Mol. Hum. Reprod. Advance Access originally published online on July 4, 2006
Molecular Human Reproduction 2006 12(9):577-585; doi:10.1093/molehr/gal058
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Prostaglandin E2 and F2
receptors in the human Fallopian tube before and after mifepristone treatment
1Division of Woman and Child Health and 2Division of Clinical Science, Intervention and Technology, Department of Obstetrics and Gynecology, Karolinska Institutet, Stockholm, Sweden
3 To whom correspondence should be addressed at: Department of Obstetrics and Gynecology, Karolinska University Hospital, Huddinge, S-141 86 Stockholm, Sweden. E-mail: kjell.wanggren{at}karolinska.se
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Prostaglandins are associated with several reproductive processes in addition to their effects on the vascular system and muscular contractility. The aim of this study was to gain information about the localization of the receptors for PGE2 (EP1–EP4) and PGF2
(FP) in the human Fallopian tube and their regulation following treatment with mifepristone. Sixteen healthy fertile women received a single dose of 200 mg mifepristone or placebo immediately after ovulation (LH+2). Laparoscopic sterilization was performed on days LH+4 to LH+6. Biopsies were taken from the Fallopian tubes bilaterally. The expression of EP1, EP2, EP3, EP4 and FP was analysed using immunohistochemistry and RT–PCR. The co-localization of prostaglandin receptors and c-kit or e-nos was analysed using confocal microscopy. The effect of progesterone, mifepristone and prostaglandin on tubal contractility was studied. The presence of EP1–EP4 and FP in the Fallopian tube was detected using immunostaining. The receptors were expressed in serosal cells, luminal epithelial cells, and the muscular wall and vessels of the Fallopian tube. Co-localization studies showed that the endothelial cells stained positive for EP1–EP4 and FP and that co-localization was seen for EP4 and c-kit. Decreased contractility was seen after progesterone treatment, whereas increased contractility was seen after PGF2 and PGE2 treatment. These data suggest that both the transport of the embryo and the communication between the embryo and the Fallopian tube involve the action of prostaglandins through EP and FP receptors in addition to the effect of prostaglandins on the vascular system and muscular contractility. Key words: embryo/Fallopian tube/fertilization/prostaglandin receptors/tubal transport
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Introduction |
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The Fallopian tube is of vital importance for successful reproduction. At this site, the sperm fertilizes the oocyte. This event is followed by cleavage and development of the pre-embryo during its transport through the Fallopian tube into the uterine cavity for hatching and implantation (Pauerstein and Eddy, 1979
). The environment in the Fallopian tube enhances the fertilization potential of the sperm and promotes embryo development. Embryos co-cultured with tubal epithelial cells have shown improved development, hatching and implantation potential (Xu et al., 2000, 2001). The transport of the spermatozoa and later the pre-embryo is believed to be aided by muscular contractions in the wall of the Fallopian tube and cilia in the tubal mucosa (Mastroianni, 1999). The regulation of muscular activity and the formation of cilia are influenced by adrenergic nerves (Helm et al., 1982), sex steroids (Helm et al., 1982), nitric oxide (Ekerhovd et al., 1997, 1999) and prostaglandins (Lindblom et al., 1979, 1983).
Prostaglandins are important regulators of many reproductive processes, such as ovulation, menstruation, fertilization, implantation and parturition. They are also involved in several pathological processes including inflammation (Minami et al., 2001), dysmenorrhoea (Ueno et al., 2001) and cancer (Sales et al., 2002). Prostaglandins are metabolites of arachidonic acid that is released from plasma membrane phospholipids or dietary fat by phospholipase and subsequently metabolized by cyclooxygenase (COX-1–COX-3) (Chandrasekharan et al., 2002; Morita, 2002). From the intermediate prostaglandin H2 (PGH2), the terminal prostaglandins are synthesized by their respective prostaglandin synthase (Narumiya et al., 1999). After biosynthesis, the prostaglandins are rapidly transported out of the cell by means of a prostaglandin transporter (Schuster, 1998).
