Mol. Hum. Reprod. Advance Access originally published online on March 11, 2005
Molecular Human Reproduction 2005 11(4):269-277; doi:10.1093/molehr/gah161
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Progesterone receptor expression in human decidua and fetal membranes before and after contractions: possible mechanism for functional progesterone withdrawal
1Laboratory for Research in Reproductive Sciences, Department of Obstetrics and Gynecology, 2Pathology Department, Ha'Emek Medical Center, Afula, Israel, 3Rappaport Faculty of Medicine, Technion-Institute of Technology, Haifa, Israel
4 To whom correspondence should be addressed at: Department of Obstetrics and Gynecology, Ha'Emek Medical Center, Afula, 18101, Israel. E-mail: shaleve{at}tx.technion.ac.il
 |
Abstract |
|---|
 Top Abstract IntroductionMethodsResultsDiscussionReferences |
|---|
In humans, progesterone levels are sustained before the onset of labour. Therefore, the mechanism for parturition that has been proposed for humans is ‘functional’ progesterone withdrawal. Immunohistochemical staining for the progesterone receptor (PR) was positive in the decidua with a decline after contractions began. Western blot analysis revealed a number of PR isoforms expressed in the decidua, with the PR-B form being dominant. After contractions began, all PR isoforms decreased sharply. PR-B and PR-A decreased by 85.8%±6.7 and 78.2%±7.1, respectively (P<0.001). Incubation of decidua with Prostaglandin F2
1.0 µg/ml decreased the expression of all forms of PR isoforms. PR-B was reduced by 64%±6.09 (P<0.01); PR-A was reduced by 77%±5.9 (P<0.05), while PR-C was reduced by 80%±7.24 (P<0.05). Progesterone (80 µg/ml) increased the PR-B, PR-C the 45 and 36 kDa isoforms to 150%±7.89, 210%±12.4, 270%±9.7 and 216%±13.5, respectively (P<0.05). In immunohistochemical studies, the PR was not identified in the amnion or in the chorion, regardless of the presence or absence of contractions. Western blot analysis demonstrated that PR-C (60 kDa) and the 36 kDa isoforms were dominant in the amnion. After contractions began, PR-A decreased significantly by 61.9%±7.1 (P<0.001). Key words: decidua/fetal membrane/induction of labor/progesterone receptor/progesterone withdrawal
|
Introduction |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Hierarchical associations, which start before the active process of labour is appreciated, characterize normal labour at term. During pregnancy, the uterus is maintained in a quiescent state owing to the action of several putative inhibitors. The process of labour begins as uterine release phenomena from these myometrial inhibitors (Challis et al., 2002
). Progesterone is known to have a relaxing effect on myometrial smooth muscles. It has already been shown that progesterone blocks the action of oxytocin and inhibits the formation of gap junctions (Garfield et al 1980; Siiteri and Seron-Ferre, 1981; Shi et al., 2003). In higher primates and humans, synthetic antiprogestins were found to stimulate myometrial contractions, whereas inhibitors of 3ß-hydroxysteroid dehydrogenase, which lower systemic progesterone levels, induce labour and delivery (Astle et al., 2003). Furthermore, in a recent clinical study, weekly injections of 17 alpha-hydroxyprogesterone caproate reduced the rate of recurrent preterm delivery in high-risk women (Meis et al., 2003). In most mammals the onset of labour is preceded by a rapid fall in the maternal progesterone levels (Smith et al., 2002), and progesterone withdrawal has been suggested in these animals to play a pivotal role in the initiation of labour. However, in humans and in higher primates, maternal, fetal and amniotic fluid concentrations of progesterone are sustained before the onset of labour. Therefore, the mechanism for parturition, which has been proposed for humans, is ‘functional’ progesterone withdrawal (Karalis et al., 1996; Rezapour et al., 1997; Allport et al., 2001). Many studies have focused on local mechanisms that might diminish or uncouple progesterone action in the myometrium or at the level of the decidua and fetal membranes, despite maintenance of high circulating progesterone levels. Because the enzymes for steroid metabolism and for the production of prostaglandins (PG), cytokines and matrix metalloproteinases (MMP), coexist within fetal membranes, it is tempting to postulate that changes in steroid action are linked in a paracrine fashion to the release of these important mediators of parturition. Recent data indicate a close association between progesterone withdrawal and the expression of MMP, the production of PG and pro-inflammatory cytokines in intrauterine tissues. Although there is strong evidence that progesterone is a potent repressor of those effecter molecules both in vivo and in vitro, there is a continuing debate regarding the importance of decidual or amnion activation as the initiating event in parturition (Roh et al., 2000; Shynlova et al., 2003). Progesterone receptors (PR) exist in human reproductive tract tissues in at least two functional isoforms (PR-A and PR-B; Kastner et al., 1990; Sartorius et al., 1994; Wen et al., 1994). Previous work has indicated that PR-A can act as a potent repressor of PR-B mediated gene transcription and that an increase in the ratio of PR-A to PR-B leads to a decrease in the ligand-mediated transcription (Vegeto et al., 1993; Pieber et al.2001aPieber et al.,b). For this reason, a switch in receptor isoform predominance from PR-B to PR-A could lead to a functional progesterone withdrawal rendering progesterone ineffective at preventing the onset of labour. This study was designed to explore this theory. The expression and profile of PR was measured in decidua and fetal membranes obtained from women at term without contractions versus women at term with contractions. Furthermore, the effect of PGF2 on the expression of PR as a model for induction of labour and the effect of progesterone as a possible preventive medicine were tested. |
Methods |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Patients
Chorion, amnion and decidua were collected from 14 women delivered by scheduled, repeat elective Caesarean section before any regular uterine contractions were recorded, and from 10 women delivered by Caesarean section while having regular contractions for more than 1 h. The local ethics committee approved the use of decidua, amnion and chorion, and each participating patient provided informed consent. All patients had uncomplicated, singleton, term (38–41 completed weeks) pregnancies and were operated on while membranes were intact, described in detail in our previous manuscript (Ulug et al., 2001
). Fetal heart rate monitoring was normal in all cases. Neither the clinical condition nor sampled specimens gave evidence for infection and in all newborns the 5-min Apgar score was above seven.
