Biol Reprod Email Content Delivery
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow My Folders
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Cudeville, C.
Right arrow Articles by Ferré, F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Cudeville, C.
Right arrow Articles by Ferré, F.
Agricola
Right arrow Articles by Cudeville, C.
Right arrow Articles by Ferré, F.
Biology of Reproduction 62, 759-765 (2000)
© 2000 Society for the Study of Reproduction, Inc.

Articles

Evidence for Progesterone Receptors in the Human Fetoplacental Vascular Tree1

Corinne Cudevillea, Françoise Mondona, Brigitte Roberta, Régis Rebourceta, Thérèse-Marie Mignota, Claudine Benassayaga, and Françoise Ferré2,a

a INSERM U. 361, Université René Descartes Pavillon Baudelocque, 75014 Paris, France


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The presence of progesterone receptors (PR) throughout the human term fetoplacental vascular tree was investigated. By reverse transcription-polymerase chain reaction (RT-PCR), we showed expression of PR mRNAs in stem villi vessels, chorionic arteries and veins, and umbilical arteries and veins. Binding studies and Scatchard analysis revealed a single class of high-affinity binding sites for 3H-R5020 (promegestone) in cytosolic extracts of all placental vessels, with Kd values in the range of 2.5–4 nM. High levels of PR were detected in placental vessels compared to other vascular tissues. Thus, maximum binding capacities of stem villi vessels, chorionic arteries and veins, and umbilical arteries and veins were 247 ± 25, 377 ± 58, 295 ± 40, 371 ± 118, and 672 ± 144 fmol/mg protein, respectively. Endothelial cell elimination in chorionic arteries did not significantly modify the number of PR. RT-PCR and binding studies also assessed PR expression in cultured placental vascular smooth muscle cells isolated from stem villi vessels. All these data suggested that most of the PR of fetoplacental vessels were from the media.

In conclusion, we report here the first evidence of the presence of PR in the muscular layer of human term fetoplacental vessels. This finding, together with the high progesterone concentrations in cord blood, suggests that the interactions between the PR and its ligand may play a role in the physiology and physiopathology of human fetoplacental vascularization.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Fetal growth and well-being mainly depend on the uterine and fetoplacental blood flows, which must be adequate to ensure exchanges between maternal and fetal compartments through the placental "barrier." Human fetoplacental vascularization is original in some respects and possesses its own morphologic, structural, and functional characteristics. One aspect of this originality is that placental and umbilical vessels lack autonomous innervation; consequently, regulation of fetoplacental vascular tone must depend on the balance between vasoconstrictive substances such as thromboxane A2 [1, 2], angiotensin [3], and endothelins (ET) [4–6] and vasodilator agents such as prostacyclin and nitric oxide (NO) [7], which are either produced locally or conveyed to their site of action through the bloodstream. Several reports also emphasize the close relationship between vasoactive substances and steroid hormones. Thus, the steroid hormone environment can modulate ET and ET receptor expression [8–10]. NO inhibits steroidogenesis, and especially progesterone synthesis, in granulosa cells [11], while ET-1 and ET-3 stimulate progesterone production in trophoblastic cells [12]. It is well known that the human placenta produces large quantities of progesterone throughout pregnancy until term (250–600 mg of progesterone daily): 75% of this production goes into the maternal circulation and 25% into the fetal placental circulation. The effect of progesterone on the myometrium and its role in the maintenance of gestation and the onset of parturition have been extensively described [13]. Although the vasoactive role of progesterone in the various vascular systems now seems well established [14], the effects of progesterone on the fetoplacental vessels remain largely unknown. However, a recent study showed that progesterone could cause rapid dose-dependent relaxation of the placental vascular smooth muscle from chorionic arteries and veins by an endothelium-independent mechanism [15]. Moreover, progesterone inhibits the proliferation of cultured human vascular smooth muscle cells of the umbilical vein induced by mitogenic agents such as ET-1 [16].

In target tissues, most of the effects of progesterone take place via nuclear receptor proteins. These receptors act as transcriptional factors that regulate specific gene expression by interacting with their cognate DNA sequence in response to binding of the steroid hormones. The presence of progesterone receptors (PR) in the arterial blood vessels of the reproductive tract [17], as well as in the human saphenous veins [18], indicates that progesterone may modulate the blood flow through a receptor-mediated pathway. In human placenta, the existence of PR has long been a matter of debate [19–24] and remains controversial [17, 25–29]. No data are currently available concerning PR on placental vascular smooth muscle.

Therefore, the aim of this study was to evaluate the distribution of PR throughout the human fetoplacental vascular tree in stem villi vessels, chorionic arteries and veins, and umbilical arteries and vein. We first analyzed the pattern of the mRNA of PR using a reverse transcription-polymerase chain reaction (RT-PCR) approach; we then determined the concentrations and affinity constants of PR in these vessels by binding studies.

We demonstrated for the first time that PR are present in fetoplacental vessels especially in the muscular layer of the vessels. These results were confirmed with cultured placental vascular smooth muscle cells (PVSMC) of stem villi vessels.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Placentas

Placentas were aseptically obtained immediately after elective cesarean section from healthy mothers, not on medication, in the 39th week of pregnancy. The cesarean sections, carried out before the onset of labor, were performed because of earlier diagnosed cephalopelvic disproportion.