Outside the cell, prostaglandins can act in an autocrine, a juxtacrine or a paracrine manner by binding to their receptors. The different prostaglandins PGE2, PGF2 and PGI2 exert their biological function through interaction with their corresponding receptor EP, FP or IP. There are four types of EP receptors (EP1–EP4), which are encoded by four separate genes (Narumiya et al., 1999). These receptors use alternate, and in some cases opposing, intracellular pathways. EP1 is coupled to diacyl glycerol/inositol triphosphate turnover and an increase in intracellular Ca2+ levels, whereas EP2 and EP4 are coupled to adenylate cyclase activation, EP3 is coupled to the inhibition of adenylate cyclase and FP is coupled to the stimulation of phospholipase C–inositol (IP3) pathway and Ca2+ mobilization (Sugimoto et al., 1992; Negishi et al., 1993; Watabe et al., 1993; Sando et al., 1994; Ashby, 1998).
The expression of COX-1, COX-2 and PGI2 is found in luminal epithelia and smooth-muscle cells of the mouse oviduct (Huang et al., 2004a). In other studies, mouse oviducts have been shown to produce PGI2 and PGE2, with the highest production, 2–3 days after coitus, corresponding to the time when mouse embryos transform from 2-cell stage to morulae. PGI2 is essential during the second and third days for the development of the mouse embryo, and later for its hatching and implantation (Huang et al., 2003, 2004b).
Previous studies have also shown that cells from human Fallopian tubes express COX-1, COX-2 and prostaglandin synthase (Huang et al., 2002) and that the human Fallopian tubes produce PGE2 and PGF2 (Ogra et al., 1974).
Progesterone is an important sex steroid, involved in several functions of female reproduction, such as ovulation and endometrial development. The physiological effects of progesterone are mediated by its two receptor isoforms PR-A and PR-B (Conneely et al., 2003).
Mifepristone is a potent antiprogestin that blocks the action of progesterone at the receptor level (Klein-Hitpass et al., 1991). The administration of 200 mg mifepristone on day LH+2 is highly effective as a contraceptive method (Gemzell-Danielsson et al., 1993). Treatment with mifepristone has been shown to increase the progesterone receptor levels in the human endometrium and Fallopian tube (Christow et al., 2002; Sun et al., 2003). This coincides with decreased levels of PGF2 in uterine fluid and decreased expression of PGF2, PGE2 and COX-1 and COX-2 in human endometrium (Gemzell-Danielsson and Hamberg, 1994; Nayak et al., 1998; Marions and Gemzell-Danielsson, 1999).
Prostaglandins and their action through their respective specific receptors have been suggested to be involved in several events, including the maternal–embryonic communication, promotion of embryo development, vasodilataion/constriction and regulation of muscular contractility. Despite the various possible actions, the presence, regulation and function of the receptors for PGE2 and PGF2 have, to our knowledge, not been extensively studied in the human Fallopian tube.
The aim of this study was to detect the presence and localization of receptors for PGE2 and PGF2 in the human Fallopian tube, in controls and after mifepristone treatment and, in addition, to study the effect of prostaglandins on Fallopian tube muscular contractility.
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Study subjects
Sixteen healthy women with a median age of 37 years (range 31–43 years), requesting tubal sterilization, were included in the study. All women were fertile and had regular menstrual cycles, median 28-day intervals (range 25–30). None of the women had received any hormonal treatment or used an intrauterine device for at least 3 months before the study. The women were randomly allocated into a study group (n = 8) receiving pretreatment with a single dose of 200 mg mifepristone immediately after ovulation (LH+2) or a control group (n = 8). Randomization was performed using opaque, sealed envelopes. Laparoscopic sterilization by placement of a silicone rubber ring over a segment of the isthmic part of the Fallopian tube (Yoon and King, 1975
) was performed on days LH+4 to LH+6. The day of the LH surge was detected by measuring LH in urine (ClearPlan, Searle Unipath, Bedford, UK) twice daily from cycle day 10 to the LH peak. In addition, all women collected daily urine during the cycle for the analysis of estrone- and pregnanediol-glucorinide and LH using enzyme immunoassay [Product code IM101, Immunometrics (UK) Ltd, London, UK]. At surgery, biopsies were taken from the Fallopian tubes bilaterally. On the right side, biopsies were taken from the isthmic part and on the left side from the ampullary part. The biopsies were immediately frozen in liquid nitrogen and used later for immunohistochemistry and RT–PCR. The expression of EP1–EP4, FP, c-kit and e-nos in the tubal biopsies was analysed using immunohistochemistry. The expression of prostaglandin receptor mRNAs was analysed using RT–PCR. For the tubal in vitro contractility experiment, four patients undergoing hysterectomy for benign causes were included in the study. The Fallopian tubes were immediately placed in ice-chilled Krebs–Ringer buffer solution (118 mM NaCl, 4.7 mM KCl, 1.0 mM CaCl2, 1.2 mM MgSO4, 24.8 mM NaHCO3, 1.2 mM KH2PO4 and 5.8 mM glucose) and transported to the laboratory.
