Molecular Human Reproduction, Vol. 8, No. 5, 465-474,
May 2002
© 2002 European Society of Human Reproduction and Embryology
Implantation and pregnancy |
Expression of MAPkinases (Erk1/2) during decidualization in the rat: regulation by progesterone and nitric oxide*
1 Institute of Anatomy, University Hospital of Essen, Essen and 2 Jenapharm, GmbH & Co. KG, Jena, Germany
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Abstract |
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The interaction between nitric oxide (NO), progesterone and the MAPkinase signalling pathway involved in decidualization was studied using immunohistochemistry during implantation in the rat. Early pregnant rats were treated with the inhibitor of nitric oxide synthesizing enzyme iNOS, aminoguanidine, either alone or in combination with the low dose antiprogestin, onapristone. The combined treatment was most effective on days 7 and 9 post coitum leading to a complete loss of embryos. The expression pattern of activated MAPkinases, Erk1/2 and iNOS appeared to be associated with the differentiation process of decidualization. A maximum staining of both enzymes was observed on day 9 post coitum in the mesometrial decidua. In addition, Erk1/2 and iNOS were highly coexpressed around the mesometrial sinusoids. Combined treatment with aminoguanidine and onapristone for 3 days led to a transient suppression of Erk1/2 and abolished Cox2 expression. Concomitantly, angiogenesis was reduced and dilated sinusoids were missing in the mesometrial decidua. In conclusion, our study suggests that (i) the member of the mitogen-activated protein kinase (MAPK) family, Erk1/2, is activated during implantation and may play an important role during the decidualization process, and (ii) this enzyme may be regulated by both progesterone and NO.
Cox2/decidualization/Erk1/2/nitric oxide/progesterone
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Introduction |
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In rodents, decidualization is a reaction to the attaching blastocyst and is related to the establishment and maintenance of a successful pregnancy. In contrast to the precise physiological role of the rodent decidual tissue, the histology of the decidualization process is very well known (O'Shea et al., 1983
). Characteristic morphological markers of decidualization in rodents include the intermediate filament desmin (Glasser and Julian, 1986) as well as high amounts of intercellular gap junction channels composed of connexin43 (Winterhager et al., 1993). The decidual tissue produces prolactin (Prigent-Tessier et al., 1999) and prolactin-related proteins (Roby et al., 1993; Hiraoka et al., 1999), but the function of these hormones for the maintenance of pregnancy is still under discussion.
The cell biological programme of decidualization and the signal cascades involved in decidualization are, at least in rodents, governed by progesterone and estrogen (Psychoyos, 1973). Withdrawal of progesterone inhibits uterine receptivity and implantation (Chwalisz and Garfield, 1997). In addition, the proliferation of stromal cells and the switch to the specific cell programme of decidual cells is missing in the absence of progesterone (Gruemmer et al., 1994; Zhang et al., 1994). Although progesterone and estrogen seem to sensitize the endometrium for the decidualization programme (Psychoyos, 1973), additional downstream mediators triggered by both progesterone and blastocyst signals are needed for the appropriate cell differentiation. There is ample evidence that these signals belong to the inflammatory cascade involving cytokines and prostaglandins (Salvemini et al., 1995; Kurusu et al., 1999; Tessier-Prigent et al., 1999). However, only limited knowledge of the precise signal cascades leading to the transformation of stromal cells into decidual cells is available.