Isolation of Placental Vessels

Umbilical arteries and veins, chorionic arteries and veins isolated before they enter into the plate, and stem villi vessels were the vessels studied and compared as described below. After identification in situ and isolation, all vessels were meticulously dissected and immediately placed in ice-cold Krebs solution (120 mM NaCl, 5.7 mM KCl, 2.5 mM CaCl2, 1.2 mM NaHCO3, and 11.5 mM glucose, pH 7.4). They were cleaned of blood traces and carefully freed from all surrounding fat and adherent connective tissues. In chorionic artery studies, some of the vessels were cleared of their endothelial cells: vascular segments were longitudinally cut, and the endothelial cell layer was removed by scraping. For stem villi vessel isolation, placental tissue (1–2 cm3) samples were swiftly removed between the decidual and chorionic plates and immersed in ice-cold Krebs solution; then branches of stem villi vessels (~200–300 µm mean internal diameter) were isolated by fine mechanical dissection in order to remove nonvascular tissue. After collection, all biopsies were either immediately dry frozen at -80°C for cytosolic and nuclear extracts for binding studies, frozen in Trizol reagent for RNA isolation and stored at -80°C until processed, or fixed in formalin for histological studies.

Histological Studies

For histological studies, fragments of chorionic arteries (3–5 mm3) were fixed in formalin and embedded in paraffin. For each vessel, one of the sections was stained with hematein-eosin-safran to assess general morphology. The presence of endothelial cells and, conversely, the removal of the endothelium, were controlled by using a CD34 mouse monoclonal antibody (Immunotech, Marseille, France), amplified and revealed with the APAAP system (Dako, Trappes, France). While a positive label was clearly detected before endothelium stripping, no label was evidenced after removal of endothelial cells.

Blood Samples

Maternal peripheral blood and cord blood samples (n = 16) were collected at the time of delivery in EDTA-containing tubes. Resulting plasmas were stored at -80°C until use.

Myometria from Nonpregnant Women

Biopsies of myometrial tissue were collected in the healthy myometrial zone from cyclic women (aged 39–51 yr) who underwent hysterectomies for benign gynecological indications such as leiomyoma. Biopsies were immediately frozen in Trizol reagent for RNA isolations and stored at -80°C.

This study was approved by the Consultative Committee of Protection of Persons in Biomedical Research Paris Cochin (France).

Culture PVSMC

PVSMC were obtained by the explant method described by Libby and O'Brien [30], with minor modifications according to Bourgeois et al. [5]. Briefly, immediately after isolation, branches of stem villi vessels were scraped so that the surrounding trophoblast, stroma, adventitia, and endothelial cells were removed. Placental vascular smooth muscles were cut into fine pieces (1–2 mm3) and placed in 6-cm Petri dishes (Costar, D. Dutscher, France), and each was covered with one drop of fetal bovine serum (FBS). The dishes were kept at 37°C in a humidified 5% CO2-95% air atmosphere. The following day, each explant was covered with growth medium (Dulbecco's modified Eagle's medium supplemented with 10% FBS) until reaching confluence in 25–30 days. The homogeneous layers of cells were then subcultured; cells were harvested with trypsin-EDTA, plated at 106 cells/75 cm2 flask, and grown to confluence in growth medium. Cells were studied between the second and the sixth passage.

In order to control and eliminate possible cell contamination, PVSMC were tested by RT-PCR for expression of the CD45 antigen mRNA, a specific marker of lymphomonocyte strain cells, and for the expression of Von Willebrand factor mRNA, a specific marker of endothelial cells [5].

RIA of Progesterone

Plasma samples (0.5 ml) were extracted three times by ethyl ether in the presence of 1000 cpm of [1,2,6,73H]progesterone (95 Ci/mmol; Amersham, Buckinghamshire, UK) as internal standard. After they were dry, the extracts were suspended in human male plasma and assayed with the radioimmunological kit 125I-progesterone Coatria (bioMerieux, Hazelwood, MO) according to the manufacturer's recommendation. The detection limit was 0.05 ng/ml, and the intra- and interassay coefficients of variation were 6% and 8%, respectively. All plasma samples were assayed in duplicate.

Preparation of Cytosolic and Nuclear Fractions for Binding Studies

Each placental vessel sample was thawed on ice and homogenized in ice-cold TEG buffer (1.2 mM Tris, 1.5 mM EDTA, 10% glycerol, and 2 mM natrium molybdate-dihydrate [NA2MOO4], pH 7.4) using a Polytron homogenizer at a tissue:buffer ratio of 1:5 (w:v). The homogenate was centrifuged at 1000 x g for 10 min at 4°C. The supernatant was then ultracentrifuged at 105 000 x g. The resulting supernatant corresponding to the cytosolic fractions was kept at -80°C before use for binding studies. Crude nuclear pellets isolated from the homogenate by 1000 x g centrifugation were drained well and extracted on ice for 1 h in TEG containing 0.5 M NaCl (v:v) with occasional vortexing. The salt extracts were then diluted in 3 volumes of TEG and ultracentrifuged at 105 000 x g at 4°C for 1 h. The supernatants corresponding to nuclear fractions were removed, aliquoted, and stored at -80°C until analyzed.