Collection of samples
All specimens were collected in aseptic conditions in the operating suit. All specimens were prepared in our laboratory as described in detail in our previous manuscript (Ulug et al., 2001). Briefly, once the newborn was delivered during the Caesarean section, the placenta was manually removed and taken in a sterile sheet. The amniotic membrane was gently separated from an area above the chorionic plate. The chorionic tissue sample was taken from the denuded area underneath, where the chorion turns from placenta to a membrane. This enabled sampling of chorionic tissue without decidual contamination. A sample was taken from the decidua vera (away from the placental implantation site) by scissors, after removal of the placenta. Following the collection of tissue samples, they were immediately immersed in pre-warmed medium without serum [human tubal fluid (HTF), Irvine Scientific, CA, USA]. The remaining tissue handling was performed under a laminar flow hood. All specimens were washed with HTF to remove the remaining blood before the beginning of the incubation period. No significant difference was found between the viability (as tested by trypan blue) after overnight incubation versus 72 h of incubation between samples, (93 ± 4.6% and 91 ± 5.5%, mean ± SE, respectively).
Preparation of samples
Similar sized samples (approximately 0.5 x 0.5 cm) were dissected from each tissue type. Each tissue sample was either fixed in 4% formalin for immunohistochemical demonstration on paraffin section or incubated within 0.5 ml HTF in 1.7 ml conical tubes with perforated cap, to allow free exchange of gases. The incubation period was 72 h in the absence or presence of progesterone (0.8–80 µg/ml) or PGF2 (0.1–1 µg/ml) in a 37 °C humidified incubator with 95% air and 5% CO2. After the incubation period, the media were collected and the final fluid volumes measured. The final wet weight of the corresponding tissue block was also determined. Then all media were stored at –20 °C until assay.
Immunohistochemical staining of PR
All specimens were fixed in 4% formalin for 24 h and embedded in paraffin. For immunohistochemical staining the endogenous peroxidase in formalin-fixed, paraffin-embedded tissue was inhibited by incubating specimens with 0.5% H2O2 for 10 min before incubation with the primary mouse monoclonal antibodies (anti-human PR-A, NCL-PGR-312, Novocastra labouratories, UK). The slides were incubated with anti-PR primary antibody at a 1:200 dilution, using the streptavidin–biotin system according to the manufacturer's instructions. Tissue sections used as positive controls in this study were endometrium. We used a rabbit immunoglobulin G antibody instead of the primary antibody as a negative control for immunohistochemistry. No specific immunoreactivity was detected in these tissue sections.
Preparation of nuclear extracts
Nuclear extract protein was prepared from the tissue samples after the incubation period. Tissues were homogenized in ice cold lysis buffer (10 mM HEPES Ph 7.9, 10 mM KCl, 1 mM EDTA, 1 mM Dithiotheitol, 1 mM PMSF, 10 µg/ml Leupeptin, 50 µg/ml Aprotinin). Suspensions were incubated at 2000 g for 15 min at 4 °C and Nonidet P-40 at 0.4% final concentration was added. The cell suspension was centrifuged at 4 °C and the pellet resuspended in the same lysis buffer but containing 400 mM NaCl instead of KCl. After 15 min of incubation, the pellet suspensions were centrifuged at 10 000 g for 5 min at 12 000 rpm in 4 °C. The nuclear extract was collected and stored at –20 °C until use.
Western blot analysis
Western blot was conducted as previously described (Goldman et al., 2003). Briefly, in order to detect PR, nuclear extract 30 µg/lane and mass marker (10 µl) were diluted with 4x sample buffer (5% SDS, 20% Glycerol in 0.4 M Tris, pH 6.8 containing 0.02% bromophenol blue without 2-Mercaptoethanol) and subjected to 10% polyacrylamide gel electrophoresis. After electrophoresis, the nuclear extract was adjusted to 20 µg/lane and were blotted from the SDS-PAGE onto 0.45 µm nitrocellulose membranes (Scheicher & Schuel, Dassel, Germany). Non-specific binding sites were blocked by incubating the nitrocellulose membranes overnight with 20% non-fat milk and Tris-buffered saline, containing 0.01% Tween-20. The membranes were then washed twice with Tris-buffered saline, containing 0.5% Tween-20, and incubated for 1 h with rabbit anti-human PR polyclonal antibody (1.0 µg/ml; Santa Cruz, CA, USA, sc-539) in 10% non-fat milk and Tris-buffered saline, containing 0.01% Tween-20. The membranes were subsequently washed with Tris-buffered saline, containing 0.5% Tween-20 and incubated for 1 h with horse-radish peroxidase-conjugated anti-rabbit goat secondary antibody (Jackson Immuno-Research, West Grove, PA, USA) in 10% non-fat milk and Tris-buffered saline, containing 0.01% Tween-20. Detection was enhanced by chemiluminescence (ECL: Amersham International) and quantified by densitometry using BioImmaging gel documentation system (Dinco & Renium, Jerusalem, Israel) endowed with TINA software (Raytest, Staubenhardt, Germany).
