, B and D. Besides these clear observations, some blood vessels remain difficult to identify as arterioles or venules. They are usually found at the terminal ramifications of an arteriole and correspond to capillaries. They form a network of capillaries that are collected by a venule. This could explain why some capillaries that seem to have the morphology of a small arteriole are negative when probed for type 2 17ßHSD expression (Fig. 5Go, G and H). Looking through several tissue sections from different placentas, we identified a total of 27 veins and 33 arteries. Of the veins identified, none contained detectable type 2 17ßHSD mRNA, whereas 82% of the blood vessels identified as arteries were positive (Table 1Go).



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  		Figure 5. Endothelial cells of arterioles are the major site of type 2 17ßHSD gene expression in human term villi. Human term villi tissue sections from three different placentas (first placenta, A, C, and E; second placenta, B, D, and F; third placenta, G and H) were hybridized with an antisense (A–D, G, and H) or a sense (E and F) type 2 17ßHSD RNA probe. Except for the negative control (E and F), tissue sections were first immunostained with hPL antiserum. Arterioles (A) and venules (V) are identified. Hybridizing signals are present on endothelial cells that composed the arterioles, whereas venules are negative. Scale bar in A, 50 µm (A–H). Magnification, x50.

 

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  		Table 1. Classification of signals obtained in function of the blood vessel type

 
To extend our study to the umbilical cord, mRNA levels of type 2 17ßHSD as well as type 1 17ßHSD and type 1 3ßHSD were compared in dissected umbilical cord arteries and veins (Fig. 6Go). Levels of type 1 and 2 17ßHSDs and type 1 3ßHSD mRNAs were very low in total cord samples compared with those observed in the villi. However, in agreement with results obtained by ISH in the villi, levels of type 2 17ßHSD mRNA were higher in the cord arterial sample (Fig. 6AGo) compared with the total cord sample. In contrast, the type 2 17ßHSD mRNA was not enriched in the cord vein, even though at least twice the amount of RNA was loaded on the gel. Therefore, endothelial cells of the cord vein, although they contain a minimum of type 2 17ßHSD mRNA, do not constitute the major site of type 2 17ßHSD gene expression in the placenta. This pattern of expression is specific to the type 2 17ßHSD gene, as type 1 17ßHSD and type 1 3ßHSD mRNA levels were not enriched in the cord artery or vein samples compared with the total cord (Fig. 6Go). The level of expression of type 2 17ßHSD mRNA per endothelial cell of the cord is very low compared with those of the villi, as ISH of umbilical cord sections are negative when cohybridized with a term villi section on the same slide (data not shown).



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  		Figure 6. Evidence of the specific association of type 2 17ßHSD gene expression with arterial cells of the human umbilical cord. Total (T) umbilical cord (UC) RNA (10 µg) and RNA samples prepared from dissected umbilical arteries (A; 10 µg) and vein (V; 20 µg) were subjected to Northern blot analysis using type 2 17ßHSD, type 1 17ßHSD, type 1 3ßHSD, and {gamma}-actin cDNA probes. The expression of the three steroidogenic genes was detected in total cord (T). Enrichment for arterial or vein material before RNA extraction led to an increase in the type 2 17ßHSD mRNA specifically in the arterial sample, but not in the vein sample. No enrichment was noted for type 1 17ßHSD and type 1 3ßHSD mRNA. The strong actin mRNA signal observed in the vein sample clearly indicates that type 2 17ßHSD is more abundant in the umbilical arteries than in the vein. Similar results were obtained with RNA preparations from the tissues of four other patients (data not shown).

 
17ßHSD activities were also studied in the cytosolic and microsomal fractions prepared from segments of the whole umbilical cord. Activities were assayed under conditions that differentiated between type 1 and type 2 17ßHSDs on the basis of their cellular localization and activity with E2 and testosterone (T). As shown in Fig. 7AGo, specific activities with E2 and T were greater in microsomal than in cytosolic fraction. Cytosolic E2 activity was not inhibited by T or 20{alpha}DHP (Fig. 7BGo), which is consistent with the presence of type 1 17ßHSD. Microsomal E2 activity was inhibited by T and 20{alpha}DHP, and that with T was inhibited by E2 and 20{alpha}DHP (Fig. 7CGo); these observations were characteristic of type 2 17ßHSD activity (33, 34). The observation in umbilical cord extracts that cytosolic 17ßHSD specific activity with E2 was lower than that of microsomes differs from the results obtained with trophoblast extracts where the cytosol to microsome activity ratio was approximately 5:1 (14).