Binding Analysis of PR

Before the binding assay, cytosolic and nuclear fractions were treated for 1 h at 4°C with a dextran-coated charcoal pellet (10% Norit-A; Merck, Nogent Sur Marne, France, and 0.1% dextran T70) to remove endogenous steroids. In saturation kinetics of the PR cytosolic fraction, duplicate aliquots of charcoal-treated cytosol (300–500 µg protein) were incubated (16 h at 4°C) with increasing concentrations (0.4–16 nM) of 17{alpha}-methyl 3H-R5020 (promegestone) (85 Ci/mmol; NEN Life Science Products, Boston, MA) in the presence or absence of a 500-fold molar excess of unlabeled progesterone, and the final volume of the incubation mixture was adjusted to 0.5 ml. After incubation, a 10-µl aliquot was taken for measurement of total radioactivity. Bound and free hormone fractions were separated by incubating in an equal volume of suspension of 1% charcoal and 0.1% dextran (w:v) for 10 min at 4°C followed by centrifugation at 1500 x g for 10 min. Aliquots of supernatants were counted in 4 ml of Ultima Gold scintillation fluid (Packard, Rungis, France) in a Beckman (Palo Alto, CA) scintillation counter. The binding parameters were estimated by Scatchard graphical analysis after subtraction of nonspecific binding.

Comparatively, the nuclear PR concentration in the various placental vessels was determined by a single saturating dose assay: duplicate aliquots of charcoal-treated nuclear fraction (300 µg protein) were incubated with a saturating concentration (12 nM) of 3H-R5020 (promegestone) for 16 h at 4°C in the presence or absence of a 500-fold molar excess of unlabeled progesterone. After incubation, the free steroid fraction was adsorbed from the sample by treatment with charcoal-dextran for 10 min (total minus nonspecific binding). In the same way, the cytosolic PR concentration was determined with the same procedures for each analyzed vessel. Values were expressed as fmol 3H-R5020 bound per gram of analyzed tissue. The relative percentages of cytosolic fraction PR and nuclear fraction PR in relation to total PR from the same placental vessel extract were calculated.

RNA Preparation and Reverse Transcription

Total RNA was extracted from placental vessels, myometria, and vascular smooth muscle cells using the Trizol reagent (Life Technologies, Cergy, France) according to the method of Chomczynski and Sacchi [31]. Briefly, placental vessels and myometria were homogenized in Trizol reagent at a tissue:buffer ratio of 1:10 (w:v) using an Ultra-Turrax homogenizer (Janke & Kunkel KG, Staufen, Germany). Scraped cells (107 cells) were resuspended in 1 ml of Trizol and homogenized by repeated pipetting. RNA-DNA and protein was separated by adding chloroform, and RNA was recovered by isopropanol precipitation.

Reverse transcription (RT) was performed using random hexanucleotides (20 µM) as primers on 4 µg of total RNA in the presence of 200 U of Moloney murine leukemia virus reverse transcriptase in a final volume of 25 µl at 39°C for 60 min according to the manufacturer's specifications (Life Technologies). The cDNA products were stored at 20°C until required for PCR.

PCR and Southern Blot Analysis

The primers used to amplify human PR cDNA were determined in the hormone-binding region: upper oligonucleotide (sense) 5'-GTGGGCGTTCCA AATGAAAGCCAAG-3' (priming site in exon 4, nucleotide 1978) as numbered previously [32] and lower oligonucleotide (antisense) 5'-AATTCA ACACTCAGTGCCCGGGACT-3' (priming site in exon 8, nucleotide 2690) (Genosys Biotechnologies, Montigny-Le-Bretonneux, France). For amplification, 1/10 of cDNA reaction mixture was used in the presence of 0.1 mM each dNTP. The concentrations, in 25 µl PCR buffer were, primers, 0.5 µM; MgCl2, 2 mM; and 2.5 U of Taq polymerase (Life Technologies). The amplification profile consisted of 34 cycles with denaturation at 92°C for 1 min, annealing at 58°C for 1 min, and extension at 72°C for 1 min with a final extension at 72°C for 10 min.

The specificity of each amplification product was checked by gel electrophoresis analysis of the predicted length, cleavage by a restriction enzyme specific to PR cDNA sequence, and hybridization with a reverse internal oligonucleotide (5'-AAAATACAGCATCTGCCAC-3'). Southern blot analysis was performed as previously described [33] except for hybridization, which was performed using an oligonucleotide labeled with fluorescein-11-deoxy-UTP by an ECL 3' oligolabeling and detection system kit (Amersham, Pharmacia Biotech, Courtaboeuf, France) according to the manufacturer's instructions. Lack of genomic DNA contamination was checked in all experiments by means of a control reaction containing mRNA without reverse transcriptase. In order to control for errors in input of cDNA used in PCR reactions, amplification of human endogenous glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA was performed in parallel using an additional pair of primers (Ozyme, Saint Quentin Yuelines, France).

Protein Concentration

The protein concentration was determined in each cytosolic and nuclear fraction by the "dye-binding assay" (Bio-Rad, Richmond, CA) with BSA as the standard [34].

Statistical Analysis

All results were expressed as the means ± SEM. Statistical comparisons between multiple means for progesterone concentrations and for binding kinetic parameters were performed using one-way ANOVA followed by Scheffe's test for differences among means. The difference was considered to be significant when P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Quantification of Progesterone in Maternal and Cord Bloods

The levels of progesterone in the peripheral maternal blood and fetal blood (umbilical arteries and vein) were measured by RIA and compared. The results indicated a differential progesterone distribution among the compartments. Table 1 shows that progesterone concentrations in maternal blood (311 ± 38 nM) were significantly lower than in umbilical arterial blood (734 ± 174 nM, P < 0.05) and umbilical venous blood (1099 ± 180 nM, P < 0.001) The progesterone concentration was significantly higher in venous than in arterial umbilical blood (P < 0.05).