RT–PCR
RT–RCR for PR-B (PR-B FWD 5'-ACACCTTGCCTGAAGTTTCG-3'; PR-B REV 5'-CTGTCCTTTTCTGGGGGACT-3' 196 bp product). For normalization we used the levels of the housekeeping gene glyceraldehyde-3-phosphatedehydrogenase (GAPDH).
GAPDH (FWD 5'-TGATGACATCAAGAAGGTGGTGAAG-3'; REV 5'-TCCTTGGAGGCCATGTGGGCCAT-3' 240 bp product) was performed. Total RNA was extracted from frozen samples with TRIzol reagent, according to the manufacturer's instructions (Life Technologies, Inc.-BRL, Grand Island, NY, USA). RNA concentrations were determined spectrophotometrically, and all RNA samples were stored at –80 °C until used for cDNA synthesis. An RT kit (SuperScript Preamplification system, Life Technologies, Inc.-BRL) was used in the synthesis and amplification of cDNA. Total RNA (5 µg) was denatured at 70 °C for 10 min and then reverse transcribed in the presence of 25 ng/µl oligo(deoxythymidine) primer (Life Technologies, Inc., Tokyo, Japan), 2.5 mM MgCl2, 0.5 mM deoxy-NTP, 10 mM dithiothreitol, and 10 U ribonuclease H-reverse transcriptase (SuperScript II RT, Life Technologies, Inc.) for 60 min at 42 °C, 5 min at 95 °C. Subsequently, 10 µl of the resulting cDNA was used as a template for PCR. PCR was set up using 3 mM MgCl2, 50 pmol of each primer and 2.5 U Taq DNA polymerase (Sigma, USA). PCR conditions were 94 °C for 2 min followed by 35 cycles of 94 °C for 30 s, 60 °C for 45 s, and 72 °C for 60 s with a 72 °C extension for 10 min. After PCR, the products were resolved on a 2.5% agarose ethidium bromide gel. Images were captured with Polaroid (Hertfordshire, UK) film under UV light. Products were quantified using PhosphorImager and ImageQuant software (Molecular Dynamics, Inc., Sunnyvale, CA, USA). In order to compare PR relative mRNA expression level, between groups, we analysed the ratio of each independent experiment between the expression level of PR and GAPDH from the same tissue under the same treatment.
Statistical methods
Each experiment was repeated at least six times as individual experiments in duplicate. All data are presented as mean ± SEM. Statistical analysis of the data was carried out with an analysis of variance or Student's t-test when comparing two groups. P<0.05 was considered significant.
|
Results |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
The expression of PR isoforms in the decidua before and after contractions
Immunohistochemical studies revealed nuclear staining for PR in the decidua. Nuclear staining of PR was observed in 100% of the decidual samples obtained from women without contractions (n=14; Figure 1). PR was almost undetected in decidua obtained from women with contractions. Seven of the 10 samples were negative for PR and the remaining three were only slightly positive (Figure 1). For the main comparative analysis in this study we chose an antibody concentration, which was well over the minimum amount needed to detect low levels of PR. Therefore, the decrease in staining intensity for PR observed in decidua taken during contractions is not a failure of the immunohistochemical method but indicates a substantial decrease in the level of PR.
|
Nuclear extracts (30 µg/lane) from 14 decidual samples obtained from women without contractions were tested with Western blot analysis. Figure 2A shows PR isoform profiles with PR-B (116 kDa) as the main isoform, PR-A (82 kDa), PR-C (60 kDa), and two smaller isoforms of 45 and 36 kDa were also detected. Nuclear extracts (30 µg/lane) from 10 decidual samples obtained from women with contractions were tested with Western blot analysis. In decidua obtained from women with contractions, Figure 2B, all isoforms of PR sharply decreased or vanished. PR-B and PR-A decreased significantly, by 85.8%±6.7 and 78.2%±7.1, respectively (P<0.001).
|
In order to further validate our results, PR-B gene expression was studied by RT–PCR. For normalization we had used the levels of the housekeeping gene GAPDH.
