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Unité de Recherche en Ontogénie et Reproduction (J.P., P.C., M.A.F.), Centre Hospitalier Universitaire de Québec, Centre de Recherche en Biologie de la Reproduction, and Département d’Obstétrique et Gynécologie (M.A.F.), Université Laval, Ste-Foy, Québec, Canada G1V 4G2; and Centre de Recherche en Reproduction Animale (J.S.), Faculté de Médecine Vétérinaire, Université de Montréal, St-Hyacinthe, Québec, Canada J2S 7C6
Address all correspondence and requests for reprints to: Dr. Michel A. Fortier, Unité de Recherche en Ontogénie et Reproduction, Centre Hostpitalier Universitaire de Québec, Université Laval, 2705 boulevard Laurier, Ste-Foy, Québec, Canada G1V 4G2. E-mail: . mafortier{at}crchul.ulaval.ca
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is the embryonic signal responsible for recognition of pregnancy. This is effected by a reduction of the production of PGF2
relative to PGE2. This may be accomplished by a decrease in PGF2 production, but a stimulation of PGE2 via the PGE synthase might also be involved. The purpose of the present study was to confirm the presence of PGE synthase (PGES) in the bovine endometrium, identify the factors affecting its expression, and compare it with that of cyclooxygenase-2 (COX-2). This was done by Northern blot analysis using primary cultures of bovine epithelial and stromal cells of the endometrium and bovine endometrial cell line. PGES mRNA expression was increased in the presence of lipopolysaccharides, TNF-, and IFN- in stromal cells and IFN- in epithelial cells. In stromal cells, IFN- induced a rapid increase of PGES and COX-2 mRNA expression. In bovine endometrial cells, phorbol 12-myristate 13-actetate increased PGES mRNA, COX-2 mRNA and PGE2 production. These results suggest that in endometrial cells, the expression of PGE synthase is correlated with that of COX-2 and is an important enzyme for the production of PGE2. Increasing this production will modulate the PGE2/PGF2 ratio and contribute to establishment of pregnancy. |
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and PGE2, are known to induce opposite effects in tissues such as kidneys, the vascular system, uterine myometrium, and corpus luteum in several species. In ruminants, PGF2 of uterine origin is responsible for luteolysis and high levels of PGF2 can disturb pregnancy at any time in several species (2). In contrast, PGE2 induces systemic effects and may act as a luteotrophic or antiluteolytic agent (3). PGE2 also exerts an immunomodulation that helps to prevent rejection of the conceptus (4). Interferon- (IFN-) is the recognized pregnancy recognition signal produced by the embryo in ruminants, and in the bovine it is produced maximally between d 15 and d 19 of pregnancy (5). IFN- may prevent luteolysis by inhibiting the estrogen-induced increase in gene expression of endometrial estrogen and oxytocin receptors (OTRs) (6). IFN- also prolongs the lifespan of the corpus luteum in vivo (7) and increases PGE2 production in vitro (8). PGE2 is a major metabolite of PG endoperoxide H2 formed by the PG synthase, also named cyclooxygenase (COX). Two isoforms of the COX enzyme, types 1 and 2, are coded by different genes and catalyze the double oxygenation and reduction of arachidonic acid (AA). We have demonstrated that the increase in PGE2 observed in the bovine endometrium in vitro was correlated with the specific induction of COX-2 but not COX-1 (8). Several previous reports demonstrated that COX-2 was an inducible enzyme associated with pathologic conditions such as inflammation and cancer. The accepted pathway for the production of PGE2 involves first the generation of PG endoperoxide H2 primarily through COX-2 and then conversion into PGE2 via PGE synthase (PGES), which has been identified in a few tissues and species in recent years.
