Hum. Reprod. Advance Access originally published online on December 8, 2005
Human Reproduction 2006 21(3):592-599; doi:10.1093/humrep/dei400
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Functional characterization of prostaglandin transporter and terminal prostaglandin synthases during decidualization of human endometrial stromal cells
1 Unité de Recherche en Ontogénie et Reproduction, Centre de Recherche du CHUL, 2705 Boul. Laurier, Ste-Foy, Québec, G1V 4G2 and 2 Département d’Obstétrique et Gynécologie, Université Laval, Ste-Foy, Québec, G1K 7P4, Canada.
3 To whom correspondence should be addressed at: Unité de Recherche en Ontogénie et Reproduction, Centre Hospitalier Universitaire de Québec (CHUL), Université Laval, 2705, Boul.Laurier, Sainte-Foy, Québec, G1V 4G2, Canada. E-mail: mafortier{at}crchul.ulaval.ca
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BACKGROUND: Decidualization of endometrial stromal cells is essential for successful implantation and pregnancy. Prostaglandins (PG) have been shown to be required for the initiation and maintenance of decidualization in animal models. The transport of PG across the plasma membrane is mediated by carriers such as prostaglandin transporter (PGT). Our recent data have shown the expression of human PGT (hPGT) in the endometrium during the menstrual cycle. The objective of the present study was to characterize hPGT in decidualized stromal cells. METHODS AND RESULTS: Human endometrial stromal cells were treated with a combination of cAMP and medroxyprogesterone acetate to induce decidualization. Decidualization was confirmed by morphological differentiation and increased secretion of prolactin. A large increase in hPGT mRNA level, as measured by real-time PCR analysis, was observed in decidual cells compared with control. Similarly, a 2-fold up-regulation of hPGT and 3–12-fold increase in PG biosynthetic enzymes were obtained at the protein level. Decidual cells exhibited a higher isotopic PGE2 uptake and greater intracellular PG levels than control. CONCLUSIONS: The higher uptake of PG by decidual cells is highly likely to be mediated via hPGT. PGT is a newly identified regulator of PG action at the cellular level and likely contributes to the regulation of PG action in female reproductive processes.
Key words: decidualization/human endometrial stromal cells/prostaglandins/prostaglandin transporter
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Decidualization of human endometrium is a process involving morphological and functional differentiations essential for successful implantation and maintenance of pregnancy (Dunn et al., 2003
). The process of decidualization involves the transformation of fibroblast-like human endometrial stromal cells (ESC) into larger and rounded decidual cells in preparation for uterine receptivity for the blastocyst (Dunn et al., 2003). The resulting decidual cells begin to express a variety of specific proteins, including prolactin, insulin-like growth factor binding protein-1, and tissue factor (Dunn et al., 2003). Decidualization is initiated around the spiral arteries during the mid-secretory phase of the menstrual cycle and eventually spreads throughout the stroma in the late secretory phase. Disruption of this process is an important cause of infertility and menstrual disorders (Dunn et al., 2003). In vitro, decidualization of ESC can be induced by progesterone, estradiol (E2), cAMP (Huang et al., 1987; Brar et al., 1997) and modulated by cytokines such as interleukin (IL)-1
that may inhibit (Frank et al., 1995) or induce the process depending on surrounding conditions (Strakova et al., 2005).
In many animal models, prostaglandins (PG) have been demonstrated to be required for the initiation and maintenance of decidualization. Infusion of PG to estrogen- and progesterone-treated animals stimulates decidualization (Kennedy and Lukash, 1982). Administration of indomethacin, a PG synthesis inhibitor, inhibits artificially stimulated decidualization (Rankin et al., 1979). Furthermore, deletion of the gene encoding cyclooxygenase (COX)-2, the rate-limiting enzyme in PG production, results in multiple reproductive impairments including failure of decidualization in mice (Lim et al., 1997). In humans, there is abundant evidence in support of the critical role of PG in female reproductive functions including ovulation, implantation, menstruation and parturition (Poyser, 1995).