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  		Figure 7. Specific 17ßHSD activity assayed with total umbilical cord fractions. A, 17ßHSD specific activity in umbilical cord in cytosol (C) and microsomes (M) assayed with [3H]E2 and [3H]T as substrates. The level of transformation of [3H]E2 and [3H]T indicates that type 1 17ßHSD is the principal activity present in cytosol, whereas in microsomes the results are characteristic of the presence of a type 2 activity. B, Effect of increasing concentrations of T and 20{alpha}-DHP on 17ßHSD activity assayed with [3H]E2 (reaction E2->E1) in the cytosol fraction of umbilical cord. The absence of inhibition by T and 20{alpha}DHP is characteristic of the presence of type 1 17ßHSD activity. C, Inhibitory effects of increasing concentrations of T, E2, and 20{alpha}DHP on 17ßHSD activity with [3H]E2 (open symbols) and [3H]T (reaction T->A; black symbols) in microsomes of umbilical cord. The inhibition observed is characteristic of the presence of type 2 17ßHSD activity.

 
Type 2 17ßHSD enzymatic activity was also studied in isolated cord veins and arteries to enable comparison with results obtained by ISH showing specific expression in arteries. Samples of cord vein and of artery from seven placentas were prepared and analyzed separately. Umbilical cord sections were taken 1 cm from the site of insertion into the placenta and vein, and arteries were carefully dissected free of surrounding tissue, washed extensively to eliminate residual cord blood, which contains high levels of type 1 17ßHSD activity (35), and fractionated into cytosol and microsomes. As shown in Table 2Go, specific activities with E2 and T were higher in microsomes than in cytosol for both artery and vein samples, with specific activity for the arterial sample approximately 2- to 4-fold greater than that for the vein sample (artery/vein activity ratios). The mean E2/T activity ratio was approximately 1.0–2.0 for both the arterial and venous microsomes. This is consistent with the presence of the type 2 17ßHSD isoform. We also compared the 17ßHSD activity found in the cord with that present in villi. The type 2 activity in the umbilical artery with T was about 16-fold lower than that observed in whole villi. Together, our results indicate that the type 2 17ßHSD gene in arterial endothelial cells is not expressed equally throughout the fetal arterial system, but, rather, is expressed along a gradient extending from the cord to the villi, where maximum expression has been detected. The expression of type 2 17ßHSD is undetectable by ISH within the venule system.


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  		Table 2. Metabolism of E2 and T in microsomes and cytosol of umbilical cord arteries and vein enriched extracts

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Although it is well established that the type 2 17ßHSD gene is expressed in villi, we failed to detect its mRNA in primary cultures of cytotrophoblasts and in syncytiotrophoblasts formed in vitro (17). Cytotrophoblasts are mitotic undifferentiated stem cells, and they can differentiate into syncytiotrophoblasts, which are mitotically inactive and constitute the most active steroidogenic placental cell type producing progesterone and E2 (1). Trophoblasts contain mRNAs encoding for P450scc (21, 22), type 1 3ßHSD (21, 22), type 1 17ßHSD (18, 22, 36), and P450 aromatase (22, 37, 38). This investigation of placental type 2 17ßHSD expression is important because this enzyme plays an important role in the control of progesterone and E2 secretion by the placenta and in the control of fetus-derived androgens. In this study we demonstrated that the type 2 17ßHSD gene is strongly expressed by endothelial cells of arterioles; this tissue specificity is consistent with a role for the enzyme in the control of progesterone, estrogens, and androgens during pregnancy.