In mice, the cyclooxygenase isoform, Cox2, seems to play a key role in decidualization as impressively demonstrated by knockout experiments. Deletion of Cox2, but not the constitutively expressed isoform Cox1, inhibits not only fertilization, but also implantation and decidualization (Lim et al., 1997). In recent years it has become evident that nitric oxide (NO), a major mediator of various vascular functions and inflammatory reactions (Moncada and Higgs, 1993; Ignarro, 1999), plays an important role during pregnancy and parturition (Natuzzi et al., 1993; Yallampalli et al., 1994; Vedernikov et al., 2000). More recently, it has been shown that NO is also involved in implantation and the establishment of pregnancy (Chwalisz and Garfield, 2000). In addition, NO seems to be required for normal preimplantation embryonic growth and development (Barroso et al., 1998; Gouge et al., 1998). NO, a short-living mediator, is generated by three isoforms of nitric oxide synthesizing enzymes (NOS). The tissue-specific and gestationally regulated expression of the cytokine-inducible form iNOS and endothelial form eNOS has been described in rodent uteri during pregnancy (Buhimschi et al., 1996; Purcell et al., 1999). In rats, the endometrial iNOS expression seems to be regulated by progesterone on both the protein and transcript levels (Ali et al., 1997). In contrast, uterine eNOS expression does not seem to change during pregnancy at least in rats (Ali et al., 1997). A marked up-regulation of both iNOS and eNOS during implantation has been previously described in mice (Purcell et al., 1999). This study showed that iNOS expression rises from day 6 to day 8, whereas eNOS is most abundantly expressed on days 6 and 7 (Purcell et al., 1999). This precise regulation of the NOS expression pattern suggests that NO may play an important role during implantation. In addition, several investigations using an inhibitor for all three isofoms, NG-nitro-L-arginine methyl ester (L-NAME), demonstrated a reduced pregnancy rate in rats (Biswas et al., 1998; Duran-Reyes et al., 1999). However, these studies do not discriminate possible sites of action. On the other hand, knockout experiments indicate that the individual NOS isoforms are not essential for the establishment of pregnancy since mice lacking iNOS (MacMicking et al., 1995) or eNOS (Huang et al., 1995) are fertile. However, studies with multiple NOS knockouts are still not available. It cannot be excluded that deletion of a single NOS isoform is accompanied by an increased NO production by the remaining NOS isoforms. In pregnant rats, uterine iNOS expression seems to be mostly regulated by progesterone as demonstrated by studies using progesterone antagonists (Yallampalli et al., 1996). Recently, we demonstrated in rats that progesterone and NO play a synergistic role during the establishment of pregnancy, predominantly acting on the decidualization process (Chwalisz et al., 1999). This study showed that the NOS inhibitors, aminoguanidine (iNOS inhibitor) or L-NAME (non-specific NOS inhibitor), in combination with the low-dose progesterone antagonist onapristone, had a marked inhibitory effect on the establishment of pregnancy due to a failure in decidualization. This effect was synergistic since administration of these compounds alone had only marginal effects.
In the present study we attempt to identify possible differentiation and signalling pathways which could explain the impressive synergistic action of progesterone and nitric oxide on decidualization. Since the mitogen-activated protein kinases (MAPK) represent a key growth factor signalling pathway and are activated by NO (Callsen et al., 1998), we examined the expression pattern of activated extracellular signal-regulated MAPK, Erk1/2. Erk1/2 constitute one member of the three subfamilies of MAPK further consisting of the c-JunNH(2)-terminal kinase (JNK) and the p38 MAPK (Ono and Han, 2000). Erk1/2 are activated by thyrosine and threonine phosphorylation by the dual specificity kinase Mek1/2 (Lander et al., 1996). This latter study demonstrated that NO activates all three MAPK members in Jurkat T cells. Here we provide further evidence that NO and progesterone may act during implantation through a similar signalling pathway leading to Erk1/2 activation. Our studies suggest that the ERK1/2 pathway may play an important role in decidualization, particularly in endometrial angiogenesis during implantation in the rat.
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Materials and methods |
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Animals
Female Wistar rats (Schering, TZH, Berlin, Germany) were used for all experiments. The animals were kept under standard conditions. The light/dark cycle was 14/10 h. After mating, the presence of sperm in the vaginal smear the following morning was designated as day 1 post coitum (p.c.).
Compounds and formulation
The specific progesterone antagonist onapristone (ZK98 299; Schering) was formulated in 0.2 ml benzylbenzoate plus castor oil (1:4 vol/vol) and injected s.c. in a concentration of 0.3 mg/rat/day (low dose). The low dose of onapristone is known to have only marginal effects on pregnancy outcome (Chwalisz et al., 1999). Aminoguanidine (Sigma Aldrich Chemie, Taufkirchen, Germany) was dissolved in water at pH 6.0 and given orally (p.o.) in a concentration of 120 mg/rat/day in 1 ml dose.
Experimental design
The experimental groups treated with aminoguanidine or onapristone alone and in combination were chosen after randomization in accordance with the experimental protocols (Figure 1a,b). For every experimental approach, a minimum of four animals was investigated.