PG are biosynthesized from arachidonic acid (AA), an essential fatty acid stored in membrane phospholipids of the cell. AA is released from phospholipids through phospholipase action and converted to the common intermediate, PGH2, by COX-1 and COX-2. COX-1 is constitutively expressed in most tissues whereas COX-2 expression is inducible (Otto and Smith, 1995). Terminal PG are subsequently produced by specific prostanoid synthase enzymes from PGH2. PGE synthase (PGES) and PGF synthase (PGFS) catalyse the isomerization of PGH2 to PGE2 and PGF2
, respectively. Our laboratory has identified an aldoketoreductase1B5 (AKR1B5) as the PGFS in the bovine endometrium (Madore et al., 2003a). In addition, we have found the expression of microsomal PGE synthase-1 (mPGES-1), mPGES-2, cytosolic PGE synthase (cPGES), and AKR1B1 (PGFS) in the human endometrium (Madore et al., 2003b; Parent et al., 2003, 2004).
PG exist as charged organic anions at physiological pH. The mechanisms responsible for the transport of newly synthesized PG out of producing cells, either by simple diffusion (Schuster, 2002) or a PG efflux transporter (Reid et al., 2003), are still in dispute. However, PG influx into cells appears to be poor and is thought to be mediated by carriers such as PG transporter (PGT) (Schuster, 2002). PGT is the first cloned PG transporter (Kanai et al., 1995). It emerges as a functional uptake-carrier of PG with high affinity for PGE1, PGE2, PGF2 and PGD2. PGT is postulated to be a 12-transmembrane protein and has a broad tissue expression (Schuster, 2002). PGT mRNA is expressed in reproductive tissues such as testis, ovary, and uterus (Kanai et al., 1995; Lu et al., 1996). Our group has cloned bovine PGT (Banu et al., 2003) and characterized PGT in the bovine reproductive system (Banu et al., 2003, 2005; Arosh et al., 2004). Recently, we have shown the expression of PGT in the human endometrium (Kang et al., 2005). However, the characterization of PGT in the human endometrium, especially during female reproductive processes, is still poorly understood.
Thus the aim of this study was to characterize human PGT (hPGT) during decidualization of human ESC. Decidualization was induced in vitro by the combination of cAMP and medroxyprogesterone acetate (MPA). We investigated both the expression and function of hPGT in decidualized stromal cells. The data presented here provide the first evidence for a role of PGT in the human endometrium during decidualization.
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Reagents
Gentamicin, transferrin, insulin, 4,4'-diisothiocyanatostilbene-2,2'-disulphonate (DIDS) and 8-bromo-cAMP were purchased from Sigma (St Louis, USA). MPA was bought from Pharmacia Canada Inc. (Mississauga, Ontario, Canada). Fetal bovine serum (FBS) was bought from Wisent Inc. (Quebec, Canada). Phenol red-free Roswell Park Memorial Institute (RPMI) 1640 medium, superscript II RT, dithiothreitol, 5' first-strand buffer, and TRIzol were purchased from Invitrogen (Burlington, Canada). Random hexamer primer was purchased from Amersham Biosciences (Montreal, Quebec, Canada). LightCycler FasterStart DNA Master SYBR Green I mix and MgCl2 were bought from Roche Diagnostics (Laval, Quebec, Canada). [3H]PGE2 was bought from Perkin Elmer® Life and Analytical Sciences (Boston, MA, USA). Goat anti-rabbit horseradish peroxidase-conjugated immunoglobulin (Ig) G was bought from Jackson Immunoresearch Laboratories (West Grove, PA, USA). Enhanced chemiluminescent system (Renaissance) was purchased from NEN, Life Science Products (Boston, MA, USA). Antibodies against hPGT and bovine human PGFS (AKR1B1) were produced in our laboratory as described previously (Kang et al., 2005
; Madore et al., 2003a,b). Rabbit polyclonal antibody against human mPGES-1 was bought from Cayman Chemical Company (Ann Arbor, MI, USA). Rabbit anti-COX-1 and COX-2 antibodies were donated by Dr Stacia Kargman (Merk-Frost, Montreal, Canada). Mouse monoclonal anti--actin antibody was purchased from Sigma (St Louis, USA).
Tissue collection
Endometrial biopsies were taken from women with regular menstrual cycles undergoing gynaecological investigation for benign conditions. Women with endometriosis at any stage were excluded. Informed consent for donation of anonymous endometrial samples was obtained before tissue collection. Biopsies were dated according to the stated last menstrual period. Histological examination of the biopsies was used to confirm the stage of the cycle according to criteria of Noyes et al. (1950) and absence of neoplasia or endometritis. The research protocol was approved by Centre Hospitalier Universitaire de Québec ethics committee on human research. The tissue samples were placed in sterile Hanks’ balanced salt solution containing 100 IU/ml penicillin, 100 µg/ml streptomycin and 0.25 mg/ml amphotericin B at 4°C and transported to the laboratory immediately.