View this table:
[in this window]
[in a new window]
  		TABLE 1. Plasma progesterone (P4) concentration in maternal peripheral vein (MV) umbilical arteries (UA) and umbilical vein (UV).*

Messenger RNA Expression of PR in Fetoplacental Vessels

The expression of the PR gene was investigated in total RNA prepared from stem villi, chorionic, and umbilical vessels. Human myometrium was used as a positive control. After RT-PCR amplification using external oligonucleotides specific for the PR sequence, a product of the predicted size (737 base pairs [bp]) was obtained in stem villi vessels (Fig. 1A, lane 1), in chorionic arteries and veins (Fig. 1A, lanes 2 and 3), in umbilical arteries and veins (Fig. 1A, lanes 4 and 5), and in the positive control (Fig. 1A, lane 6) as revealed by ethidium bromide staining after agarose gel electrophoresis. When reverse transcriptase was omitted, no amplification product was observed (Fig. 1A, RT-). Positive amplification of the GAPDH gene led us to assert the integrity of our placental vessel mRNA preparations and the effectiveness of RT (Fig. 1B). The resulting DNA fragment of 737 bp obtained from all fetoplacental vessels was digested with restriction endonuclease HindIII, which has a specific site in the PR sequence. After Southern blot and hybridization with an internal oligonucleotide specific for the PR sequence, the undigested 737-bp (Fig. 2, HindIII-) and the 455-bp HindIII-treated (Fig. 2, HindIII+) amplification products were labeled.



View larger version (52K):
[in this window]
[in a new window]
  		FIG. 1. A) Expression of PR mRNA analyzed by RT-PCR in stem villi vessels (lane 1), chorionic arteries and veins (lanes 2 and 3), umbilical arteries and veins (lanes 4 and 5), and positive control, human myometrium (lane 6). The amplification reaction consisted of 34 cycles of the following sequential steps: 92°C for 1 min, 58°C for 1 min, 72°C for 1 min. Lanes 1–6: Ethidium bromide-stained gel. Reverse transcriptase was added (RT+) or omitted (RT-). Lane 7 corresponds to the 123-bp DNA ladder (Life Technologies). Data were representative of at least four similar experiments performed with preparations of four distinct placentas. B) Expression of GAPDH mRNA analyzed by RT-PCR (30 cycles: 1 cycle 2 min at 92°C, 1 min at 59°C, and 1 min at 72°C) in stem villi vessels (lane 1), chorionic arteries and veins (lanes 2 and 3), and umbilical arteries and veins (lanes 4 and 5)



View larger version (26K):
[in this window]
[in a new window]
  		FIG. 2. Southern blot analysis of RT-PCR products probed with a fluorescein-labeled forward reverse internal PR oligonucleotide. RT-PCR products for PR of stem villi vessels (lane 1), chorionic arteries and veins (lanes 2 and 3), umbilical arteries and veins (lanes 4 and 5), and positive control human myometrium (lane 6) mRNA expression were digested (HindIII+) or not (HindIII-) by restriction endonuclease HindIII. Data were representative of two similar experiments performed on two distinct placentas

These data demonstrated the expression of PR mRNAs in all fetoplacental vessels studied.

PR Binding Studies in Fetoplacental Vessels

Specific binding of increasing concentrations of 3H-R5020 (promegestone) to cytosolic PR of fetoplacental vessels was saturable whatever the vessel studied. Scatchard analysis of each vessel revealed a single class of high-affinity binding sites. The apparent dissociation constant (Kd) and the maximum binding capacity (Bmax) in each case are summarized in Table 2. The Kd values for each type of vessel did not differ significantly (2.7 ± 0.5 to 3.8 ± 1.8 nM). No statistical difference was established between the number of binding sites from stem villi vessels, chorionic arteries, chorionic veins, and umbilical arteries (247 ± 25, 377± 58, 294 ± 40, and 371 ± 118 fmol/mg protein, respectively). In comparison, the cytosolic PR concentration in the umbilical veins (672 ± 144 fmol/mg protein) was found to be significantly higher (P < 0.05). Binding studies using a single saturating dose assay concentration of 3H-R5020 were developed for comparing the relative percentages of PR in the cytosolic and nuclear extracts from the different placental vessels. The percentages of cytosolic and nuclear PR with respect to total receptors were calculated. The data indicated that, whatever the type of vessel analyzed, the majority of PR were found in the cytosol, and the nuclear fraction represented only about 8–10% of total PR.


View this table:
[in this window]
[in a new window]
  		TABLE 2. Apparent affinities (Kd) and binding capacities (Bmax) of cytosolic progesterone receptors of the different feto-placental vessels

PR Binding Studies on Chorionic Arteries After Removal of Endothelium

The real participation of the media in PR binding was determined after vessel dissection for removing endothelial cells. Scatchard plots determined by saturation binding studies of 3H-R5020 on cytosolic PR from chorionic arteries with or without endothelium (Fig. 3) indicated that the Kd values were in the same range of magnitude (respectively, 3.8 ± 1.8 and 3.3 ± 1.2 nM). Likewise, after removal of endothelial cells, the number of cytosolic PR for chorionic arteries was not significantly altered (Bmax: 377 ± 58 fmol/mg protein for arterial vessel with endothelium; 358 ± 65 fmol/mg protein without endothelium), showing the presence of these receptors mainly in the muscular layer.



View larger version (16K):
[in this window]
[in a new window]
  		FIG. 3. Scatchard analysis of 3H-R5020 binding to chorionic arteries with (solid circles) or without (open circles) endothelium. Cytosolic preparations were incubated with increasing concentrations of 3H-R5020 (0.4–16 nM) in the presence or absence of a 500-fold molar excess of unlabeled progesterone. Each point represents the mean of duplicate determinations obtained from a single representative experiment; four separate experiments were performed from four distinct placentas

Messenger RNA Expression and Binding Studies of PR in PVSMC from Stem Villi Vessels

Expression of the PR gene was investigated in total RNA prepared from confluent PVSMC. After RT-PCR amplification using an oligonucleotide specific to the PR sequence, a product of the predicted size (737 bp) was obtained in PVSMC (Fig. 4, lane 1) and in human myometrium (Fig. 4, lane 2). When reverse transcriptase was omitted (RT-), no amplification product was observed.