Ethics
All women gave their written informed consent before entering the study. The study was approved by the Karolinska Hospital Ethics Committee.
Immunohistochemistry
The biopsies were mounted in an embedding medium (OTC Compound; Miles Inc., Elkhart, IN, USA) and serially sectioned to 9 µm using a Reichert-Jung Cryocut 1800 (Cambridge Instruments GmbH, Nussloch, Germany). The sections were mounted on glass slides and immersed in 2% paraformaldehyde in phosphate-buffered saline (PBS) for 20 min and thereafter washed in PBS. The mounted sections were then wrapped in parafilm and stored at –70°C until analysis. Before immunostaining, the slides were thawed and rinsed for 3 x 5 min in PBS, incubated in darkness for 30 min in H2O2 (0.3% in methanol) to block endogenous peroxidase activity and then washed 3 x 5 min with PBS/BSA (Albumin, Bovine 0.05%). Thereafter, the slides were blocked with 1.5% goat serum for EP1–EP4 and FP and horse serum for c-kit and e-nos (in PBS/BSA) in humidified chamber for 30 min. The sections were then incubated with the primary antibody overnight in a humidified chamber at 4°C. The antibodies for EP1, EP2, EP3 and EP4 receptors were rabbit polyclonal antibodies raised against synthetic peptides from the human EP1, EP2, EP3 and EP4 receptors, respectively (catalogue number 101740, 10750, 10760 and 10770; Cayman Chemical, Ann Arbour, MI, USA). The antibody for FP was an affinity-purified rabbit antibody (catalogue number P 8622, Sigma, St Louis, MO, USA) raised against a synthetic peptide from the human PGF2 receptor. Mouse monoclonal antibodies were used to detect c-kit protein (catalogue number PC34; Calbiochem, Merck KGaA, Darmstadt, Germany) and e-nos (Transduction Laboratories, Lexington, KY, USA). As a negative control for EP1, recombinant EP1 protein (catalogue number 301740; Cayman Chemical) was added to neutralize the antibody before analysis. As negative control for EP2–EP4, FP and c-kit, the primary antibody was replaced with PBS/BSA. The slides were washed in PBS/BSA three times for 5 min each and thereafter incubated with the secondary antibody diluted 1:300 for 30 min at room temperature, goat anti-rabbit for EP1–EP4 and FP and horse anti-mouse for c-kit. The slides were then washed with PBS/BSA three times for 5 min each before incubation with avidin–biotin–peroxidase (ABC) complex (Vectastain Elite ABC; Vector Laboratories, Burlingame, CA, USA) according to the manufacturer’s instructions. After washing three times with PBS/BSA, freshly prepared diaminobenzidine-hydrogen peroxide solution (DAB Kit, Vector Laboratories) was added to the slides that were thereafter rinsed with distilled water. The slides were counterstained with 10% Mayer’s haematoxylin (VWR, Stockholm, Sweden), then washed for 10 min in cold water and mounted with glycerol-gelatine (VWR).
Two persons evaluated the immunohistochemical staining independently, blinded to the identity of the samples. The staining intensity was graded on a scale of 0 = no staining of cells, + = faint staining, ++ = moderate staining and +++ = strong staining. To quantify the number of vessels in the muscular wall of the Fallopian tube, we counted the vessels in six fields of views in each slide. The average number of vessels per field was thereafter calculated.
Confocal microscopy
To identify which type of cells were seen, partly as ‘cell clusters’, in the muscular wall of the Fallopian tube, and a possible relation to c-kit, we performed co-localization studies with c-kit as a marker for intestinal cells of Cajal (ICC), mast cells or stem cells and e-nos as a marker of endothelial cells.