Isolation and culture of human ESC
Four endometrial biopsies from different patients were used, one in menstrual phase, two in proliferative phase and one in secretory phase. The isolation and culture of human ESC were performed as described previously (Kang et al., 2004). Cells were maintained in RPMI 1640 containing 50 ng/ml gentamicin, 10 mg/ml insulin, 5 mg/ml transferrin and 10% FBS at 37°C in an atmosphere of 5% CO2/95% air. After reaching confluence, cells were passaged and experiments were carried out before the fourth cell passage.
In vitro decidualization of human ESC
Decidualization was induced as described by Brosen et al. (1999). Briefly, confluent stromal cells were treated in Phenol Red-free RPMI 1640 medium containing 2% dextran-coated charcoal-treated FBS (DCC-FBS), and 1% penicillin–streptomycin in the presence or absence of the combination of 0.5 mmol/l 8-bromo-cAMP and 1 mmol/l MPA. Cells treated in the absence of 8-bromo-cAMP and MPA were used as control. The medium was changed every 2 days. Culture supernatants (48 h) were collected and stored at –20°C for PG and prolactin measurements. Cells were either used for influx experiments or recovered for protein assay, RNA extraction and intracellular PG extraction.
Prolactin assays by enzyme-linked immunosorbent assay (ELISA)
Prolactin levels were measured by ADVIA Centaur® immunoassay system (Bayer HealthCare LLC, USA). The lower detection limit of the measurement was 0.3 mg/l. The coefficients of variation of intra- and inter-assays were 1.9–4.4% and 2–5.3% respectively.
RNA extraction, RT and real-time PCR (LightCycler)
Total RNA was extracted from cells with TRIzol according to the manufacturer’s instructions. From each sample, 2.5 mg of total RNA was reverse-transcribed using random hexamer primers and Superscript II Reverse Transcriptase in a final volume of 20 ml. Real-time quantitative RT–PCR (LightCycler) was performed using fluorescent SYBR Green I according to the manufacturer’s instructions as described recently (Kang et al., 2005). 18S was used as an internal standard. Standard RT–PCR products of hPGT and 18S were ligated into pEF6/V5-His TOPO vector. The plasmids containing the appropriate complementary DNA (cDNA) inserts were used as standards in LightCycler. Each reaction mixture contained FastSTART DNA Master SYBR Green 1, MgCl2 (3 mmol/l), forward and reverse primers and 2 ml of template (cDNA or standard) in a total volume of 20 ml. The reaction mixture was denatured for 5 min at 95°C and subjected to 50 cycles for hPGT, or 32 cycles for 18S in a three-step PCR consisting of a denaturation step (95°C for 10 s), annealing step (57°C for 5 s) and extension step (72°C/20 s). The concentrations of hPGT and 18S were determined from the copy number using standard curves. The resulting concentrations of hPGT mRNA were then expressed as a ratio of 18S to monitor changes in expression.
Western blot analysis
Protein extraction, quantification and western blot analysis were performed as described previously (Kang et al., 2004). Aliquots of 10 mg protein of each sample were separated on 10% sodium dodecyl sulphate (SDS)–polyacrylamide gel electrophoresis for hPGT, COX-1, COX-2 and -actin and 12.5% for AKR1B1 and mPGES-1. Proteins were then transferred onto nitrocellulose membranes. The membranes were blocked overnight at 4°C in phosphate-buffered saline (PBS) containing 5% fat-free dry milk and 0.05% Tween 20. The membrane was subsequently probed with rabbit anti-hPGT (1:10 000), or rabbit anti-COX-1 (1:6000), or rabbit anti-COX-2 (1:6000), or rabbit anti-AKR1B1 (1:2000), or rabbit anti-mPGES-1 (1:1000) and goat anti-rabbit horseradish peroxidase-conjugated IgG (1:10 000). -Actin (1:5000) was measured as an internal control. Immunoreactive proteins were visualized with an enhanced chemiluminescent system. The density of the signals was determined by computer-assisted densitometry (BioImage, Visage 110s; Genomic Solutions Inc., Ann Arbor, MI, USA).