Figure 3 shows significant decrease in the relative mRNA level of PR-B in the decidua after contractions began (lane 4). In order to compare PR relative mRNA expression level, among groups, we analysed the ratio of each independent experiment between the expression level of PR and GAPDH from the same tissue under the same treatment. The expression level was decreased by 99% (P<0.001), ratio of 0.48 ± 0.047 without contractions versus 0.003 ± 0.0015 with contractions.
|
PGF2
reduced PR isoform levels in the term decidua without contractionsThe effect of PGF2
on the expression of the PR as a model for induction of labour was tested. Six samples (30 µg/lane) of decidua at term without contractions were incubated for 72 h in HTF medium in the absence or presence of PGF2 0.1 or 1.0 µg/ml. The incubation period and concentrations chosen were in accordance with our previous manuscript (Ulug et al., 2001). Figure 4 shows that incubation of decidua with PGF2 1.0 µg/ml caused a significant decrease in the expression of all forms of PR isoforms as compared to the control (P<0.05). Western blot analysis showed that the expression of PR-B was reduced by 64%±6.09 (P<0.01), PR-A by 77%±5.9 (P<0.05) while PR-C by 80%±7.24 (P<0.05). PGF2 1.0 µg/ml caused a reduction by 34%±5.7 and 40%±5.4 for PR-45 and 36 kDa expression, respectively (P<0.05). In order to further validate our results, PR-B gene expression was studied by RT–PCR. For normalization we used the levels of the housekeeping gene GAPDH. Figure 5 shows significant decrease in the relative mRNA level of PR-B in the decidua after incubation with PGF2 1.0 µg/ml (lane 4). In order to compare PR relative mRNA expression level, between groups, we analysed the ratio of each independent experiment between the expression level of PR and GAPDH from the same tissue under the same treatment. The expression level was decreased by 98% (P<0.001), ratio of 0.48 ± 0.047 without PGF2 versus 0.009 ± 0.002 with PGF2.
|
|
Progesterone increased PR isoform levels in the decidua without contractions
The effect progesterone had at different concentrations (0.8–80 µg/ml) on MMP secretion in the decidua was tested in a related study. The maximal effect was observed at a concentration of 80 µg/ml (data not shown). We chose this concentration as our working concentration in this study. Six decidual samples were incubated in the presence of progesterone (80 µg/ml) for 72 h. Nuclear extracts (30 µg/lane) were run on 10% SDS-PAGE gel for Western blot analysis. Progesterone (80 µg/ml) significantly increased the expression of PR-B, PR-C the 45 and 36 kDa isoforms to 150%±7.89, 210%±12.4, 270%±9.7 and 216%±13.5, respectively (P<0.05) as seen in Figure 6B. Addition of progesterone (80 µg/ml) to decidua with contractions not only failed to increase PR-B levels but also increased PR-A levels, Figure 6D. In order to further validate our results, PR-B gene expression was studied by RT–PCR. For normalization we have used the levels of the housekeeping gene GAPDH. Figure 7 shows a significant increase in the relative mRNA level of PR-B in the decidua after incubation with progesterone 80 µg/ml (lane 4). In order to compare PR relative mRNA expression level, among groups, we analysed the ratio of each independent experiment between the expression levels of PR and GAPDH from the same tissue under the same treatment. The expression level was increased by 87.5% (P<0.001), ratio of 0.48 ± 0.047 without progesterone versus 0.9 ± 0.26 with progesterone.
|
|
The expression of PR isoforms, in the amnion before and after contractions
Using immunohistochemistry, PR could not be identified in the amnion, regardless of the presence or absence of contractions (amnion without contractions n=14, amnion with contractions n=10; Figure 8). Western blot analysis of 14 nuclear extracts (30 µg/lane) obtained from amnion before contractions began, demonstrated (Figure 9A) that several PR isoforms are expressed in the amnion. PR-C (60 kDa) and the 36 kDa isoform were dominant. After contractions began (Figure 9 gel B), all PR isoforms sharply decreased or vanished. PR-A decreased significantly, by 61.9%±7.1 (P<0.001). In the presence of 80 µg/ml progesterone in the amnion without contractions (Figure 10B), 36 kDa isoform was significantly increased by 82.5%±37.2, (P<0.05). No change in the level of PR isoforms was observed following the addition of 80 µg/ml progesterone to the media in the amnion with contractions, as seen in Figure 10D.