Two forms of PGES have been identified. Watanabe et al. (9, 10) have reported the purification of a protein from bovine heart with a PGES activity independent of reduced glutathione (GSH). This activity was studied in various bovine tissues including the uterus. Another group has identified a different form of PGES in human tissues (11). The latter enzyme is microsomal, inducible, and GSH dependent and shares 38% identity in amino acid sequence with microsomal GSH S-transferase 1, which belongs to the membrane-associated proteins in eicosanoid and GSH metabolism superfamily (12). Low levels of this microsomal PGES were detected in human uterus. Homologous PGES sequences in mouse (84% homology to human) (13) and rat (82% homology) (14, 15) have also been characterized. Recently, bovine PGES was identified and cloned (85% homology to human) in ovarian follicles, and its expression was modulated by human chorionic gonadotropin (16). In bovine endometrial cells, we have demonstrated that IFN- stimulates PGE2 production and COX-2 but not COX-1 or phospholipase A2) expression (8). This increase in COX-2 expression was greater in stromal than in epithelial cells. Another study also demonstrated that, in vitro, human chorionic gonadotropin-induced human stromal cell differentiation via an up-regulation of COX-2 correlated with an increase in PGE2 production (17). Together the previous observations suggest that PGES may be an important rate-limiting enzyme for PGE2 biosynthesis in the bovine endometrium.
Over the last years, our observations led us to hypothesize that recognition and establishment of pregnancy depends on the regulation of the balance between PGF2 as the luteolytic signal and PGE2 as the antiluteolytic or luteotrophic signal. In this respect, the relative production of PGE2 and PGF2 may be more important than the absolute production of each PG. The PGE2/PGF2 ratio can be modulated in favor of PGE2 and establishment of pregnancy by three different pathways: 1) the well-accepted hypothesis of a decrease in the production of PGF2; conversion of PGE2 into PGF2 by a putative 9-keto-prostaglandin E2 reductase previously described by our group (18); and 3) an increase in PGE2 through the stimulation of PGE synthase. We have shown that PGE2 production is increased in both types of endometrial cells following treatment with recombinant ovine IFN- (roIFN-) (19, 20). In epithelial cells, the alteration in PG production was such that the primary PG produced was changed from PGF2 to PGE2 (19). We have shown recently that the expression of PGES mRNA closely followed that of COX-2 during the bovine estrous cycle (21). However, to date, no study has reported the presence of PGES in bovine endometrial cells in relation with expression of COX-2 and production of PGE2.
The objectives of the present study were to: 1) confirm the presence and identity of PGES in the bovine endometrium; 2) determine whether the expression of PGES is regulated by known modulators of PGE2 production in endometrial cells; and 3) compare the expression of PGES with that of COX-2 (coexpression) during modulation of PGE2 production.
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Materials and Methods |
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, and lipopolysaccharides (LPS) were purchased from Sigma (St. Louis, MO). BrightStar Plus membrane and ULTRAhyb solution were purchased from Ambion, Inc. (Austin, TX). [-32P]dCTP radioactivity was purchased from Perkin-Elmer Life Sciences (Markham, Ontario, Canada). Topo TA cloning kit was purchased from Invitrogen Corp. (Carlsbad, CA). The roIFN- was kindly provided by Dr. Fuller Bazer; the antiviral activity was 1 x 108 U/mg protein.
Preparation of tissues
To evaluate the presence of PGES in the endometrium and other tissues, different tissues were collected at a local slaughterhouse within 15 min of death of animals. Some tissues were collected from one cow in the early stage of the estrous cycle (d 1–3) (ovary, oviduct, endometrium, myometrium, heart, liver, lung, muscles, and kidney) and others from one bull (testis and seminal vesicles). Tissues were transported on ice to the laboratory where they were rapidly cut in small pieces, frozen in liquid nitrogen, and stored at -80 C until RNA extraction.
Isolation and culture of endometrial cells
Preparation of epithelial and stromal cells was done according to the procedure described by Fortier et al. (22). Briefly, bovine uteri were collected from animals in early days of the estrous cycle (d 1–5), and the myometrium was dissected out, keeping the tubular structure of the endometrium and endometrial cells separated by selective digestion. In this study, a total of four uteri were used to generate four different cell preparations. Endometrial epithelial and stromal cells were cultured in 6-well plates in RPMI-1640 supplemented with 10% FBS depleted of steroids by dextran-charcoal extraction in a humidified atmosphere of 5% CO2: 95% air at 37 C. The medium was changed every 2 d until the cells were used. Confluency of the two cell types was observed after 7–8 d in culture.