Influx
Influx experiments were performed as described by Chan et al. (2002). Briefly, cells grown in 6-well culture plates were washed twice with cold RPMI 1640. Cells were then assigned to three treatment groups. In group 1, cells were incubated with 1 nmol/l [3H]PGE2 for 0 min. In group 2, cells were incubated with 1 nmol/l [3H]PGE2 for 20 min. In group 3, cells were preincubated with organic anion transport inhibitor DIDS (1 mmol/l) for 30 min and then incubated with 1 nmol/l [3H]PGE2 for 20 min. Influx was carried out at room temperature and terminated by removal of incubation media followed by two washes with cold 5% FBS. Cells were harvested and mixed with liquid scintillation mixture and analysed by liquid scintillation counting. The [3H]PGE2 transient was defined as the radioactivity at 20 min minus radioactivity at time zero.
Intracellular PG extraction
Cells in 24-well plates were washed with PBS and lysed in 200 ml of lysis buffer (10 mmol/l Tris–HCl, pH 7.4, 1% SDS, 1 mmol/l dithiothreitol and 1 mmol/l phenylmethylsulphonylfluoride). Cell lysates were collected into microtubes. A total of 600 ml methanol, 200 ml chloroform, and 500 ml water were added successively. After centrifugation, chloroform phase was collected and evaporated under nitrogen. The residue was reconstituted in the culture medium and kept at –20°C for PG measurement.
Enzyme immunoassays (EIA) for PG
An EIA technique was used for PGE2 and PGF2 measurement as described previously (Asselin et al., 1996), which utilized acetylcholinesterase-linked PG tracers (Cayman Chemical Company, Ann Arbor, MI, USA). The inter- and intra-assay coefficients of variation were 16 and 10% respectively.
Statistical analysis
Data are presented as the mean ± SEM. Statistical analysis was performed using analysis of variance (ANOVA) followed by Fisher’s protected least significant difference, Duncan’s new multiple range and Student–Newman–Keuls multiple comparison tests. In addition, for comparison of extra- and intracellular PG levels, the paired Student’s t-test was used (Super ANOVA; Abacus Concepts, Berkeley, CA, USA).
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Morphology of decidualized human ESC and secretion of prolactin by stromal cells during decidualization
Cultured human ESC undergo differentiation in response to the combination of 8-bromo-cAMP and MPA as described previously (Brosens et al., 1999
). Decidualization was first identified by the morphology of the cells. The decidualized stromal cells changed their morphology from a fibroblast-like spindle shape to a round, flattening appearance with large nuclei and abundant cytoplasm (Figure 1A). There is no difference in the morphology of decidualized stromal cells obtained from the proliferative or secretory phase of the cycle (data not shown). Decidualization was further confirmed by the measurement of prolactin, a marker of decidualization. Prolactin concentrations in the culture medium were assayed by ELISA. As illustrated in Figure 1B, control cells secreted a basal level of prolactin. The treatment of cells with the combination of MPA and the stable cAMP analogue 8-bromo-cAMP induced a modest increase in prolactin concentrations in the first 6 days. A significant stimulation of prolactin (P < 0.05) was observed thereafter. Among the treatments used, we have observed the highest levels of prolactin at 10 days.
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Up-regulation of hPGT mRNA level during decidualization
After 10 days of treatment with or without the combination of 8-bromo-cAMP and MPA, total RNA was extracted from cells and hPGT mRNA levels were determined using real-time PCR. Figure 2 shows the fluorescence graph and hPGT mRNA levels of control and decidualized stromal cells. Human PGT mRNA expression was increased 9-fold in decidual cells (mean ± SEM = 11.6 ± 0.6) compared with control (mean ± SEM = 1.25 ± 0.4) (P < 0.05).
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Up-regulation of hPGT, COX, and terminal PG synthase protein expression during decidualization
Proteins were extracted from cells after the treatment with or without the combination of 8-bromo-cAMP and MPA for 10 days. In decidualized stromal cells, hPGT protein expression was increased 2-fold compared with control. COX-1, COX-2, PGF2 synthase (AKR1B1), and mPGES-1 protein levels were measured in parallel. Western blot analysis showed a 13.8-fold increase in COX-1, a 3.8-fold increase in COX-2, a 3.9-fold increase in AKR1B1, and a 12-fold increase in mPGES-1 expression in decidualized stromal cells compared with control (Figure 3).