|
|
|
The expression of PR isoforms, in the chorion before and after contractions
Using immunohistochemistry, PR could not be identified in the chorion, regardless of the presence or absence of contractions (chorion without contractions n=14, chorion with contractions n=10; Figure 11). In Western blot analysis of the chorion, with and without contractions, PR was not observed (data not shown).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
In humans, despite the lack of a fall in progesterone levels in the maternal plasma or uterine tissue before the initiation of labour, administration of the PR antagonist RU486 leads to an increased uterine activity and the induction of labour (Frydman et al., 1992
; Tenore, 2003). Moreover, the use of prophylactic progesterone reduced the frequency of uterine contractions and the rate of preterm delivery (Astle et al., 2003; Meis et al., 2003). In order to study the theory of ‘functional progesterone withdrawal’ in humans, we established a set of experiments to examine the expression of PR in all three compartments of the fetal maternal interface (i.e. decidua, amnion and chorion). Immunohistochemical staining for PR revealed the existence of PR in decidua but not in the fetal membranes. More important was the finding of a dramatic decline in PR in the decidua after contractions began. Since the major PR form tested with the immunohistochemistry was PR-A, we further conducted Western blot analysis with polyclonal antibody in order to evaluate all forms of PR and to confirm our results obtained with the immunohistochemistry. Western blot analysis revealed a wide spectrum of PR isoforms expressed in the decidua, with PR-B dominating before contractions began. Studies on human PR indicate that two PR isoforms, namely PR-A and PR-B, mediate the effects of progesterone (Vegeto et al., 1993; Giangrande et al., 2000; Pieber et al.2001aPieber et al.,b). These isoforms are transcribed from a single gene using distinct estrogen-inducible promoters and differ only by the presence of 164 amino acids in the amino-terminal. Detailed structure/function studies on these PR isoforms indicate that PR-B in all cellular contexts in vitro, functions as a ligand-dependent transactivator of progesterone-responsive genes in contrast to PR-A, which in some contexts, acts as a ligand-dependent transcriptional repressor of PR-B as well as of other steroid hormone receptors (Kastner et al., 1990; Vegeto et al., 1993; Sartorius et al., 1994; Wen et al., 1994). In the decidua obtained from women with contractions, PR-B levels, together with all other isoforms (PR-A, PR-C) observed by Western blot analysis, were reduced dramatically consistent with the immunohistochemistry studies. We also found that progesterone can increase the PR-B level in the decidua obtained from women without contractions, indicating a possible autocrine or paracrine mechanism. This could be an additional mechanism by which progesterone may prevent preterm labour, where the high progesterone level maintains a low PR-A/PR-B ratio. These results support the hypothesis of a ‘functional progesterone withdrawal’ in humans. Several studies have described similar results, most of them from the myometrium. A decrease in PR immunostaining in the myometrium at term has been reported with a shift from PR-B expression to PR-A expression (Khan-Dawood and Dawood, 1984; Padayachi et al., 1990; Pieber et al.2001aPieber et al.,b; Mesiano et al., 2002; Winkler et al., 2002; Condon et al., 2003). This shift to PR-A dominance has also been observed in human myometrium using Western blot analysis and RT–PCR. Numerous groups have investigated the levels of PR in non-pregnant or pregnant myometrium at different gestational ages with conflicting results (Haluska et al., 2002; Condon et al., 2003). Padayachi and colleagues (Padayachi et al., 1990), using ligand binding and enzyme-immunoassay, found higher levels of PR in myometrium and endometrium from non-pregnant women and in decidua during the first trimester than in tissues obtained at term. This is consistent with protein data from others who found a decrease towards term (How et al., 1995; Rossmanith et al., 1997; Shanker and Rao, 1999; Haluska et al., 2002). It was also suggested that a decline in PR co-activator expression and in histone acetylation, in the uterus near term, may impair PR function by causing a functional progesterone withdrawal (Condon et al., 2003). Furthermore, a 9-fold decrease in the binding of PR to its response element was observed in decidua obtained after the onset of labour (Henderson and Wilson, 2001). These results are consistent with our results, where dramatic decline in the PR-B level was documented after contractions began. In the present study, after initiation of contractions, a sharp decline in PR-B shifts the PR-A/PR-B ratio toward PR-A dominant. Since progesterone acts mostly via its PR-B receptor, it is more than likely that genes inhibited by progesterone, such as cyclo-oxygenase-2, which induce PG production, will increase their expression level as a result of functional progesterone withdrawal. In this study we have found that PGF2 reduced PR-B levels shifting PR-A/PR-B ratio toward PR-A. Another support for the capability of PG to regulate PR came from the study of Madsen et al. in which differential control of myometrial PR-A and PR-B expression has been observed by PGE2 and PGF2 and by specific intracellular signalling pathways (Madsen et al., 2004). The authors conclude that PG acting via the protein kinase C (PKC) pathway facilitates functional progesterone withdrawal, by increasing the myometrial PR-A/PR-B expression ratio. To the best of our knowledge, the present study is the first to report on the effect of PG on PR in the human term decidua. PG is known to be involved in the cascade of events leading to cervical softening, contractions and labour. The production of PGE2 and PGF2 by amnion and chorion have been shown to be more prominent in patients during spontaneous labour compared to that in patients undergoing elective Caesarean section (Brennand et al., 1998). Thus, the provision of PGF2 to the respective cultures could be considered as a stimulus that mimics the onset of labour. In this study we have shown that PGF2 reduced PR isoform expression in the decidua. PG is formed from arachadonic acid released from membrane stores by the action of PG hydrogenase synthase (PGHS), which catalyses a cyclo-oxygenation–peroxidation reaction to form the PG intermediate, PGH2 (Brennand et al., 1998). PGH2 is then converted to a variety of primary PG through the action of different PG isomerases (Mitchell and Trautman, 1993; Wimsatt et al., 1993; Hirst et al., 1995; Brennand et al., 1998). There are two PGHS isoforms. Type I is a constitutively expressed enzyme with a wide tissue distribution that is responsible for cellular housekeeping functions (Hirst et al., 1995). PGHS type II is a highly inducible enzyme that is responsible for mediating acute-phase reactions, including inflammation. Although both PGHS-I and PGHS-II have been identified within the human and ovine intrauterine tissues, it is the expression and activity of PGHS-II that increases towards the end of gestation and has been directly correlated with the onset of labour (Wimsatt et al., 1993). Our finding, that PGF2 reduced PR isoform expression in the decidua can further support this hypothesis. Thus, we may speculate that the increase in the expression of PG reduces PR expression leading to an increase in tissue sensitivity to contractile stimulus.