View larger version (56K):
[in this window]
[in a new window]
  		FIG. 4. Expression of PR mRNA analyzed by RT-PCR in PVSMC (lane 1) and positive control, human myometrium (lane 2). The amplification reaction consisted of 34 cycles of the following sequential steps: 92°C for 1 min, 58°C for 1 min, 72°C for 1 min. Lanes 1–6: Ethidium bromide-stained gel. Reverse transcriptase was added (RT+) or omitted (RT-). Lane 3 corresponds to the 100-bp DNA ladder (Life Technologies). Data were representative of two similar experiments performed with two distinct PVSMC cultures

Using a single saturation dose assay of 3H-R5020, the presence of PR was confirmed in PVSMC. Indeed, specific binding sites in the same range of concentration (299 fmol/mg protein) as those of stem villi vessels (247 ± 25 fmol/mg protein) were found.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This study is the first to show evidence for the presence of PR in human fetoplacental vessels in term pregnancy. We demonstrate that the muscular layer of fetoplacental vessels is a potential target tissue for progesterone.

The lower progesterone concentrations measured in the peripheral maternal venous blood as compared to the blood of the umbilical vein and arteries are in agreement with previous reports [35–37]. The higher progesterone rate found in the umbilical vein in comparison to umbilical arteries may be related to the fact that the trophoblast is the major site of progesterone production. A fraction of this hormone reaches the fetus, where it is used as a precursor for fetal steroidogenesis. The presence of high concentrations of progesterone in fetoplacental circulation also warranted investigation with respect to its possible role in regulation of vascular tree functions via a receptor-mediated process.

Literature on the existence of PR in the human placenta shows many discrepancies. When receptors were found, results were contradictory concerning cellular localization as well as their quantity throughout placental development [19–29]. Using RT-PCR and primers to the hormone binding region of the PR gene, we show that PR mRNAs were detected in every type of fetoplacental vessel. In parallel, hormone binding assays attesting to the functionality of the PR hormone binding domain were performed. It is well known that steroid hormone receptors are classically nuclear receptors that continually shuttle between the nucleus and the cytoplasm. These receptors are hormone-activated transcription factors that regulate the expression of specific genes by binding to steroid-responsive elements. As described by other authors in different tissues, specific progesterone binding sites representative of the nuclear untransformed receptors were mainly characterized (90%) in the cytosolic extracts of fetoplacental vessels; only 8–10% of PR remain in the nuclear extracts. The binding characteristics of cytosolic PR throughout the placental vasculature revealed specific high-affinity binding sites for 3H-promegestone, with Kd values in the nanomolar range for all studied vessels. High levels of PR (247 to 672 fmol/mg protein) were detected in placental vessels compared to other vascular tissues [18, 38, 39].

The unaltered PR concentration in the chorionic arteries after removal of endothelial cells strongly suggested that the majority of these receptors were in the media of the fetoplacental vessels. However, this study did not rule out the existence of PR in endothelial cells—all the more so because the muscular layers represent higher cell numbers than the monolayer endothelium. The presence of PR in endothelial cells is not well established in the vascular system, for example in saphenous vein [18], but PR have been localized in endothelial cells from decidual vessels [40]. The confirmation that PR were present in the placental vessel muscular layer was provided by our experiments with vascular smooth muscle cells isolated from stem villi vessels (PVSMC). Indeed, we demonstrated by RT-PCR the presence of PR transcripts, and we identified specific progesterone binding sites in these cells. These results are in agreement with recent work localizing PR in the muscular layer of various human vessels [14].

The vasoactive properties of progesterone alone or in combination with other steroid hormones, especially estradiol-17ß, are now well documented in other vascular systems [41]. Thus it has been reported that progesterone may be responsible for endothelium-independent vessel relaxation [42]. It is still not fully understood whether all the effects of progesterone on the vascular system are totally dependent on classical intracellular PR. Indeed, in human placental vessels, relaxation occurring rapidly after progesterone exposition may be mediated by a receptor localized on the membranes of the smooth muscle cells [15]. Such nongenomic effects of steroid hormones have been shown in many tissues [43], in particular in reproductive tissues [44]. In the placenta, genomic and nongenomic actions of steroid hormones via nuclear and membrane types of PR may synergize, leading to adequate uterine and fetoplacental blood flows necessary for ensuring fetal growth and well-being throughout pregnancy. In addition to its vasoactive effects, progesterone might inhibit proliferation of vascular smooth muscle cells [9, 45].