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Influx
Influx experiments were carried out after induction of decidualization for 10 days. At 20 min, decidualized stromal cells showed a higher uptake level in comparison with control (Figure 4A). In addition, this uptake was inhibited by pre-treatment with 1 mmol/l of an organic anion transporter inhibitor DIDS (Figure 4A), indicating that the greater uptake of [3H]PGE2 by decidual cells is mediated by an organic anion transporter. Given the up-regulation of hPGT mRNA and protein expression in decidualized stromal cells which we have observed and described above, the higher uptake of [3H]PGE2 by decidual cells is highly likely to be regulated by hPGT.
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Extra- and intracellular PG measurements
We have developed a new technique by which we are able to extract intracellular PG as we described in Materials and methods. This approach was verified using [3H]PGE2. Over 99% PG was retained in the chloroform phase whereas <1% stayed in the upper water phase. After the treatment of cells with or without the combination of 8-bromo-cAMP and MPA for 10 days, the culture supernatants were collected. Cells were lysed and intracellular PG were extracted from the cell lysate. PG concentrations were analysed by EIA. Data were then converted to the total amount of PG according to the volume of the culture medium added. Figure 4B illustrates the amount of PG inside and outside control and decidualized stromal cells. In control cells, the extracellular PGF2 levels were higher (P < 0.05) than inside whereas both concentrations were the same for PGE2. In decidualized stromal cells, the distributions of intracellular and extracellular PGE2 and PGF2 concentrations were opposed, higher for the former and lower for the second PG. Finally, total PGF2 accumulation was increased (P < 0.05) in decidualized compared to control cells.
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The human endometrium is a unique tissue which undergoes extensive regeneration and maturation during the menstrual cycle and in preparation for embryo implantation. The role of PG in the human endometrium has been implicated in reproductive functions such as implantation, decidualization, and menstruation (Poyser, 1995
) and also associated with reproductive pathology, including menorrhagia, dysmenorrhoea and endometriosis (Sales and Jabbour, 2003). PGT, the uptake carrier of PG for PG signal termination and metabolic clearance (Schuster, 2002), constitutes an important part of PG signal transduction cascade and contributes to the regulation of local PG concentrations (Nomura et al., 2005). The expression of hPGT in the human endometrium has been described in our recent study (Kang et al., 2005). However, the regulation of hPGT remains unclear. We have tried different factors including TNF- (1 ng/ml), IL-1 (1 ng/ml), LPS (10 mg/ml), PMA (100 nmol/l), H2O2 (10 mmol/l), E2 (1 nmol/l) and progesterone (10 nmol/l) to treat ESC, but no obvious difference was found at the hPGT protein level (data not shown). In order to establish the role of hPGT in female reproductive events, we investigated the characterization of hPGT during decidualization of human ESC in the present study.
Substantial evidence for decidualization is based on the in vitro data. Previous studies have shown the involvement of AA (Tessier-Prigent et al., 1999), HCG (Nemansky et al., 1998), PGE2 (Frank et al., 1994), progesterone and E2 (Huang et al., 1987) in promoting the decidual transformation and suggested a critical role of cAMP in this differentiation process (Gellersen and Brosens, 2003). In the present study, we used the combination of MPA and 8-bromo-cAMP, a stable cAMP analogue. Our data showing morphological differentiation and prolactin accumulation in the culture medium (Figure 1) confirmed decidualization in our culture system and supported other studies (Brosens et al., 1999). Western blot analysis was performed to detect hPGT protein expression after decidualization for 6 and 10 days respectively. There was no appreciable difference at the hPGT protein level between control and decidual cells after 6 days of treatment (data not shown). However, an apparent up-regulation was observed in decidual cells after 10 days, when we have also observed maximal prolactin levels. Consequently, the cells treated with or without 8-bromo-cAMP and MPA for 10 days were used as the in vitro study model. We have found a significant increase in hPGT at both mRNA (Figure 2) and protein levels (Figure 3) in decidual cells compared with control. The observed increase was much higher at the mRNA (9-fold) than at the protein (2-fold) level, suggesting regulatory steps at both the transcription and the translation–maturation levels.
Coincident with the increase in hPGT, a sharp up-regulation of COX-1, COX-2, AKR1B1 and mPGES-1 protein expression was also detected in decidualized stromal cells (Figure 3). Expression of COX, cPGES and mPGES has been described in decidual cells or tissues of mouse uterus during decidualization (Scherle et al., 2000; Ni et al., 2002, 2003). In humans, the expression of COX gene and protein has also been illustrated in the endometrium during the implantation period or in decidua (Shaw et al., 1994; Marions and Danielsson, 1999). Our data, however, represent the integrated study on the regulation of COX and terminal synthase protein expression in decidualized human ESC in vitro. Microsomal PGES-1, a membrane-associated and inducible enzyme, is preferentially coupled with COX-2 for delayed PGE2 production (Jakobsson et al., 1999; Murakami et al., 2000). AKR1B1 was identified to be the most likely PGFS in the human endometrium by previous studies from our group (Madore et al., 2003b). So far, there is no information available on either mPGES-1 or AKR1B1 in association with decidualization in the human endometrium. These findings in our study provide the first evidence for the regulation of these terminal PG synthases during decidualization.