In decidua without contractions, an additional two bands were observed, 45 and 36 kDa. Although one cannot exclude the possibility that these two forms are degradation products, several publications have reported on the expression of PR isoforms with small molecular weight that differ in the structure and mechanisms of action from the classical nuclear receptor (Duffy et al., 1996; Luconi et al., 1998; Rae et al.1998aRae et al.,b; El-Hefnawy et al., 2000). A non-classical form of PR involved in the opening of calcium channels in the mitochondrial and cellular membranes has been characterized in the cytoplasmic membranes of human sperm (Luconi et al., 1998). Expression of the non-classical forms of PR has been demonstrated also in the ovary, particularly in granulosa cells (Duffy et al., 1996). Expression of an unusual PR isoform has been described in porcine granulosa cells, which mediates its actions through mobilization of Ca2+ from the endoplasmic reticulum through the activation of phospholipase C (Rae et al.1998aRae et al.,b). Ca2+ is a known regulator of many enzymes, including PKC. Activated PKC, in turn, serves as an interacting signal in the regulation of gene expression.
It is possible that the amnion is a critical component in the initiation and propagation of labour. In the amnion, the dominant forms were PR-C and 36 kDa. This could explain the lack of staining in immunohistochemistry. Although the possible role and mechanism of these forms in decidua and fetal membrane is still unclear the low molecular weight PR isoforms (36 and 45 kDa) could be mediators of rapid progesterone effect via non-genomic signalling pathway.
The faint expression of the two nuclear forms (PR-A and PR-B) suggests a different role for progesterone in each compartment. In the amnion, as in the decidua, PR isoform expression dramatically declined after contractions began. The low level of PR-B might suggest that the amnion is less affected by progesterone concentrations and is more vulnerable to changes in PG concentration. The rise in the level of PG towards labour will result in the further decrease of PR-B, inducing a cascade of events leading to the propagation of labour.
A recent clinical study shows that weekly injections of 17 alpha-hydroxyprogesterone caproate reduced the rate of recurrent preterm delivery in high-risk women (Meis et al., 2003). In order to investigate if the preventive effect of progesterone involves auto-regulation on the PR-B receptor, we incubated the decidua in the presence of progesterone. In the decidua without contractions, progesterone significantly increased PR-B expression. In the decidua with contractions progesterone not only failed to increase its receptor B level but also increases the PR-A/PR-B ratio by increasing PR-A levels. This might explain why progesterone may be good at preventing preterm labour but not at stopping labour once the process has begun.
In summary, the present study has shown a different PR profile between decidua and amnion, which in both compartments decline after contractions begin. We also found that PGF2 reduced PR levels in term decidua. This may indicate a functional progesterone withdrawal with a decrease in expression of PR responsive genes in the decidua and amnion eventually leading to an increased sensitivity to contractile stimuli.
|
References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Allport VC, Pieber D, Slater DM, Newton R, White JO and Bennett PR (2001) Human labour is associated with nuclear factor-kappaB activity which mediates cyclo-oxygenase-2 expression and is involved with the ‘functional progesterone withdrawal’. Mol Hum Reprod 7, 581–586.
Astle S, Slater DM and Thornton S (2003) The involvement of progesterone in the onset of human labour. Eur J Obstet Gynecol Reprod Biol 108, 177–181.[CrossRef][Web of Science][Medline]
Brennand J, Leask R, Kelly R, Greer I and Calder A (1998) The influence of amniotic fluid on prostaglandin synthesis and metabolism in human fetal membranes. Acta Obstet Gynecol Scand 77, 142–150.[CrossRef][Web of Science][Medline]
Challis JRG, Matthews SG, Gibb W and Lye SJ (2002) Endocrine and paracrine regulation of birth at term and preterm. Endocr Rev 21, 514–550.[CrossRef]
Condon JC, Jeyasuria P, Faust JM, Wilson JW and Mendelson CR (2003) A decline in the levels of progesterone receptor coactivators in the pregnant uterus at term may antagonize progesterone receptor function and contribute to the initiation of parturition. Proc Natl Acad Sci USA 100, 9518–9523.
Duffy DM, Molskness TA and Stouffer RL (1996) Progesterone receptor messenger ribonucleic acid and protein in luteinized granulosa cells of rhesus monkeys are regulated in vitro by gonadotropins and steroids. Biol Reprod 54, 888–895.[Abstract]
El-Hefnawy T, Manna PR, Luconi M, Baldi E, Slotte JP and Huhtaniemi I (2000) Progesterone action in a murine Leydig tumor cell line (mLTC-1), possibly through a nonclassical receptor type. Endocrinology 141, 247–255.
Frydman R, Taylor S, Paoli C and Pourade A (1992) RU 486 (mifepristone): a new tool for labour induction women at term with live fetus. Contracept Fertil Sex (Paris). 20, 1133–1136.[Medline]
Garfield RE, Kannan MS and Daniel EE (1980) Gap junction formation in myometrium: control by estrogens, progesterone, and prostaglandins. Am J Physiol 238, C81–C89.[Web of Science][Medline]
Giangrande PH, Kimbrel EA, Edwards DP and McDonnell DP (2000) The opposing transcriptional activities of the two isoforms of the human progesterone receptor are due to differential cofactor binding. Mol Cell Biol 20, 3102–3115.
Goldman S, Weiss A, Eyali V and Shalev E (2003) Differential activity of the gelatinases (matrix metalloproteinases 2 and 9) in the fetal membranes and decidua, associated with labour. Mol Hum Reprod 9, 367–373.