Little is known of the mechanism responsible for the physiological adaptation of fetoplacental vascularization during pregnancy. The noticeable presence of PR in placental vessels provides a potential cellular mechanism for their participation in blood vessel formation or remodeling. The demonstration of PR in human PVSMC isolated from stem villi vessels suggests a role for progesterone in the control of proliferation and/or differentiation of these cells, important events in placental vascular physiology and pathology. The role of progesterone and PR isotypes in sustaining vascular placental blood flow and proliferation, together with the action of other vasoactive substances such as estrogen, ET, NO, and eicosanoids, requires further clarification. Moreover, as previously discussed [46], the differential expression of PR isoforms (A/B) might generate a system of tissue-specific regulation of progesterone action. In this work, the primers used did not enable discrimination between PR isoforms A and B. Thus, further studies are needed to elucidate the proportional representation of PR isotypes throughout the placental vascular tree, especially since specific functions are devoted to each vessel type in the placental vascular tree. We have previously shown that fetoplacental vessels have a particular profile of contractile protein expression in comparison to smooth muscle adult tissues. The composition of the contractile apparatus changes gradually from umbilical to stem villi vessels, suggesting regional modulation of fetoplacental blood flow [47]. Indeed, comparison of the relative response from diverse vasoactive agents between the fetoplacental vessels indicated markedly different sensitivity to these substances, suggesting specific contribution from each vessel to the maintenance of placental vascular tone [48]. In particular, some evidence indicated that stem villi vessels represent the resistance vessels of the fetoplacental vasculature [49]. During pregnancy, a progressive decrease in fetoplacental vascular resistance normally occurs [50], leading to an increase in fetoplacental blood flow that is necessary for healthy growth of the fetoplacental unit. But several hypertensive diseases such as preeclampsia are associated with abnormally high resistance of fetoplacental vessels and reduction of placental perfusion, increasing the incidence of intrauterine growth retardation.

In conclusion, the present study indicates that progesterone may well contribute to the regulation of fetoplacental vascular smooth muscle cell phenotype by receptor-mediated processes during physiological and physiopathological development of the fetoplacental unit.


   ACKNOWLEDGMENTS
 
This work was supported by grants from the Foundation for Medical Research, Paris, from the Institut National de la Santé et de la Recherche Médicale and from the Université René Descartes Paris V (UFR, Cochin Port-Royal). We are grateful to Madame Vacher-Lavenu and Madame Hagnéré for the histological study. We thank G. Delrue (SC6 INSERM) for photographic work and Madame M. Verger for her secretarial aid. The authors are grateful to J. Bram for editorial help.


   FOOTNOTES
 
First decision: 7 September 1999.

1 Supported by Institut National de la Santé et de la Recherche Médicale and by University René-Descartes. Back

2 Correspondence: F. Ferré, INSERM U. 361, Pavillon Baudelocque, 123, Boulevard de Port-Royal, 75014 Paris, France. FAX: 33 143 26 44 08; u361{at}cochin.inserm.fr Back

Accepted: October 29, 1999.