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To evaluate the most sensitive phase of NO and progesterone acting on pregnancy outcome, aminoguanidine in the presence and absence of low-dose onapristone was administered during the peri-and post-implantation phase between 7 and 10 days p.c. (Figure 1a
). Onapristone or aminoguanidine alone or in combination were given for 4 days on days 7, 8, 9 and 10 p.c. In the following procedures both compounds were administered in combination, since separate treatment had only marginal effect on pregnancy outcome (Figure 2a). Onapristone and aminoguanidine were given in combination: (i) for 3 days on days 7, 8 and 9 p.c.; (ii) for 2 days on days 7 and 8 p.c. or days 8 and 9 p.c.; (iii) as a single injection on day 8 or 9 p.c. respectively.
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To evaluate the pregnancy outcome, autopsy was performed on day 19 p.c. (term 21–22 days p.c.). Empty uteri were stained with 10% ammonium sulphide to identify early resorptions.
For histological and immunohistochemical investigations, animals were treated with onapristone or aminoguanidine alone as well as in combination for 3 days on days 7–9 p.c. (Figure 1b). In order to investigate the time course of cell biological changes in the decidual tissue, the rats were autopsied 2, 6 and 10 h after the last treatment on day 9 p.c. at 10:00, 14:00 and 18:00 (Figure 1b). During autopsy on day 9 p.c., uteri were removed, implantation sites were evaluated, photographed and then processed for morphological and immunohistochemical investigations.
Morphology and immunohistochemistry
Post-implantation rat embryos were obtained by dissecting the implantation chambers at 9 days p.c.; the specimens were quickly frozen in liquid nitrogen and stored at –80°C for later cryosectioning and immunostaining. For morphology and immunohistochemistry, implantation chambers were fixed in 4% paraformaldehyde overnight and routinely embedded in paraffin after dehydration in a graded series of alcohols. For morphology, serial sections were stained with azan or haematoxylin–eosin.
For detection of CD 90 (Thy-1) and Cox2, we used freshly frozen material. Cryostat sections (10 µm) were fixed for 10 min in ice-cold 96% ethanol. After rinsing twice for 5 min in phosphate-buffered saline (PBS), the sections were incubated with 0.5% bovine serum albumin (BSA; Sigma) in PBS for 30 min to reduce non-specific staining. The sections were incubated for 60 min at room temperature with the primary antibodies (Table I), then rinsed with PBS containing 0.5% BSA (3x10 min). For detection of CD 90 (Thy-1), we used Cy3-conjugated donkey anti-mouse IgG, and for the detection of Cox2, we used pig anti-goat IgG (Table II). After incubation with the secondary antibody for 60 min at room temperature, the sections were rinsed in PBS (3x10 min) and were then covered with Vectashield to avoid fading of the fluorescence.
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For detection of the diphosphorylated mitogen-activated kinase Erk1/2 and of iNOS we used the LSAB (labelled streptavidin biotin) method on paraffin sections. Paraffin sections were deparaffinized and rehydrated in a graded series of alcohols. After rinsing with PBS (3x10 min), the endogenous peroxidase was blocked. The sections were incubated for 90 min with antibodies against activated Erk1/2 or iNOS (Table I
). After incubation with the primary antibody, the sections were rinsed in PBS (3x10 min), then a secondary biotinylated antibody, and after that with streptavidin–biotin–peroxidase complex for 10 min. Staining was visualized with 3,3'-diaminobenzidine. For double immunolabelling, cryostat sections were postfixed for 10 min in ice-cold 96% ethanol and incubated with antibodies of activated ERK1/2 and iNOS as described above. Sections were stained using a Cy3-conjugated donkey anti-mouse IgG followed by a fluorescein isothiocyanate-conjugated pig anti-rabbit secondary antibody. Photographs were taken with an Axiophot microscope equipped with epifluorescence (Zeiss, Oberkochem, Germany).
Statistical analysis
For statistical analysis of the effects of onapristone and aminoguanidine on pregnancy outcome (day 19 p.c.), the Wilcoxon test for comparison between the groups was applied.