Haluska GJ, Wells TR, Hirst JJ, Brenner RM, Sadowsky DW and Novy MJ (2002) Progesterone receptor localization and isoforms in myometrium, decidua, and fetal membranes from rhesus macaques: evidence for functional progesterone withdrawal at parturition. J Soc Gynecol Investig 9, 125–136.[Web of Science][Medline]
Henderson D and Wilson T (2001) Reduced binding of progesterone receptor to its nuclear response element after human labour onset. Am J Obstet Gynecol 185, 579–585.[CrossRef][Web of Science][Medline]
Hirst JJ, Teixeira FJ, Zakar T and Olson DM (1995) Prostaglandin endoperoxide-H synthase-1 and -2 35 messenger ribonucleic acid levels in human amnion with spontaneous labour onset. J Clin Endocrinol Metab 80, 517–523.[Abstract]
How H, Huang ZH, Zuo J, Lei ZM, Spinnato JA, II and Rao CV (1995) Myometrial estradiol and progesterone receptor changes in preterm and term pregnancies. Obstet Gynecol 86, 936–940.[CrossRef][Web of Science][Medline]
Karalis K, Goodwin G and Majzoub JA (1996) Cortisol blockade of progesterone: a possible molecular mechanism involved in the initiation of human labour. Nat Med 2, 556–560.[CrossRef][Web of Science][Medline]
Kastner P, Krust A, Turcotte B, Stropp U, Tora L and Gronemeyer H (1990) Two distinct estrogen-regulated promoters generate transcripts encoding the two functionally different human progesterone receptor forms A and B. EMBO J 9, 1603–1614.[Web of Science][Medline]
Khan-Dawood FS and Dawood MY (1984) Estrogen and progesterone receptor and hormone levels in human myometrium and placenta in term pregnancy. Am J Obstet Gynecol 150, 501–505.[Web of Science][Medline]
Luconi M, Bonaccorsi L, Maggi M, Pecchioli P, Krausz CS, Forti G and Baldi E (1998) Identification and characterization of functional nongenomic progesterone receptors on human sperm membrane. J Clin Endocrinol Metab 83, 877–885.
Madsen G, Zakar T, Ku CY, Sanborn BM, Smith R and Mesiano S (2004) Prostaglandins differentially modulate progesterone receptor-A and -B expression in human myometrial cells: evidence for prostaglandin-induced functional progesterone withdrawal. J Clin Endocrinol Metab 89, 1010–1013.
Meis PJ, Klebanoff M, Thom E, Dombrowski MP, Sibai B, Moawad AH, Spong CY, Hauth JC, Miodovnik M, Varner MW et al. (2003) Prevention of recurrent preterm delivery by 17 alpha-hydroxyprogesterone caproate. N Engl J Med 348, 2379–2385.
Mesiano S, Chan EC, Fitter JT, Kwek K, Yeo G and Smith R (2002) Progesterone withdrawal and estrogen activation in human parturition are coordinated by progesterone receptor A expression in the myometrium. J Clin Endocrinol Metab 87, 2924–2930.
Mitchell MD and Trautman MS (1993) Molecular mechanisms regulating prostaglandin action. Mol Cell Endocrinol 93, C7–C10.[CrossRef][Web of Science][Medline]
Padayachi T, Pegoraro RJ, Rom L and Joubert SM (1990) Enzyme immunoassay of oestrogen and progesterone receptors in uterine and intrauterine tissue during human pregnancy and labour. J Steroid Biochem Mol Biol 37, 509–511.[CrossRef][Web of Science][Medline]
Pieber D, Allport VC and Bennett PR (2001) Progesterone receptor isoform A inhibits isoform B-mediated transactivation in human amnion. Eur J Pharmacol 427, 7–11.[CrossRef][Web of Science][Medline]
Pieber D, Allport VC, Hills F, Johnson M and Bennett PR (2001) Interactions between progesterone receptor isoforms in myometrial cells in human labour. Mol Hum Reprod 7, 875–879.
Rae MT, Menzies GS and Bramley TA (1998a) Bovine ovarian non-genomic progesterone binding sites: presence in follicular and luteal cell membranes. J Endocrinol 159, 413–427.[Abstract]
Rae MT, Menzies GS, McNeilly AS, Woad K, Webb R and Bramley TA (1998b) Specific non-genomic, membrane-localized binding sites for progesterone in the bovine corpus luteum. Biol Reprod 58, 1394–1406.
Rezapour M, Backstrom T, Lindblom B and Ulmsten U (1997) Sex steroid receptors and human parturition. Obstet Gynecol 89, 918–924.[CrossRef][Web of Science][Medline]
Roh CR, Oh WJ, Yoon BK and Lee JH (2000) Up-regulation of matrix metalloproteinase-9 in human myometrium during labour: a cytokine-mediated process in uterine smooth muscle cells. Mol Hum Reprod 6, 96–102.
Rossmanith WG, Wolfahrt S, Ecker A and Eberhardt E (1997) The demonstration of progesterone, but not of estrogen, receptors in the developing human placenta. Horm Metab Res 29, 604–610.[Web of Science][Medline]
Sartorius CA, Melville MY, Hovland AR, Tung L, Takimoto GS and Horwitz KB (1994) A third transactivation function (AF3) of human Progesterone receptors located in the unique N-terminal segment of the B-isoform. Mol endocrinol 8, 1347–1360.