Controlling the levels of active sex steroids within the maternal-feto-placental unit is crucial to many processes occurring during pregnancy, including the onset of labor, the timely maturation of some fetal organs, and the preparation of the maternal reproductive system for delivery. This could be particularly true for the control of the balance between progesterone and E2 because of their opposing actions on uterine contractility. More than 2 decades ago, Albrecht and Pepe clearly established the importance of the regulation of placental steroid production to normal fetal development. They identified E2 as an important signal in the process of maturation of the hypothalamus-pituitary-adrenal axis (9, 39).

Our data indicate that cells expressing the type 1 17ßHSD in the villi are immunoreactive to hPL and not vimentin (or cadherin-5). This is in agreement with previous in vivo studies that identified the syncytium as the predominant site of E2 synthesis (22, 40). From our results, the expression of the type 2 17ßHSD gene is an exclusive property of endothelial cells. No hybridization signal was detected elsewhere in the villi, including in trophoblasts. These observations are also in line with those of Takeyma’s study (40), in which immunoreactivity against the type 2 17ßHSD protein was associated with the endothelium of blood vessels. More importantly, our data unequivocally showed that all endothelial cells of the villi do not express the type 2 17ßHSD at similar levels, which is, in fact, characteristic of the endothelial cells that form the arterioles. All the more convincing, positive arteriole endothelial cells and negative venous endothelial cells colocalized to the same tissue section. A recent in situ hybridization study reported the presence of type 2 17ßHSD mRNA in villous cytotrophoblasts of human placenta (41). This contradicts both previous observations (42) and our present observations. It should be noted that here positive signals were strong and clearly above the background observed with the negative control. In addition, the hybridization results presented here are in agreement with the study of activities in umbilical cord. In fact, the magnification showed in that particular study (41) should discourage any absolute conclusions as to the site of expression. In our study we present ISH photographs at magnifications of x250, making endothelial cells easily distinguishable from other cell types, including trophoblasts. Moreover, we have based the cell type identification on both the morphology of blood vessels and specific phenotypic markers.

Barely detectable signals for type 2 17ßHSD mRNA were also observed by ISH in the umbilical cord (data not shown). These data are in agreement with the lower levels of mRNA observed by Northern blot analysis and activity detected in the cord arteries compared with the villi (this study and Ref. 17). The molecular mechanism regulating this difference is unknown, but it would be interesting to determine whether the type 2 17ßHSD gene is subject to regulation, whether distinct subpopulations of arterial endothelial cells express the gene differently, or both. In fact, the existence of a difference in type 2 17ßHSD gene expression between the cord and the placenta argues in favor of the presence of a regulatory factor(s).

Northern blot indicated that the type 2 17ßHSD mRNA is also present in the venous system. However, based on the activity detected in the cord vein (~3-fold less than the cord arteries and about 48- to 80-fold less than the villi), its expression level is very low. If the level of activity and expression present in the venous system represents a physiological condition, a possible function of type 2 17ßHSD enzyme in the vein could be in the regulation of estrogen action on vascular tone during pregnancy (43, 44).

This work opens up a whole new way of looking at regionalization and localization of steroidogenesis in the villi and could be indicative of intricate communication between trophoblasts and endothelial cells in the regulation of sex steroid release by the placenta. Such tissue and site specificity of type 2 17ßHSD gene expression is likely to have many important implications and suggests the existence in the villi of a control in the production of progestins, estrogens, and androgens during pregnancy in human.


   Acknowledgments
 
We thank Dr. Cindy Goodyer for her help in collecting tissues, Dr. Jodell Allen for identification of arteries and veins, Mrs. Denise Ramsey for technical assistance with the determination of activities, and Dr. Serge Rivest for helpful discussions.


   Footnotes
 
1 This work was supported by the Medical Research Council of Canada (to Y.T.; MT14365) and Grant N609 from HealthPartners Research Foundation (to C.H.B.). A preliminary report was presented at the 81st Annual Meeting of The Endocrine Society, San Diego, CA, 1999. Back

2 Recipient of a senior scholarship from le Fonds de la Recherche en Santé du Québec. Back

Received February 15, 2000.

Revised June 23, 2000.

Revised August 15, 2000.

Accepted September 1, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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