The same initial treatment was used for confocal microscopy as for immunohistochemistry, but the peroxidase treatment was omitted. As second antibody, Alexa 546 conjugated goat anti-rabbit antibody (Molecular Probes, Eugene, OR, USA) for EP1–EP4 and FP or Alexa 488 goat anti-mouse (Molecular Probes) for c-kit and e-nos was used. The slides were incubated for 1 h at room temperature and thereafter washed in PBS and subsequently, mounted in Vectashield hard set mounting medium from Vector Laboratories (H-1400). Confocal microscopy recordings were made, using Leica TCS SP inverted confocal laser microscope.
RNA preparation and RT–PCR
RT–PCR was used to study the mRNA of EP1, EP2, EP3, EP4 and FP in the isthmic part of the Fallopian tube in six different subjects, four controls and two mifepristone-treated women. Total RNA was extracted using Trizol® Reagent (Invitrogen, Stockholm, Sweden) according to the manufacturer’s protocol. One microgram of the total RNA samples was reverse transcribed using deoxynucleotide triphosphates (10 mM each), random hexamer (250 ng/ml), ribonuclease inhibitor (40 U/µl) and Superscript reverse transcriptase (200 U/µl; using the SuperscriptTM II RNase H– Reverse Transcriptase Kit (Invitrogen). Two microlitres of the RT product was further amplified by PCR in a Mastercycler® gradient (Eppendorf-Netheler-Hinz GmbH, Hamburg, Germany) using Master Taq Kit (Eppendorf). For EP1–EP4, known primer sequences used are summarized in Table I (Timoshenko et al., 2003; Spinella et al., 2004). Thirty cycles of amplification were performed for EP1–EP4 and FP and 19 cycles for 28S, under the following conditions: a 3-min hot start at 95°C, followed by melting at 95°C for 30 s for EP1–EP4 and FP and 94°C for 28S; annealing at 58°C for EP1, 2, 3 and 28S, 62°C for EP4 and 59°C for FP for 60 s; and extension at 72°C for 60 s. The PCR products were analysed by electrophoresis on a 1.5% agarose gel. To verify that the amplified products originated from mRNA and not genomic DNA, we performed a PCR, excluding cDNA and including RNA. A negative control PCR without template cDNA was performed in each experiment to rule out any other contamination (not shown in the figure). 28S was used as an internal positive control. The PCR products were separated by electrophoresis and visualized in a 1.5% agarose gel stained with ethidium bromide solution, identified in relation to a 100-bp ladder. The image was captured using ChemiDoGel Documentation System with the software Quantity One version 4.4.0 from BioRad (Hercules, CA, USA).
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Fallopian tube contractility experiments
The ampullary isthmic portion of the Fallopian tube was resected after being identified with a 1-mm probe inserted into the Fallopian tube (Lindblom et al., 1978
). Longitudinal tissue strips (size 
7 x 1 x 1 mm) consisting of the tubal muscular wall and mucosa were prepared under a stereomicroscope. Each strip was placed in an organ chamber, filled with Krebs–Ringer buffer solution at a constant temperature of 37°C and oxygenated with a gas mixture consisting of 95% O2 and 5% CO2. The strips were mounted under tension with a load equivalent to 1 g. Contractions were recorded with a Grass FT03C force-displacement transducer and registered on a Grass model 7 and 79D polygraph. Experiments were carried out after 30 min of equilibration. The test substances (progesterone, mifepristone, PGF2 and PGE2) were administered to the chambers at 20-min intervals after being dissolved in a buffer solution.
Statistics
Kruskal–Wallis one-way analysis of variance on ranks was used for statistical analysis of the staining intensity in the immunohistochemical methods. For comparison of the number of vessels stained, Mann–Whitney rank sum test was chosen.
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Expression of EP1 in the Fallopian tube
Immunostaining of EP1 was seen in luminal epithelial cells (Figure 1, A1 and A2). EP1 staining seemed to be more intense in the isthmic than in the ampullary part of the Fallopian tube. Decreased staining was seen after mifepristone treatment, P = 0.019 (Table II). Staining of EP1 was also found in the serosal epithelium (Figure 1, A5 and A6). The same staining intensity was seen in the ampullary as well as in the isthmic part of the Fallopian tube. There were no differences between mifepristone-treated and untreated subjects (Table III). Co-localization studies showed that the staining in the muscular layer was predominantly seen in endothelial cells. This co-localization was seen in endothelial cells in all compartments (Figures 1, A3 and A4 and 2). A reduced number of stained vessels/field were seen in the muscular layer after mifepristone treatment (Figure 3A; Table IV). The mRNA for EP1 was not detected (data not shown), although two different and previously published (Timoshenko et al., 2003
; Spinella et al., 2004) primer pairs were used in this study.