Shanker YG and Rao AJ (1999) Progesterone receptor expression in the human placenta. Mol Hum Reprod 5, 481–486.
Shi L, Shi SQ, Given RL, von Hertzen H and Garfield RE (2003) Synergistic effects of antiprogestins and iNOS or aromatase inhibitors on establishment and maintenance of pregnancy. Steroids 68, 1077–1084.[CrossRef][Web of Science][Medline]
Shynlova O, Mitchell JA, Tsampalieros A, Langille BL and Lye SJ (2003) Progesterone and Gravidity Differentially Regulate Expression of Extracellular Matrix Components in the Pregnant Rat Myometrium. Biol Reprod [Epub ahead of print].
Siiteri PK and Seron-Ferre M (1981) Some new thoughts on the feto-placental unit and parturition in primates. In Novy MJ and Resko JA (eds) Fetal endocrinology. Academic Press, New York, pp. 1–34.
Smith R, Mesiano S and McGrath S (2002) Hormone trajectories leading to human birth. Regul Pept 108, 159–164.[CrossRef][Web of Science][Medline]
Tenore JL (2003) Methods for cervical ripening and induction of labour. Am Fam Physician 67, 2123–2128.[Web of Science][Medline]
Ulug U, Goldman S, Ben-Shlomo I and Shalev E (2001) Matrix metalloproteinase (MMP)-2 and MMP-9 and their inhibitor, TIMP-1, in human term decidua and fetal membranes: the effect of prostaglandin F(2alpha) and indomethacin. Mol Hum Reprod 7, 1187–1193.
Vegeto E, Shahbaz MM, Wen DX, Goldman ME, O'Malley BW and McDonnell DP (1993) Human progesterone receptor A form is a cell- and promoter-specific repressor of human progesterone receptor B function. Mol Endocrinol 7, 1244–1255.
Wen DX, Xu YF, Mais DE, Goldman ME and McDonnell DP (1994) The A and B isoforms of the human progesterone receptor operate through distinct signaling pathways within target cells. Mol Cell Biol 14, 8356–8364.
Wimsatt J, Nathanielsz PW and Sirois J (1993) Induction of prostaglandin Endoperoxide synthase isoform-2 in ovine cotyledonary tissues during late gestation. Endocrinology 133, 1068–1073.
Winkler M, Kemp B, Classen-Linke I, Fischer DC, Zlatinsi S, Neulen J, Beier HM and Rath W (2002) Estrogen receptor alpha and progesterone receptorA and B concentration and localization in the lower uterine segment in term parturition. J Soc Gynecol Investig 9, 226–232.[CrossRef][Web of Science][Medline]
Submitted on January 8, 2005; accepted on February 2, 2005.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  A. Merlino, T. Welsh, T. Erdonmez, G. Madsen, T. Zakar, R. Smith, B. Mercer, and S. Mesiano Nuclear Progesterone Receptor Expression in the Human Fetal Membranes and Decidua at Term Before and After Labor Reproductive Sciences, April 1, 2009; 16(4): 357 - 363. [Abstract] [PDF]
|
|
|
|
 |
|
 A. Samalecos and B. Gellersen Systematic Expression Analysis and Antibody Screening Do Not Support the Existence of Naturally Occurring Progesterone Receptor (PR)-C, PR-M, or Other Truncated PR Isoforms Endocrinology, November 1, 2008; 149(11): 5872 - 5887. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
O. Bukulmez, D. B. Hardy, B. R. Carr, R. A. Word, and C. R. Mendelson Inflammatory Status Influences Aromatase and Steroid Receptor Expression in Endometriosis Endocrinology, March 1, 2008; 149(3): 1190 - 1204. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. M. Yellon, C. A. Ebner, and Y. Sugimoto Parturition and Recruitment of Macrophages in Cervix of Mice Lacking the Prostaglandin F Receptor Biol Reprod, March 1, 2008; 78(3): 438 - 444. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Schumacher, R. Guennoun, A. Ghoumari, C. Massaad, F. Robert, M. El-Etr, Y. Akwa, K. Rajkowski, and E.-E. Baulieu Novel Perspectives for Progesterone in Hormone Replacement Therapy, with Special Reference to the Nervous System Endocr. Rev., June 1, 2007; 28(4): 387 - 439. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. B. Zaragoza, R. R. Wilson, B. F. Mitchell, and D. M. Olson The Interleukin 1beta-Induced Expression of Human Prostaglandin F2alpha Receptor Messenger RNA in Human Myometrial-Derived ULTR Cells Requires the Transcription Factor, NFkappaB Biol Reprod, November 1, 2006; 75(5): 697 - 704. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. H. Taylor, P. C. McParland, D. J. Taylor, and S. C. Bell The Progesterone Receptor in Human Term Amniochorion and Placenta Is Isoform C Endocrinology, February 1, 2006; 147(2): 687 - 693. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Welsh, C. M. Mitchell, W. A. Walters, S. Mesiano, and T. Zakar Prostaglandin H2 synthase-1 and -2 expression in guinea pig gestational tissues during late pregnancy and parturition J. Physiol., December 15, 2005; 569(3): 903 - 912. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||