To prove the function of hPGT in decidualized stromal cells, we performed influx experiments and studied extra- and intracellular PG distribution. Results from influx experiments indicated a stronger capacity of decidual cells to take up [3H]PGE2 (Figure 4A), and this uptake was inhibited by DIDS, an organic anion inhibitor (Figure 4A). It suggests that the greater uptake of PG by decidualized stromal cells is mediated by an organic anion transporter. General studies on PG output focus on the assay of PG which are released from the cells and accumulated in the culture medium. The approach we have developed to extract intracellular PG allows us to measure the relative levels of PG distributed inside and outside the cells and provides a useful tool in PG trafficking study. Our data showed the increased intracellular PGE2 and PGF2 levels (Figure 4B) and decreased extra- to intracellular PGE2 and PGF2 ratios (calculated from data shown) in decidual cells compared with control. Thus both influx and PG distribution studies have confirmed the greater uptake of PG by decidualized cells compared to control. Members of organic anion transporting polypeptide and organic anion transporter gene families have been suggested to play a role in the transport of PG, but with limited efficiency compared to PGT (Schuster, 2002). Given the increased hPGT mRNA and protein levels in decidual cells, we postulate that hPGT at least contributes, if not being the sole contributor, to the greater uptake of PG observed during decidualization. The total secretion of extra- and intracellular PGF2 is significantly higher in decidualized cells, a finding correlating with the increased terminal PGF2 synthase AKR1B1 protein expression (Figure 3). Studies on the total PGE2 generation yielded contradictory results. There is no marked difference in total PGE2 production by control and decidual cells. However, we observed a significant elevation of mPGES-1 protein expression in decidual cells as described above. One possibility is that there is a potential conversion of PGE2 into PGF2 by 9-keto-reductase (Oshige et al., 1992).
In humans, the role of PG in the process of decidualization remains unclear. Previous studies showed that PGE2 is an activator of adenylyl cyclase, producing elevated cAMP concentrations through EP2 or EP4 receptors. High intracellular cAMP concentrations appear to be required to sensitize the cells to the action of progesterone. A recent study (Dimitriadis et al., 2005) also reported that relaxin and PGE2 regulate IL-11, a cytokine directly associated with decidualized stromal cells (Dunn et al., 2003). From the point of angiogenesis/permeability, PGE2 is known to be vasodilating and PGF2 vasoconstricting. We hypothesize that PG may also play a role in the process of decidualization by contributing to the changes in vascular permeability, which accompany decidualization to promote leukocyte invasion and favour uterine receptivity.
Our understanding of the molecular mechanisms involved in decidualization of the human endometrium is far from complete. The increase in hPGT mRNA and protein is one of the characteristics we have observed in the decidualized stromal cells in vitro (day 10). We do not know the significance of having higher PGT at day 10 than at day 6. PGE2 is reported to be associated with proliferation and apoptosis. However, since total PGE2 production (extra- plus intracellular) in decidual cells is similar to that in control cells, we do not think the increase in PGT is more involved in preventing/promoting apoptosis. Total PGF2 production (extra- plus intracellular) is significantly higher in decidual than in control cells and this may be related to the angiogenic response associated with decidualization. Indeed, PGF2 and F-series prostanoid receptor were recently confirmed as important angiogenic factors in the endometrium (Sales et al., 2005).
In conclusion, we have demonstrated an up-regulation of hPGT mRNA and protein expressions as well as the primary PG biosynthetic enzymes during decidualization of human ESC in vitro. Influx experiments and increased intracellular PG levels in decidual cells strongly suggest that the greater uptake of PG by decidual cells is mediated by PGT. These data provide additional evidence on the contribution of PGT in the regulation of PG action in the female reproductive processes.
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This study was supported by a grant from the Canadian Institute of Health Research (CIHR) to M.A.F.
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Submitted on August 26, 2005; resubmitted on October 18, 2005; accepted on October 28, 2005.
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