Received: August 5, 1999.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Poston L, McCarthy AL, Ritter JM. Control of vascular resistance in the maternal and feto-placental arterial beds. Pharmacol Ther 1995; 65:215–239.[CrossRef][Medline]
  2. Boura ALA, Walter WAW, Read MA, Leicht IM. Autacoids and control of human placental blood flow. Clin Exp Pharmacol Physiol 1994; 21:737–748.[Medline]
  3. Kalenga MK, de Gasparo M, Thomas K, de Hertog R. Angiotensin II and its different receptor subtypes in placenta and fetal membranes. Placenta 1996; 17:103–110.[Medline]
  4. Mondon F, Malassiné A, Robaut C, Vial M, Bandet J, Tanguy G, Rostene W, Calvero I, Ferré F. Biochemical characterization and autoradiographic localization of [125I] endothelin-1 binding sites on trophoblast and blood vessels of human placenta. J Clin Endocrinol Metab 1993; 76:237–244.[Abstract]
  5. Bourgeois C, Robert B, Rebourcet R, Mondon F, Mignot TM, Duc-Goiran P, Ferré F. Endothelin-1 and ETA receptor expression in vascular smooth muscle cells from human placenta: a new ETA receptor messenger ribonucleic acid is generated by alternative splicing of exon 3. J Clin Endocrinol Metab 1997; 82:3116–3123.[Abstract/Free Full Text]
  6. Sabry S, Mondon F, Levy M, Ferré F, Dinh-Xuan AT. Endothelial modulation of vasoconstrictor responses to endothelin-1 in human placental stem villi small arteries. Br J Pharmacol 1995; 115:1038–1042.[Medline]
  7. Myatt L, Brockman DE, Eis AL, Pollock JS. Immunohistochemical localization of nitric oxide synthase in the human placenta. Placenta 1993; 14:487–495.[CrossRef][Medline]
  8. Maggi M, Vannelli GB, Peri A, Brandi ML, Fantoni G, Giannini S, Torrisi C, Guardabasso V, Barni T, Toscano V, Massi V, Serio M. Immunolocalization, binding and biological activity of endothelin in rabbit uterus: effect of ovarian steroids. Am J Physiol 1991; 260:292–305.
  9. Morey AK, Pedram A, Razandi M, Prins BA, Ren-Ming Hu, Beisiada E, Levin ER. Estrogen and progesterone inhibit vascular smooth muscle proliferation. Endocrinology 1997; 138:3330–3337.[Abstract/Free Full Text]
  10. Fomin VP, Cox BE, Word RA. Effect of progesterone on intracellular Ca2+ homeostasis in human myometrial smooth muscle cells. Am J Physiol 1999; 276:379–385.
  11. Dave S, Farrance DP, Whitehead SA. Evidence that nitric oxide inhibits steroidogenesis in cultured rat granulosa cells. Clin Sci 1997; 92:277–284.[Medline]
  12. Mignot TM, Mallassiné A, Cronier L, Ferré F. Endothelin increases progesterone release by human trophoblast in culture. In: Baldi E, Maggi M, Cameron IT, Dunn MJ (eds.), Endothelin in Endocrinology: New Advances. Frontiers in Endocrinology, Vol. 15: Ares-Serono Symposia Publications; 1995: 277–283.
  13. Ferré F. Molecular mechanisms of parturition. Infect Dis Obstet Gynecol 1997; 5:98–105.
  14. Bergqvist A, Bergqvist D, Ferno M. Estrogen and progesterone receptors in vessel walls. Acta Obstet Gynecol Scand 1993 72:10–16.
  15. Omar HA, Ramirez R, Gibson M. Properties of a progesterone-induced relaxation in human placental arteries and veins. J Clin Endocrinol Metab 1995; 80:370–373.[Abstract]
  16. Morey AK, Razandi M, Pedram A, Hu RM, Prins BA, Levin ER. Oestrogen and progesterone inhibit the stimulated production of endothelin-1. Biochem J 1998; 330:1097–1105.
  17. Perrot-Applanat M, Deng M, Fernandez H, Lelaidier C, Meduri G, Bouchard P. Immunohistochemical localization of estradiol and progesterone receptors in human uterus throughout pregnancy: expression in endometrial blood vessels. J Clin Endocrinol Metab 1994; 78:216–224.[Abstract]
  18. Perrot-Applanat M, Cohen-Solal K, Milgrom E, Finet M. Progesterone receptor expression in human saphenous veins. Circulation 1995; 92:2975–2983.[Abstract/Free Full Text]
  19. Younes MA, Besch NF, Besch PK. Estradiol and progesterone binding in human term placental cytosol. Am J Obstet Gynecol 1981; 141:170–174.[Medline]
  20. Padayachi T, Pegorato RJ, Hofmeyr J, Joubert SM, Norman RJ. Decreased concentrations and affinities of oestrogen and progesterone receptors of intrauterine tissue in human pregnancy. J Steroid Bio-chem 1987; 26:473–479.[CrossRef][Medline]
  21. Rivera J, Cano A. Oestrogen and progesterone receptors in human term placenta. Measurement by binding assays and immunological methods. Placenta 1989; 10:579–588.[Medline]
  22. McCormick PD, Razel AJ, Spelsberg TC, Coulam CB. Absence of high-affinity binding of progesterone (R5020) in human placenta and fetal membranes. Placenta 1981; 3(suppl):123–132.
  23. Roussev RG, Higgins NG, McIntyre JA. Phenotypic characterization of normal human placental mononuclear cells. J Reprod Immunol 1993; 25:15–29.[CrossRef][Medline]
  24. Wang JD, Zhu JB, Shi WL, Zhu PD. Immunocytochemical colocalization of progesterone receptor and prolactin in individual stromal cells of human decidua. J Clin Endocrinol Metab 1994; 79:293–307.[Abstract]
  25. Patricio B. Estrogen and progesterone receptors in the human placenta. Rev Fr Gynecol Obstet 1994; 89:145–147.[Medline]
  26. Taddei G, Noci I, Periti E, Borri P, Torricelli F, Tozzi P, Moncini D, Paglierani M, Branconi F. Chorion villosum does not express progesterone and estrogen receptors during the first trimester of pregnancy. Gynecol Obstet Invest 1996; 42:77–79.[CrossRef][Medline]
  27. Gopi Shanker Y, Sharma SC, Jagannadha Rao A. Expression of progesterone receptors mRNA in the first trimester human placenta. Biochem Mol Biol Int 1997; 42:1235–1240.[Medline]
  28. Khan-Dawood FS, Dawood MY. Estrogen and progesterone receptor and hormone levels in human myometrium and placenta in term pregnancy. Am J Obstet Gynecol 1984; 150:501–505.[Medline]
  29. Rossmanith WG, Wolfahrt S, Ecker A, Eberhardt E. The demonstration of progesterone, but not of estrogen, receptors in the developing human placenta. Horm Metab Res 1997; 29:604–610.[Medline]
  30. Libby P, O'Brien KV. Culture of quiescent arterial smooth muscle cells in a defined serum-free medium. J Cell Physiol 1983; 115:217–223.[CrossRef][Medline]
  31. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 1987; 162:156–159.[Medline]
  32. Misrahi M, Atger M, Auriol L. Complete sequence of the human progesterone receptor deduced from cloned cDNA. Biochem Biophys Res Commun 1987; 143:740–748.[CrossRef][Medline]
  33. Robert B, Malassiné A, Bourgeois C, Mignot TM, Cronier L, Ferré F, Duc-Goiran P. Expression of endothelin precursor genes in human trophoblast in culture. Eur J Endocrinol 1996; 134:490–496.[Abstract/Free Full Text]
  34. Bradford MM. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976; 72:247–248.[CrossRef]
  35. Dawood MY, Helmkamp F. Human umbilical arterial and venous progesterone concentrations: effect of fetal sex, weight and mode of delivery. Obstet Gynecol 1977; 50:450–455.[Medline]
  36. Shaxted EJ, Maynard PV. Plasma progesterone in the umbilical vessels at term an overview. Br J Obstet Gynaecol 1983; 90:801–810.[Medline]
  37. Aisien AO, Towobola OA, Otubu JAM, Imade GE. Umbilical cord venous progesterone at term delivery in relation to mode of delivery. Int J Gynecol Obstet 1994; 47:27–31.[CrossRef][Medline]
  38. Perrot-Applanat M, Groyer-Picard MT, Garcia E, Lorenzo F, Milgrom E. Immunocytochemical demonstration of estrogen and progesterone receptors in muscle cells of uterine arteries in rabbits and humans. Endocrinology 1988; 123:1511–1519.[Abstract/Free Full Text]
  39. McGill HC Jr, Sheridan PJ. Nuclear uptake of sex steroid hormones in the cardiovascular system of the baboon. Circ Res 1981; 48:238–244.[Abstract/Free Full Text]
  40. Wang Jd, Zhu Jb, Fu Y Shi Wl, Qiao Gm, Wang Yq, Chen J, Zhu Pd. Progesterone receptor immunoreactivity at the materno-fetal interface of first trimester pregnancy: a study of the trophoblast population. Hum Reprod 1996; 11:413–419.
  41. Mabry White M, Zamudio S, Stevens T, Tyler R, Lindenfeld J, Leslie K, Moore LG. Estrogen, progesterone, and vascular reactivity: potential cellular mechanisms. Endocr Rev 1995; 16:739–751.[Abstract/Free Full Text]
  42. Jiang CW, Sarrel PM, Lindsay DC, Poole-Wilson PA, Collins P. Progesterone induces endothelium-independent relaxation of rabbit coronary artery in vitro. Eur J Pharmacol 1992; 211:163–167.[CrossRef][Medline]
  43. Wehling M. Specific, nongenomic actions of steroid hormones. Annu Rev Physiol 1997; 59:365–393.[CrossRef][Medline]
  44. Revelli A, Massobrio M, Tesarik J. Nongenomic actions of steroid hormones in reproductive tissues. Endocr Rev 1998; 19:3–17.[Abstract/Free Full Text]
  45. Lee WS, Harder JA, Yoshizumi M, Lee ME, Haber E. Progesterone inhibits arterial smooth muscle cell proliferation. Nat Med 1997; 3:1005–1008.[CrossRef][Medline]
  46. Kastner P, Krust A, Turcotte B, Stropp U, Tora L, Gronemeyer H, Chambon P. Two distinct estrogen-regulated promoters generate transcripts encoding the two functionally different human progesterone receptor forms A and B. EMBO J 1990; 9:1603–1614.[Medline]
  47. Cavaillé F, Kacémi A, Mondon F, Fournier T, Ferré F. Contractile proteins in human fetoplacental vessels. Am J Obstet Gynecol 1995; 173:1793–1799.[CrossRef][Medline]
  48. Tulenko TN. Regional sensitivity to vasoactive polypeptides in human umbilico-placental vasculature. Am J Obstet Gynecol 1979; 135:629–636.[Medline]
  49. Allen J, Forman A, Maigard S, Jespersen LT, Anderson KE. Effect of endogenous vasoconstrictors on maternal intramyometrial and fetal stem villous arteries in pre-eclampsia. J Hypertens 1989; 7:529–536.[CrossRef][Medline]
  50. Myatt L. Current topic: control of vascular resistance in the human placenta. Placenta 1992; 13:329–341.[Medline]