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Results |
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Effects of aminoguanidine and low-dose onapristone on implantation and pregnancy outcome
To test the most sensitive phase for the synergistic action of the both compounds, pregnancy outcome was recorded after different times of treatment during peri-implantation (Figure 2a,b
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As shown in Figure 2a, combined administration of onapristone and aminoguanidine for 4 days (7–10 days p.c.) led to a complete loss of fetuses, whereas the individual substances had only marginal effects. For further investigations, only the combined treatment was chosen.
As shown in Figure 2b, low-dose onapristone and aminoguanidine given in combination when reduced to 3 days of treatment between 7 and 9 days p.c. led to a complete failure of pregnancy outcome. Treatment with both compounds, once on day 9 or for 2 days on days 7 and 8 p.c. or 8 and 9 p.c., resulted in a reduction of live-born pups; however, the difference was not significant due to high variations. A single administration of both components on day 8 p.c. had no effect on successful implantation (Figure 2b).
After treatment for 3 days (7–9 p.c.) to obtain the most effective results, gross morphological observations of the implantation chambers on day 9 p.c. demonstrated a degeneration process of the decidualized tissue of the implantation chambers. Two hours after treatment with both compounds on day 9 p.c. there was regression of the implantation chamber in a few animals compared to the controls (Figure 3a,b). Ten hours after the last administration of both compounds, a nearly complete regression of the implantation chambers in all animals was observed (Figure 3c,d).
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Expression of iNOS and Erk1/2 on days 7–9 p.c.
In previous studies, it has been shown that iNOS increases until day 8 p.c. of pregnancy (Purcell et al., 1999
) and seems to play a major role in the differentiation of the decidua. To further investigate the iNOS expression pattern and its association with progress in decidualization, we extended our investigation to day 9 p.c. On day 9 p.c., iNOS immunostaining spread out to the mesometrial side but decreased in the primary decidual zone as well as in the antimesometrial part of the decidual tissue (Figure 4a,b). This indicates that iNOS follows the spatial and temporal pattern of the differentiation process of the stromal into decidual cells and switches to the mesometrial side at this time. In order to investigate whether MAPK are implicated in this differentiation system, we have examined activated Erk1/2 expression in the developing decidua of untreated animals between days 7 and 9 p.c.
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On day 7 of pregnancy, activated MAPK, Erk1/2, was found in the antimesometrial part of the developing decidua (data not shown). Expression increased on day 8 p.c. in the mesometrial decidua and was strongest in cells which are in the process of decidualization at the border with the myometrium. Cells which had already differentiated into decidua expressed less Erk1/2. Most staining could be seen in the stromal cell population on the border with the myometrium (Figure 4c
). In addition, spreading out of the activated Erk1/2 to the mesometrial side in the shape of a curve around the uterine lumen was observed (Figure 4c). On day 8 p.c., iNOS and Erk1/2 expression pattern was nearly identical (compare Figure 4a and c). On day 9 p.c., staining of Erk1/2 was enhanced in the mesometrial part of the decidua in parallel with a decrease in the antimesometrial decidua except from the primary decidual zone (Figure 4d). Like iNOS, the activated Erk1/2 followed the spatial and temporal pattern of decidualization, but with some variations: Erk1/2 expression was stronger in the mesometrial part and stayed in the primary decidual zone (compare Figure 4b and d).
Extensive angiogenesis and sinusoid formation is characteristic for the developing mesometrial decidua. Double immunolabelling of the decidual sinusoids for iNOS and activated Erk1/2 on day 9 p.c. demonstrated staining of the sinusoidal endothelium as well as the surrounding decidual cells in the same area (Figure 5a–d).
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Effects of aminoguanidine and low-dose onapristone on Erk1/2
As mentioned above, 3 days of treatment with aminoguanidine and onapristone led to an impairment of the decidualization process after 10 h. During this time period, we selected the animals with a modest reaction to the compounds 2–6 h after the last treatment. In order to demonstrate the importance of this marker gene for the maintainance and progression of the decidualization, we investigated activated Erk1/2 protein expression in these animals compared to that in decidua of control animals (Figure 6a
). After treatment with onapristone alone for 3 days (7–9 p.c.), the staining intensity of the Erk1/2 was decreased (Figure 6b). A similar but even more enhanced effect was observed after treatment for 3 days with aminoguanidine alone. Aminoguanidine application inhibited Erk1/2 expression predominantly at the antimesometrial side (Figure 6c). Treatment with both compounds for 3 days (7–9 p.c.) reduced the expression intensity predominantly in the antimesometrial decidua and to some extent in the junctional zone when animals were autopsied 2 h after the last treatment (Figure 6d). A complete loss of Erk1/2 staining was observed 6 h after the last application (Figure 6e). Expression recovered 10 h after the last administration, but re-expression was not observed in the decidual zone between the mesometrial and antimesometrial part of the decidua (Figure 6f).
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Effects of aminoguanidine and low-dose onapristone on angiogenesis and Cox2
In the region where the mesometrial and antimesometrial part of the decidua meet, trophoblast invasion took place and large sinusoids were formed. Most of the sinusoids were oriented towards the ectoplacental cone (Figure 7a
). After treatment with both compounds the large sinusoids were reduced and were no longer directed towards the ectoplacental cone (compare Figure 7a and b). After using the endothelial marker Thy-1, which is highly specific for endothelial cells during angiogenesis in the endometrium (Lee et al., 1998), this effect became more obvious. Although this marker was still expressed in the endothelial cells of animals treated for 3 days with aminoguanidine and onapristone, the number of sinusoids seemed to be reduced and a disorientiation of the sinusoids was observed (compare Figure 7c and d). Higher resolution revealed that the sinusoids were smaller in width (Figure 7h) compared to controls (Figure 7e). The treatment with onapristone or aminoguanidine alone had no obvious effect on sinusoid formation (Figure 7f,g).
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Interestingly, after 3 days of treatment with both compounds, Cox2 staining, which showed an expression only in the region of the closure reaction overlaying the ectoplacental cone, was nearly completely lost on day 9 p.c. (Figure 7i,j
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Discussion |
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The action of NO and progesterone is most critical for successful decidualization. By inhibition of the inducible NO and by a slight withdrawal of progesterone action, we could show here that the most sensitive phase for the synergistic effect is between day 8 and 9 p.c., when the mesometrial part of the decidua is formed. A marked worsening of pregnancy outcome is recorded when the iNOS-specific inhibitor aminoguanidine in combination with a low dose of onapristone are given during these days. The immunohistochemical observations in the present study demonstrated that Erk1/2 expression is correlated to the spatio-temporal pattern of decidua formation and is coexpressed with iNOS in the decidual cells surrounding the sinusoids. The expression of the Erk seems to be an essential step in the differentiation and maintenance of the decidua since suppression of MAPK was associated with an impairment of the decidualization process. This enzyme is not only regulated by progesterone but also by NO. This may explain the synergistic effect of aminoguanidine and low-dose onapristone on the regression of the decidual tissue. These findings support the proposed pathway for NO signalling, resulting in the MAPK activation in other cell systems (Beck et al., 1999
; review). Here we add the information that progesterone seems to act through the same signalling route for the differentiation of the decidua. The loss of iNOS activity and reduced progesterone action led to a drastic loss of MAPK activity and resulted not only in a regression of the decidua but in a loss of appropiate sinusoid formation. This could explain that the most effective phase for the action of both compounds correlates with the process of angiogenesis in the mesometrial decidua. Furthermore, Erk1/2 is re-expressed with decreasing serum levels of both compounds, but obviously not in the critical junctional zone between both parts of the decidua in the direct neighbourhood of the invading trophoblast. In a former publication of our group (Purcell et al., 1999), it has been shown that iNOS is also expressed in the ectoplacental cone. Thus, the suppression not only of NO derived from decidual cells but also from cells of embryonic origin may have differentiation effects on this decidual region invaded by trophoblast cells, and the loss of both may affect this part of the decidua more drastically. Thy-1, which here has been shown to be an ideal marker for decidual angiogenesis, is normally found in endothelial cells of newly formed vessels induced by the inflammatory cascade (Lee et al., 1998). This marker is not abolished after suppression of NO and progesterone action; however, differences in angiogenesis are detected. Sinusoid formation, which occurs most impressively in the region between the two parts of the decidua, was impaired resulting in reduced, non-dilated and disoriented vessels. Angiogenesis, however, was not completey inhibited in the decidua; this could be explained by the presence of eNOS in the decidual tissue which could compensate for this (Purcell et al., 1999). However, it has to be considered that aminoguanidine is only moderately selective for iNOS compared to that observed when it is tested in vitro (Boer et al., 2000). Reduced angiogenesis and the missing dilated sinusoids could restrict nutritition for the development of the decidua and the embryo leading to a breakdown of both. Further investigations are needed to investigate whether growth factors such as basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF) or epidermal growth factor (EGF), which are known to be involved in the implantation reaction and angiogenesis (Srivastava et al., 1998), act upstream of NO and are affected by the iNOS inhibitor and/or low-dose antigestagens. In other cell systems, such a correlation has been shown. Using specific inhibitors for NO synthesis and for Erk1/2, it was found that Erk are essential for VEGF-induced cell proliferation of cultured microvascular endothelial cells (Parenti et al., 1998). Another study showed that VEGF and bFGF activate MAPK in a brain capillary endothelial cell line (D'Angelo et al., 1995). Furthermore, we could demonstrate that Cox2 expression, which is very well known to be crucial for decidualization (Lim et al., 1997), is abolished after treatment with both compounds. Thus, we have identified four of the players necessary for maintaining decidualization, progesterone and NO, which synergistically activate the downstream pathways, Erk1/2 and Cox2 expression. The question arises of how these mediators are arranged in this differentiation cascade of decidualization. Recently, p44 MAPK (Erk1) knockouts have been generated. p44 MAPK-deficient mice were viable and fertile, thus p44 alone is not responsible for this process of decidualization and p42, Erk2, may compensate for this loss (Pages et al., 1999). Up to now it is not known if the decidualization process needs both kinases for appropriate differentiation. NO is probably acting through activation of Cox2 resulting in induced or increased prostaglandins and prostacyclins (Salvemini et al., 1995). But NO generated from iNOS is not the only mediator for decidualization and angiogenesis since iNOS gene deficiency does not interfere with the implantation process (MacMicking et al., 1995). Thus, a not-yet-understood network of compensating and interacting signals has to be postulated. However, prostaglandins, leukotrienes and cytokines are needed for implantation and decidualization in rodents (Sharkey, 1998). By using leukaemia inhibitory factor (LIF) gene-deficient mice, which fail to decidualize (Stewart and Cullinan, 1997), it has been demonstrated that the heparin-like growth factor as well as Cox2 are aberrantly expressed in the endometrium surrounding the blastocyst (Song et al., 2000). This points to the fact that growth factors and prostaglandins are acting downstream from the cytokine, LIF. A defined role for decidualization has been demonstrated for Cox2. Using Cox2 mutant mice (Lim et al., 1997), it was shown that the Cox2-derived prostacyclin is the essential prostaglandin for decidualization (Lim et al., 1999). There is ample evidence that NO interacts with Cox2 expression in other systems. It has been demonstrated, by application of a Cox2 inhibitor, that the prostaglandin production in an estrogenized rat uterus by EGF acts via NO produced by iNOS (Ribeiro et al., 1999). Using a similar experimental design, one study (Franchi et al., 1998) demonstrated that a Cox2 inhibitor as well as a NO antagonist prevent the elevation of prostaglandins produced by IL-1
. Since it has been shown that NO stimulates Cox2 (Salvemini et al., 1995), iNOS should most likely act upstream of Cox2 in these systems. To conclude, both progesterone and NO derived from iNOS act on the expression of Erk1/2 probably followed by the expression of Cox2. All mediators seem to play a crucial role in the induction and maintenance of decidualization for successful pregnancy. To unravel the different pathways and cross-talks between these and other mediators of the signalling pathway is a great challenge and is needed to establish novel therapies in reproduction.
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Acknowledgements |
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We are grateful to Dr E.Schillinger for helpful discussions. We thank Ms B.Bragulla for excellent technical help, Dave Kittel for photographic work and Claudia Hoffmann for correcting the manuscript. This work was supported by the Schering AG, Berlin.
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Notes |
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* This work was presented in part at the 47th Annual Meeting of the Society for Gynecological Investigation in Chicago, IL, USA, March 2000.
3 Present address: TAP Pharmaceutical Products Inc., Lake Forest, IL, USA
4 Institute of Anatomy, University Hospital of Essen, Hufelandstr. 55, 45122 Essen, Germany. E-mail: e.winterhager{at}uni-essen.de
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Submitted on November 29, 2000; resubmitted on November 2, 2001; accepted on February 19, 2002.
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