The commercial BEND (CRL-2398) was purchased from ATCC (Manassas, VA). BEND cells were used as an alternate in vitro model in which the primary PG produced is PGE2. The culture and propagation of BEND cells was done as described in the instructions provided by ATCC. Briefly, cells were grown in a 1:1 mixture of Ham’s F12 and Eagle’s MEM with Earle’s BSS (D-valine modification) with 1.5 mM L-glutamine, 1.5 g sodium bicarbonate containing 0.034 g/liter D-valine, 10% FBS, and 10% horse serum. In all experiments using 6-well plates, BEND cells (passage 21) were plated at a 1/3 split ratio and grown at 37 C under a humidified atmosphere containing 95% O2 and 5% CO2. When the cells were fully attached (24 h), the culture medium was replaced with fresh medium supplemented with 10% FBS and horse serum depleted of steroids by dextran-charcoal extraction. Under those conditions, cells reached confluence 2 d later.
Experimental protocols for endometrial cells in culture
Epithelial and stromal cells were grown to confluency, and the medium was replaced with RPMI-1640 without FBS. Cells were then stimulated for 16 h with different factors known to influence PG production: IFN- (2 µg/ml or 20 µg/ml), TNF- (10-9 M), OT (10-7 M), and LPS (0.01 µg/ml). BEND cells were treated for 24 h with PMA (10-7 M) and LPS (0.01 µg/ml) in serum-free medium. Three wells were used for each treatment. For all experiments at the end of incubation time, the culture medium was recovered for PGE2 measurement and stored at -20 C until further processing. Total RNA was extracted directly from epithelial, stromal, and BEND cells with 0.8 ml/well TRIzol reagent, according to the manufacturer’s instructions. RNA samples were resuspended in water containing diethyl pyrocarbonate (0.05% vol/vol) and stored at -80 C. Before use, RNA was quantified by measurement of absorbance at 260 nm.
RNA isolation from frozen tissues
Frozen tissues kept at -80 C were placed in liquid nitrogen and further pulverized in a mortar. The resulting powder was weighed and TRIzol was added at a 1 ml/100 mg ratio. The suspension was homogenized three times on ice and RNA was extracted and processed as described above.
RT-PCR
Total RNA extracted from tissues was used as follows: total RNA samples (1 µg) were reverse-transcribed at 37 C with MMLV-RT (200 U) and oligo(dT) primers (0.2 µg) in a final volume of 20 µl. Reaction volume was then brought to 70 µl with diethyl pyrocarbonate water. Each PCR amplification was run with 5 µl reverse transcription template or negative control and Taq polymerase (1.5 U) in a final volume of 50 µl. Primers used were the same used for ovarian tissues described in a recent publication (16) (PGES sense: 5'-GAATGACATGGAGACCATCTACCC-3' and antisense: 5'-TATCAATCGTGACGGTCCGTCTC-3'; ß-actin sense: 5'-AACTGGGACGACATGGAGAAGATCTGGCA-3' and antisense: 5'-GAGGATCTTCATGAGGTAGTCTGTCAGGTC-3'). PCR amplification was achieved as follows: 94 C for 30 sec, 55 C for 1 min, and 72 C for 1 min during 40 cycles for PGES and 35 cycles for ß-actin. PCR products were loaded on 1.2% agarose gel and visualized with ethidium bromide. Bands were quantified by image analysis using the AlphaImager 2000 software (Alpha Innotech Corp., San Leandro, CA).
Northern blot analysis
Equal amounts (15 µg) of total RNA were loaded on a 1.2% wt/vol formaldehyde agarose gel, electrophoresed, and transferred onto a nylon membrane. Complementary DNA probes for bovine PGES and bovine COX-2 were generated by PCR amplification with specific primers (PGES: sense 5'-AAACATATGCCTGCCCACAGCCTGGTGATG-3' and antisense 5'-AAACATATGTCACAGGTGGCGGGCTGCCTC-3'; COX-2: sense 5'-TCTTTGACTGTGGGAGGATACA-3' and antisense 5'-TCCAGATCACATTTGATTGACA-3'). The cDNA fragments of bovine PGES and COX-2 were obtained by EcoR1 digestion of recombinant clones, thus liberating a 466-bp fragment for PGES and a 449-bp fragment for COX-2 ready to be labeled. These recombinant clones came from a cloning in Topo cloning kit (Invitrogen). The same membrane was used for both messengers, one at a time. Probes were labeled with dCTP and [32P]dCTP and purified by precipitation (23). Prehybridization was performed at 45 C in UltraHyb solution for 4–5 h, then the labeled probe was added, and hybridization was performed overnight at 45 C. Washings were done at room temperature 2 x 5 min and at 68 C 1 x 15 min in saline sodium citrate (2x) supplemented with 0.1% SDS and then 2 x 15 min in saline sodium citrate (0.2x) supplemented with 0.1% SDS. Signals were detected by autoradiography on X-omat (Kodak, New York, NY) at -80 C after exposure for 1–7 d before development. Bands were quantified by image analysis using the AlphaImager 2000 software (Alpha Innotech Corp.). Intensity of each band was normalized to the intensity of corresponding 18S as seen on the gel.
ELISA for PGs
For PGE2 measurement, an enzyme immunoassay was used, using acetylcholinesterase-linked PG tracers as described previously (20). We used a fully characterized rabbit anti-PGE2 (20, 24) antibody. The inter- and intraassay coefficients of variation (n = 12) were 16% and 10%, respectively.
Statistical analysis
Ratios of PGES/18S, COX-2/18S, and PGE2 levels were analyzed by ANOVA using super-ANOVA software (ABACUS Concepts Inc., Berkeley, CA). Sources of variations included effects because of cell preparations, treatments, and their interactions. Individual comparisons of means were made using Fisher’s protected least significant differences, Duncan new multiple range, Student-Newman-Keuls, and Scheffé’s S tests. Differences were considered statistically significant when P was less than 0.05.
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. All tissues tested expressed PGES albeit at different levels, even though the amount of RNA used for the PCR was equivalent as revealed by ß-actin amplification. This expression is higher in ovary, myometrium, testis, seminal vesicles, and lung. PGES mRNA is present in the endometrium at low levels, but as we have reported recently, this is due to low expression on early days of the estrous cycle (21).
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), PGE2 production was stimulated by all treatments (P < 0.05). PGES mRNA was expressed in all conditions and the expression was increased 2-fold by IFN- (P < 0.05). The expression of COX-2 closely matched that of PGES being stimulated following treatment with IFN- (P < 0.05). In stromal cells (Fig. 3), the production of PGE2 followed the expression of PGES mRNA and was stimulated following treatments with TNF- (4.6-fold), LPS (5-fold), and IFN- (7-fold) (P < 0.05). Because stromal cells do not respond to OT (20), LPS was used to stimulate PGE2 production by a pathway different from TNF-.
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was studied in primary stromal cells by Northern blot analysis (Fig. 4). Stromal cells were stimulated with a dose of IFN- in the upper physiological range (20 µg/ml) for 6, 12, and 24 h. There is no visible signal for PGES at 6 h under control conditions, but treatment with IFN- results in a detectable signal at all times from 6 to 24 h with a maximal induction at 12 h (>5-fold) (P < 0.05). The pattern of expression of COX-2 is similar to that of PGES under both control and stimulated conditions (r = 0.922).
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) (r = 0.976). The effect of LPS on BEND cells is comparable with what was observed in stromal cells (Fig. 3
). Because BEND cells do not respond to OT, PMA was used to stimulate the protein kinase C (PKC) pathway and induced a strong stimulation of PGE2 production and expression of both PGES and COX-2.
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Discussion |
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(8, 19) is associated with an increase in mRNA levels of both PGES and COX-2. PGES activity has been identified first in the human lung cancer cell line (25), and more recently bovine PGES was cloned from follicular cDNA (16). Another group (26) found PGES in the human endometrium. We have shown recently that PGES was expressed and regulated in the bovine endometrium during the estrous cycle (21). The present study clearly confirms the presence of PGES in bovine endometrial cells and its modulation under conditions known to influence PG production.
In this report, the GSH-dependent and not the GSH-independent type of PGES reported by Watanabe et al. (9, 10) and Tanikawa et al. (27) was studied because of its known association with COX-2 and PGE2 production (13). We have already demonstrated that IFN- increased PGE2 production and COX-2 but not COX-1 expression (8). Therefore, we assumed that the cytosolic type of PGES linked to COX-1 (28) was less important for the production of PGE2 in endometrial cells, but we do not exclude this possibility.
The presence of PGES in the endometrium and in other bovine tissues is illustrated in Fig. 1. Similar results were already reported for human (11) and different bovine tissues (16). The presence of PGES in the endometrium was investigated further in vivo, and we have found that it was regulated during the estrous cycle in a pattern closely matching that of COX-2 (21). PG synthase expression without distinction of the two isoforms was studied in vivo, and it was found to be modulated throughout the cycle with variations similar in the different cell types forming the endometrium (29).
Previous studies (25) have reported the effects of different agents like IL-1ß, LPS, TNF-, and PMA on PGES expression in vitro in A549 human lung cancer cells. In primary endometrial cells, PGES expression was induced by IFN- in epithelial cells (Fig. 2), whereas TNF- and OT did not show any effect. In stromal cells, IFN-, TNF-, and LPS up-regulated PGES mRNA expression (Fig. 3). In these cells, PGE2 production was modulated in parallel with induction of PGES and COX-2 expression in all conditions. In epithelial cells, the increase in PGE2 in response to OT and TNF- was observed in absence of a detectable effect on PGES and COX-2 mRNA. We have already characterized the stimulation of PG production in primary epithelial and stromal cell cultures in response to different factors (30). In that study OT response was found only in epithelial cells, and it was suggested that stimulation of PGF2 and PGE2 production was effected through a PKC-mediated pathway. The involvement of phospholipase C and PKC in the regulation of PG production was demonstrated further in epithelial cells following treatment with OT (31). In a more recent study, we have confirmed that in vivo, OTRs were expressed primarily in luminal epithelial cells of the bovine endometrium (32). Therefore, it is possible to increase PGE2 production simply by increasing available AA in the presence of COX-2 and PGES proteins before a noticeable increase in COX-2 or PGES mRNA. Indeed, we have shown that NS-398, a selective COX-2 inhibitor, was able to inhibit both basal and stimulated PG production in cultured endometrial cells (31). Moreover, even though COX-1 is expressed in these cells, it does not appear to contribute to PG production, suggesting that low levels of COX-2 can sustain the relatively high basal levels of PG production in cultured cells (33). This is further supported by the reported high sensitivity of COX-2 to low levels of AA, compared with COX-1 (34). It is likely, however, that agents increasing PGE2 production also increase both COX-2 and PGES mRNA and proteins either at a time anterior or subsequent to the 16 h used in the present experiment. This time was chosen before the maximal effect on PG production because the measurement of mRNA by Northern analysis was the only quantitative tool available for PGES at the time of experimentation. We and others have shown that maximal stimulation of COX-2 mRNA and proteins is short lived both in vitro and in vivo. In BEND cells for instance, in which PGES and COX-2 mRNA were measured at 24 h and correlated with the accumulation of PGE2 over the 24-h period, we see a very good correlation between the expression of the two enzymes and PGE2 production in cells treated with PMA, compared with untreated cells.
BEND cells, bovine epithelial endometrial cells derived from d 14, were used as an alternate, commercially available in vitro model. Although the exact lineage of those cells is not clear, BEND cells produce primarily PGE2 and respond to IFN- consistently in our laboratory, making them ideally suitable to study PGE synthase. Stimulation of expression of PGES and COX-2 occurred only with addition of PMA that resulted in increased PGE2 production. No effect on PGES expression occurred in presence of LPS. These results are different from those obtained with primary endometrial cells, either epithelial or stromal. We have observed several differences in properties of BEND cells, compared with primary endometrial cells, and we have described and discussed them extensively (35).
At the time of recognition of pregnancy, stimulation of PGES may be one way to increase the PGE2/PGF2 ratio to favor establishment of pregnancy. We and others use IFN- in studies in vitro to mimic the effect of the embryo and understand the mechanisms involved at the cellular and molecular levels. We have used primary stromal cells to study the time course of IFN- action (Fig. 4) because it caused a 7-fold increase in PGES mRNA level after 16 h (Fig. 3). Figure 4 illustrates that in the presence of IFN-, PGES expression is increased, compared with control without IFN-. Maximal expression of both PGES and COX-2 occurred at 12 h, but effects were detected as soon as 6 h after stimulation and were still visible at 24 h. These results suggest that IFN- can induce a quick and simultaneous stimulation of COX-2 and PGES expression. In vivo, IFN- is secreted locally by the conceptus for a short period of time, between d 15 and d 24 of gestation (5), and it was suggested that exposure for at least 3 d was necessary to induce a reduction in OTRs and sensitivity (36). On the other hand, it has been demonstrated that a bovine embryo can be transferred as late as d 16 and induce pregnancy at a time when functional oxytocin receptors are present (37, 38). That indicates that IFN- can induce establishment of pregnancy in less than 24 h. A quick induction of PGES and COX-2 is then very important to produce PGE2 to favor establishment of pregnancy by increasing the PGE2/PGF2 ratio.
Whereas the role of PGF2 as the luteolytic factor is clearly proven, the antiluteolytic or luteotrophic action of PGE2 is still debated. Similarly, the action of IFN- on endometrial cells to reduce PGF2 production and/or stimulate PGE2 production depends on the dose and type of IFN used. Different concentrations of roIFN- have been used in epithelial and stromal cells in culture. Our initial report of a stimulating action of IFN- on COX-2 and PGE2 production appeared in conflict with other reports describing an inhibition. This apparent discrepancy could be explained by the use of different doses and isoforms (33) of IFN- and is now considered as reflection of the complex interactions of IFN- with its receptor(s) (39). Indeed, we have shown that different doses of IFN- could produce a biphasic effect. At low doses (<1 µg/ml), no significant variation of PGE2 production (19) and or a reduction of PGF2 was observed (8, 19). With higher concentrations of IFN-, a significant modulation of the ratio PGE2/PGF2 was seen, and a 27-fold stimulation of PGE2 production occurred with the maximal dose (20 µg/ml). Accordingly, the maximal dose was used to study the time course of PGES mRNA expression in stromal cells. This dose is still 20 times lower than the maximal concentration found in the vicinity of the embryo in vivo.
Recent reports suggested that TNF- can act as a luteolytic factor by increasing PGF2 in bovine endometrial cells (40, 41). Our results demonstrate that TNF- also can induce PGE2 production in epithelial cells in primary culture and even more in stromal cells. PGES mRNA expression was also induced by TNF- in stromal cells. Therefore, a better characterization of the balance between induction of PGF2 and induction of PGE2 has to be done to conclude that TNF- induces luteolysis.
In conclusion, our results demonstrate a good correlation between COX-2 and PGES mRNA expression and PGE2 production in bovine endometrial cells. Similar correlations have been reported before in various tissues of different species, but to the best of our knowledge, it is the first time that this is demonstrated in the endometrium of any species. We already showed a modulation of COX-2 with an increased PGE2 production (8). We now demonstrate that PGES can be modulated at the same time as COX-2 to explain preferential increase in PGE2 production. In reproduction, it has already been shown that PGES is essential in the preovulatory follicle before ovulation (16). We speculate that other reproductive processes that need PGE2 production like fertilization, decidualization, and implantation will be associated with the regulation of PGES.
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Acknowledgments |
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; Christian Villeneuve for his help with the primary culture cells; Joe A. Arosh for his help with the statistical analysis; and Éric Madore for helpful discussions. |
Footnotes |
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Abbreviations: AA, Arachidonic acid; COX, cyclooxygenase; -dCTP, DNA labeling kit; FBS, fetal bovine serum; GSH, reduced glutathione; IFN-, interferon-; LPS, lipopolysaccharides; MMLV-RT, Moloney murine leukemia virus-reverse transcription; OT, oxytocin; OTR, oxytocin receptor; PG, prostaglandin; PGES, PGE synthase; PKC, protein kinase C; PMA, phorbol 12-myristate 13-actetate; roIFN-, recombinant ovine IFN-; RT, reverse transcription.
Received February 6, 2002.
Accepted for publication April 23, 2002.
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