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Expression of EP2 in the Fallopian tube
Strong immunostaining was seen in the apical and basolateral sides of the luminal epithelial cells (Figure 1, B1 and B2). There was no difference between the staining intensity in luminal epithelial cells in the isthmic and ampullary parts of the Fallopian tube (Table II). EP2 immunostaining was also seen in the serosal cells of the Fallopian tube (Figure 1, B5 and B6). There were no significant differences between the two parts of the Fallopian tube or between the two treatments (Table III). EP2 staining was seen in the muscular layer, mainly co-localized with the endothelial cells (Figure 2B). This co-localization was seen in endothelial cells in all compartments. There was no significant difference in the number of stained vessels after mifepristone treatment, P = 0.091 (Figure 3B; Table IV). RT–PCR analysis confirmed the expression of EP2 mRNA in the Fallopian tube (Figure 4).
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Expression of EP3 in the Fallopian tube
EP3 staining was seen in the luminal epithelium, being most intense in the apical part of the luminal epithelial cells (Figure 1, C1 and C2; Table II). Faint immunostaining of EP3 was generally present in the serosal surface (Figure 1, C5 and C6; Table III). The stained cells in the muscular layer were mainly co-localized with e-nos, showing that these cells were endothelial cells (Figures 1, C3 and C4 and 2C). This co-localization was also seen in endothelial cells in other compartments. The number of stained vessels/field was the same in all groups studied (Table IV). The expression of EP3 mRNA was confirmed using RT–PCR (Figure 4).
Expression of EP4 in the Fallopian tube
EP4 showed moderate to strong staining in the luminal and the serosal epithelia (Figure 1, D1, D2, D5 and D6). The staining intensity did not differ between the groups (Tables II and III). The EP4-positive cells co-localized mainly with e-nos, showing that these cells mainly were endothelial cells (Figure 2D). There were some cells co-localizing with c-kit in the muscular layer, whereas other EP4-stained cells in the muscular wall did not co-localize with any of the tested antibodies (Figure 2D and F). There was no difference in the number of stained vessels either between the isthmic and the ampullary parts or between the treatment groups (Table IV). RT–PCR analysis confirmed the expression of EP4 mRNA in the Fallopian tube (Figure 4).
Expression of FP in the Fallopian tube
FP was expressed in luminal epithelial cells and serosal cells (Figure 1, E1, E2, E5 and E6). Immunostaining of FP was also seen in cells in the muscular wall of the Fallopian tube (Figure 1, E3 and E4). Co-localization studies showed that the stained cell clusters in the muscular layer were mainly endothelial cells (Figure 2E). Pretreatment with mifepristone significantly (P = 0.002) reduced the number of stained endothelial cells in the muscular wall (Figure 3; Table IV). RT–PCR confirmed the expression of FP in the Fallopian tube (Figure 4).
Tubal contractility experiments
Spontaneous contractions of the muscular strips occurred within 20 min in all patients included in the experiment. Progesterone administration resulted in a reduced frequency and amplitude of the contractions. Mifepristone administration had limited effect on the contractions. After administration of PGF2, tubal contractions were more frequent and had higher amplitude. PGE2 also had a stimulating effect on the amplitude of the contractions (Figure 5).
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Discussion |
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The human Fallopian tube expresses all four receptors for PGE2 (EP1–EP4) as well as the receptor for PGF2
(FP). All receptors showed similar distribution and were seen in the epithelial cells of the tubal lumen, in serosal surface and in vessels in the muscular wall. EP1 and FP are involved in the activation of phospholipase C and mobilization of the IP3 pathway. EP2 and EP4 activate the adenylate cyclase and cAMP/protein kinase A pathway, whereas EP3 can both inhibit adenylate cyclase and activate phospholipase C (Coleman et al., 1994). EP3 reduces intracellular secondary messenger cAMP, which can inhibit cellular responses initiated through other receptors. The co-localization of different receptors in the same cells may act to regulate the balance of the response to prostaglandins.
The human embryo has been shown to produce PGE2 in vitro (Holmes et al., 1990). The presence of prostaglandin receptors in the tubal epithelial cells in the Fallopian tube indicates a communication between the embryo and the maternal side during early embryo development. Recent studies show cross communication in the endometrium between EP2, EP4 and a receptor of the EGF family (HER1), possibly through the release of HB-EGF or action through non-receptor kinases such as c-Src (Sales et al., 2004a,b). Recent studies also demonstrated the expression of HB-EGF and its receptors HER1 and HER4 in the human Fallopian tube (Sun et al., 2006). Therefore, it is possible that communication between the embryo and the Fallopian tube to some extent uses the same pathways as in the endometrium.
PGE2 is known to regulate mucus secretion in gastric mucus cells. EP1 has been shown to mediate exocytosis evoked by PGE2 in guinea-pig antral mucous cells (Ohnishi et al., 2001). In this study, the expression of EP1 was found in the luminal epithelial cells of the human Fallopian tube. This leads to the speculation that the secretion of mucus, which might be favourable for embryo development, into the Fallopian tube involves the action of PGE2 through EP1.
EP2 receptors were localized at the apical and basolateral sides of the luminal epithelium of the Fallopian tube. It has previously been shown that female mice deficient in EP2 receptors are infertile, due to inhibited fertilization of the ova. However, in these mice, it was possible to perform IVF (Tilley et al., 1999). It was therefore suggested that the action of PGE2 through EP2 receptors creates a microenvironment in the Fallopian tube essential for fertilization (Tilley et al., 1999). It has been speculated that one mechanism behind infertility is due to inhibited PGE2 receptor-mediated cumulus expansion that would inhibit fertilization in the oviduct (Hizaki et al., 1999).
Our findings show that EP2 receptors are present in the luminal epithelium of the human Fallopian tube. The role of EP2 could be to regulate the composition of the excreted tubal fluid, important for cumulus expansion and facilitation of an optimal milieu for the embryo during its passage through the Fallopian tube.
EP4 is present in the rabbit and mouse oviducts (Segi et al., 2003). PGE2 has been shown to stimulate cilia activity and glycoprotein secretion through EP4 in the epithelium of the rabbit oviducts (Segi et al., 2003). Thus, the function of EP4 in the human Fallopian tube might be to regulate glycoprotein secretion and cilia beat in a similar way as in the rabbit and mouse oviducts.
PGF2 facilitates contractile signals via FP, whereas PGE2 can either contract via EP1 and EP3 or relax via EP2 and EP4 (Senior et al., 1993; Negishi et al., 1995). EP1 and FP induce Ca2+ release via a G-coupled receptor, which leads to smooth-muscle contraction, whereas EP2 and EP4 increase the accumulation of intracellular cAMP leading to muscle relaxation (Jabbour and Sales, 2004). EP3 is generally associated with a decline in cAMP and a stimulation of smooth-muscle contraction (Jabbour and Sales, 2004). The effect of the prostaglandin receptors on muscular contractility varies depending on the type of muscle. In the porcine uterus, EP1/IP agonist treatment showed an excitatory response in both circular and longitudinal muscles (Cao et al., 2002). The action through EP2 varied with the dose of the agonist, whereas EP4 was not involved in the muscular contractility (Cao et al., 2002). The EP3 receptor generally stimulates smooth-muscle contraction (Narumiya et al., 1999), and in the porcine uterus, it was shown that EP3 agonist contracted longitudinal muscles, whereas there was almost no effect on circular muscles (Cao et al., 2002).
In the myometrium, the muscular activity is regulated by prostaglandins. IL-1ß was shown to increase COX-2 in myometrial cells followed by increased levels of EP4 (Erkinheimo et al., 2000). In vitro studies of the human Fallopian tube have shown an excitatory effect of PGF2 and an inhibitory effect of PGE2 (Lindblom et al., 1978, 1979, 1983).
Earlier studies (Lindblom et al., 1978) as well as our experiments show a stimulatory effect of PGE2 and PGF2 on the longitudinal smooth muscle of the human Fallopian tube. Our experiment also indicates an inhibitory effect of progesterone on the Fallopian tube muscle activity. These receptors activate different intracellular pathways. EP1 and EP3 receptors are coupled to calcium mobilization and inhibition of adenylate cyclase, respectively, both of which enhance contraction. In contrast, EP2 and EP4 receptors both stimulate adenylate cyclase and tend to decrease smooth-muscle contractility (Coleman et al., 1994).
Specialized c-kit-positive cells called intestinal cells of Cajal (ICC) have been found in the smooth-muscle cells of the human gut and recently also in the human Fallopian tube (Popescu et al., 2005; Shafik et al., 2005). It has been suggested that the ICC act as pacemakers by the generation of electric waves (Rumessen et al., 1992). In the present study, we can detect co-localization solely between c-kit and EP4. This might indicate a regulatory effect of ICC on contractility in the Fallopian tube. The c-kit-positive cells found in the Fallopian tube could, however, also consist of other c-kit protein-positive cells such as mast cells or stem cells (Shakoory et al., 2004).
EP3 is a regulator of vascular function on ocular tissue (Sennlaub et al., 2003), whereas EP4 and FP have been reported to be present in perivascular cells of endometrial adenocarcinoma (Jabbour et al., 2001; Sales et al., 2004a,b).
It has previously been shown that treatment with mifepristone increases progesterone receptor expression in the Fallopian tube (Sun et al., 2003). The results of the present study indicate that the expression of PGF2 and PGE2 receptors in contrast to progesterone receptors rather seem to diminish after mifepristone treatment. The co-localization studies of EP1-4 and FP with e-nos showed that the cell clusters seen in the muscular wall consist of vessels. The number of EP1, EP2 and FP stained vessels in the muscular wall of the Fallopian tube decreased after mifepristone treatment. This could indicate that prostaglandin receptors in the Fallopian tube are directly or indirectly regulated by progesterone and might thereby be involved in the prostaglandin-mediated effects on the Fallopian tube.
Vascular endothelial growth factor (VEGF), a known mediator of angiogenesis and vascular permeability (Ferrara and Davis-Smyth, 1997), is present in the Fallopian tube (Lam et al., 2003). There are studies showing that PGE2 coupling to EP2 can induce the expression of VEGF in pancreatic cancer cells and endometrial adenocarcinoma cells (Eibl et al., 2003; Sales et al., 2004b). It has also been shown that angiogenesis is important for the normal function of the Fallopian tube throughout the menstrual cycle (Shweiki et al., 1993; Lam et al., 2004). In the present study, we show the presence of EP2 in vessels, which supports the discussion in the article by Jabbour and Sales (2004), that prostaglandin action through the prostaglandin receptors is involved in angiogenesis in reproductive tissues.
One of the contraceptive effects of mifepristone, in addition to other known mechanisms in the endometrium, might be the inhibition of fertilization and transport within the Fallopian tube mediated by prostaglandins. COX-1 and COX-2 are present in the endometrial epithelium throughout the menstrual cycle (Stavreus-Evers et al., 2005). Treatment with mifepristone during the luteal phase has been shown to affect the levels of both COX-1 and COX-2 in the luminal epithelium of the endometrium leading to the impairment of endometrial receptivity (Marions and Gemzell-Danielsson, 1999). In the Fallopian tube, mifepristone might also play a role by disturbing the synthesis of prostaglandins.
The characterization of the physiology of the normal human Fallopian tube is of importance for understanding of events leading to successful reproduction. The knowledge of the mechanism of action and regulation of prostaglandin receptors is essential in understanding the possible effects of different drugs such as non-steroidal anti-inflammatory drugs (NSAIDs), antiprogestins and prostaglandins on fertility and could in addition be used in future designing of new more effective and less harmful contraceptives or improved IVF treatments. The expression of PGE2 and PGF2 receptors in the human Fallopian tube suggests an importance of the Fallopian tube not only for transport of the embryo to the uterine cavity but also for the signalling between the embryo and Fallopian tube, which facilitates optimal embryo development.
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Acknowledgements |
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We are grateful to Dr Alexander Christow for taking the biopsies, Margareta Hellborg, research nurse and the staff at the gynaecological wards at the Karolinska University Hospital, Stockholm, Sweden, for taking excellent care of the patients and Anna Hildenbrand for linguistic control. The study was supported by grants from the Swedish Medical Research Council (6392) and Center for Health Care Sciences.
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Submitted on May 15, 2006; accepted on May 30, 2006.
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