This article has been cited by other articles:


Home page
 Mol. Pharmacol.Home page
H. Wang, E.-W. Lee, L. Zhou, P. C. K. Leung, D. D. Ross, J. D. Unadkat, and Q. Mao
Progesterone Receptor (PR) Isoforms PRA and PRB Differentially Regulate Expression of the Breast Cancer Resistance Protein in Human Placental Choriocarcinoma BeWo Cells
Mol. Pharmacol., March 1, 2008; 73(3): 845 - 854.
[Abstract] [Full Text] [PDF]

Home page
 Endocr. Rev.Home page
J. L. Turgeon, M. C. Carr, P. M. Maki, M. E. Mendelsohn, and P. M. Wise
Complex Actions of Sex Steroids in Adipose Tissue, the Cardiovascular System, and Brain: Insights from Basic Science and Clinical Studies
Endocr. Rev., October 1, 2006; 27(6): 575 - 605.
[Abstract] [Full Text] [PDF]

Home page
 J. Clin. Endocrinol. Metab.Home page
F. A. Patel, J. W. Funder, and J. R. G. Challis
Mechanism of Cortisol/Progesterone Antagonism in the Regulation of 15-Hydroxyprostaglandin Dehydrogenase Activity and Messenger Ribonucleic Acid Levels in Human Chorion and Placental Trophoblast Cells at Term
J. Clin. Endocrinol. Metab., June 1, 2003; 88(6): 2922 - 2933.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Endocrinol.Home page
K. W. Cheng, C.-K. Cheng, and P. C. K. Leung
Differential Role of PR-A and -B Isoforms in Transcription Regulation of Human GnRH Receptor Gene
Mol. Endocrinol., December 1, 2001; 15(12): 2078 - 2092.
[Abstract] [Full Text] [PDF]

Home page
 Mol Hum ReprodHome page
D. Pieber, V. C. Allport, F. Hills, M. Johnson, and P. R. Bennett
Interactions between progesterone receptor isoforms in myometrial cells in human labour
Mol. Hum. Reprod., September 1, 2001; 7(9): 875 - 879.
[Abstract] [Full Text] [PDF]

Home page
 Biol. Reprod.Home page
C. Benassayag, I. Souski, T.-M. Mignot, B. Robert, J. Hassid, P. Duc-Goiran, F. Mondon, R. Rebourcet, L. Dehennin, E.-A. Nunez, et al.
Corticosteroid-Binding Globulin Status at the Fetomaternal Interface During Human Term Pregnancy
Biol Reprod, March 1, 2001; 64(3): 812 - 821.
[Abstract] [Full Text]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow My Folders
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Cudeville, C.
Right arrow Articles by Ferré, F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Cudeville, C.
Right arrow Articles by Ferré, F.
Agricola
Right arrow Articles by Cudeville, C.
Right arrow Articles by Ferré, F.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS