Cyclooxygenase-2 Unlike Cyclooxygenase-1 Is Highly Expressed in ...

BIOLOGY OF REPRODUCTION 57, 1032-1040 (1997)

Cyclooxygenase-2 Unlike Cyclooxygenase-1 Is Highly Expressed in Ovine Embryos during the Implantation Period' Gilles Charpigny,23 Pierrette Reinaud, 3 Jean-Philippe Tamby, 3 Christophe Creminon, 4 and Michel Guillomots Laboratoire de Physiologie Animale,3 Institut National de la Recherche Agronomique, 78352 Jouy-en-Josas Cedex, France Service de Pharmacologie et d'lmmunologie, 4 Commissariat I'Energie Atomique, Centre d'Etudes de Saclay, 91191 Gif-sur-Yvette Cedex, France Unite de recherches associe 1291 CNRS, laboratoire de Physiologie Animale, Institut National de la Recherche Agronomique, 78352 Jouy-en-Josas Cedex, France ABSTRACT In this study we investigated expression of the two isoforms of the prostaglandin-forming enzyme, cyclooxygenase-1 (Cox-1) and cyclooxygenase-2 (Cox-2), in sheep embryos. Using Western blot and immunohistochemical analyses, we demonstrated that Cox-2 was highly expressed in embryos from Day 8 to Day 17 of development whereas Cox-1 was undetectable during this time. The expression of Cox-2 was developmentally regulated. It was maximal between Days 14 and 16. There was a 30-fold increase in Cox-2 content per protein extract between Day 10 and Day 14, corresponding to a 50 000-fold increase in the whole embryo. The expression of Cox-2 declined after Day 16 to become undetectable by Day 25 of pregnancy. Cox-2 was localized in the trophoblastic cells and was not detected in the inner cell mass. The [3 H]arachidonic acid metabolites synthesized by Cox2-rich conceptuses were analyzed by HPLC after short-term embryo culture. Day 14 conceptuses released mainly cyclooxygenase metabolites and to a lesser extent lipoxygenase derivatives. Cyclooxygenase products were 6-keto-prostaglandin (PGF)l, 18.2% ( 4.2), thromboxane-B 2 22.51% ( 15.9), PGF 2. 21% (+ 11), PGE2 14.5% ( 7.4), and PGD 2 2.7% ( 2.6). Taken together, these results suggest an important role for the Cox-2dependent cyclooxygenase metabolites during embryo development. INTRODUCTION Prostaglandins are involved in the process of blastocyst implantation (reviewed in [1]). They are responsible for the increase in endometrial vascular permeability and subsequent decidualization (reviewed in [2]). Both the endometrium and blastocyst are capable of producing prostaglandins, but the endometrium rather than the blastocyst is thought to be the major source of the prostaglandins involved in implantation [3]. The prostaglandins produced by the embryos have been considered to be responsible for blastocoelic fluid accumulation and for hatching [4]. In sheep, prostaglandins of blastocyst origin have been evoked in trophoblast elongation [5]. Prostaglandins are converted from arachidonic acid by the key enzyme prostaglandin synthase. The prostaglandin synthase enzyme exists under two isoforms encoded by separate genes (reviewed in [6, 7]). The first one, referred to as cyclooxygenase-1 (Cox-l), is expressed constitutively and is relatively unresponsive to stimuli. The second one, Accepted June 30, 1997. Received May 14, 1997. ° 'This work was supported by grants from European Union (n B102CT92-0067). 2Correspondence. FAX: 33-1-34652364; e-mail: [email protected]

cyclooxygenase-2 (Cox-2), which is the more recently identified isoform, can be induced by a wide variety of factors including cytokines, growth factors, and tumor promoters [8-101. However, there are only few reports evaluating the expression of prostaglandin synthase enzymes in embryos. Prostaglandin synthase has recently been demonstrated in mouse and bovine embryos. In mice, the blastocysts express lower levels of prostaglandin synthase than do embryos at earlier stages of development [11, 12]. In the bovine embryo, cyclooxygenase expression appears transient and is associated with the first cleavages, since a decrease in enzyme concentration occurs at the morula stage [13]. These reports suggest an important physiological role for this enzyme in early embryonic development prior to implantation. A second burst of prostaglandin synthase expression is suggested in ruminant embryos, however, since ovine [4, 14] and bovine conceptuses [15, 16] produce large and increasing amounts of prostaglandins from the time of hatching to implantation. The initial objective of the present investigation was to establish the prostaglandin synthase expression in hatched ovine embryos during the elongation and the implantation periods. Secondly, since Cox-2 was recently identified in the ovine placenta at term [17] and the cellular localization of Cox-2 has not yet been determined precisely [18, 19], we decided to examine the localization of both Cox-I and Cox-2 expression in late pregnancy. In the present study we demonstrate that the cyclooxygenase-2 isoform was highly expressed in the trophoblastic cells of the ovine conceptus before implantation, whereas cyclooxygenase- was undetectable. Since Cox-2 was expressed to a large extent in ovine embryos and was developmentally regulated, an important function for the resulting prostanoids produced by embryos was suggested. Therefore, this study also investigated the pattern of eicosanoid synthesis by ovine embryos. MATERIALS AND METHODS Animals and Collection of Embryos, Uteri, and Placentomes All procedures relating to the care and use of animals were approved by the French Ministry of Agriculture according to the French regulations (instruction 19/04/1988) for animal experimentation. Ewes of the Pr6alpes-du-Sud breed were used. Estrus was synchronized using intravaginal sponges containing 60 mg 6a-methyl-17cL-acetoxyprogesterone for 14 days as previously described by Peterson et al. [20]. On the day of

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COX-1 AND COX-2 IN OVINE EMBRYOS sponge withdrawal, the ewes received one injection i.m. of 500 IU eCG. Estrus was observed 48 h later (Day 0), and the ewes were mated twice. On Days 8, 10, 12, 13, 14, 15, 16, and 17 of pregnancy the ewes were slaughtered. The uterus was removed. Except for Day 17 embryos, which were recovered by dissection of the uterus, the embryos were flushed from the uterus with PBS at 37C. On Day 25 of pregnancy the uterus was removed, sectioned in pieces 1 cm long, frozen in liquid nitrogen, and stored at -80°C. Placentomes with adjacent intercotyledonary area were obtained from Day 130 pregnant ewes under general anesthesia. Placentomes were immediately frozen in liquid nitrogen and stored at -80°C until further analysis. Western Blot After collection, the embryos were washed three times in 0.1 M phosphate buffer, pH 7.3 (PBS), and individually frozen until Western blot analysis. Embryo tissues were sonicated in 20 mM Tris-HCl, pH 7.5, containing 5 mM EDTA, 16 mM 3-[(3-cholamidopropyl)-dimethylammonio]-l-propane-sulfonate (CHAPS), 1 [Lg/ml leupeptin, 10 Vg/ml soybean trypsin inhibitor, and 1 mM benzamidineHC1. The homogenates were clarified by centrifugation (3000 x g, 15 min, 4C). Protein from the homogenates was separated on 10% SDS-PAGE and electroblotted onto 0.1-pxm nitrocellulose membranes as previously described [21]. Immunoblots were performed in duplicate, one for Cox-1 analysis and the other for Cox-2 analysis. Cox-2 was detected with the mouse monoclonal antibody (mAb294) raised against the peptide C-NASSSRSGLDDINPTVLLK (Cox-2 peptide), which is present in the carboxyl-terminus of human Cox-2 and absent in Cox-1 protein. Cox-1 was detected with the rabbit anti-Cox-1 polyclonal antibody (L855) raised against Cox-l purified from ram seminal vesicles. A more detailed description of anti-Cox-1 and antiCox-2 antibodies has been previously reported [21, 22]. Membranes to be probed with the anti-Cox-2 antibody were saturated overnight at 4C in 50 mM Tris-HCl (pH 7.5), 250 mM NaCl, 0.1% Tween 20 (TBS-T buffer) containing 5% fat-free dry milk. Membranes were then incubated for 1 h with the anti-Cox-2 mAb294 at a concentration of 3 pIg/ml in TBS-T buffer containing 5% fat-free dry milk. Membranes to be probed with the anti-Cox- 1 antibody were saturated overnight at 40C with TBS-T buffer containing 1% Tween 20. Membranes were then incubated for 1 h with the anti-Cox-1 L855 at 1:3000 dilution in TBS-T containing 1% Tween 20. After extensive washes in TBS-T buffer, blots were further incubated for 1 h at room temperature with goat horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG at 1:3000 dilution in TBS-T buffer. Excess of the second antibody was eliminated by extensive washes in TBS-T buffer (five consecutive 10-min washes). Chemiluminescent substrate was used according to the manufacturer's instructions (ECL Kit; Amersham, Les Ulis, France), and immunoreactive proteins were visualized after 0.5- to 10-min exposure to hyperfilm-ECL (Amersham). Protein concentrations of the samples were determined by Bio-Rad (Richmond, CA) protein assay based on the Bradford dyebinding procedure [23]. Immunoreaction signals were quantified on the films by scanning densitometry using an image analysis system (M-Lecphor, Biocom, France). The concentration of Cox-1 and Cox-2 in the embryonic extracts was determined by comparison to known amounts of each cyclooxygenase isoform loaded onto the gel. Standards for Cox-1 and Cox-2

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were purchased from Cayman Chemical (Spi-Bio, Massy, France). Densitometric analysis indicated that the intensity of the immunostaining was proportional to the quantities of Cox-2 loaded onto the gel from 15 ng to 250 ng of standard Cox-2 protein and from 0.5 pxg to 5 Lg of total embryo proteins. The relationship between the intensity of the immunostaining and the standard Cox-1 protein ranged from 6 ng to 100 ng. A total of 3 to 10 embryos of each stage were used. Immunohistochemistry After collection, conceptuses from Day 8, Day 10, and Day 14 were washed three times with PBS and were fixed for 1 h in 0.1 M PBS containing 4% paraformaldehyde. Placentomes as well as uterine horns containing implanted Day 17 and Day 25 conceptuses were sectioned in 6- to 8-[tm slices using a Frigocut cryostat (Reichert-Jung, Nossloch, Germany). Sections were collected on microscope slides, dried in air, and stored at -20 0 C. Frozen tissue slices were fixed for 15 min in a cold 4% paraformaldehyde solution and washed twice for 5 min with 0.1 M PBS (pH 7.2), 0.1 M glycine for rehydration. Day 8, Day 10, and Day 14 conceptuses that were not sectioned were permeabilized with 0.5% Triton X-100 in 0.1 M PBS for 10 min prior to the immunodetection procedure. Nonsectioned tissues and sections were then treated in the same conditions except that all buffers used for nonsectioned tissues contained 0.05% saponin. Samples were then incubated for 1 h with nonimmune goat serum (1:10 dilution) in 0.1 M PBS containing 0.2% BSA. Tissues and sections were then incubated for 1 h at room temperature in a humidified atmosphere with the anti-Cox-2 monoclonal antibody mAb294 (2.5 pxg/100 pl) or anti-Cox- polyclonal antibody L855 (1:20 dilution), which were diluted in 0.1 M PBS containing 2% BSA. Controls were incubated with the primary antibodies adsorbed with the respective antigen or were incubated with nonimmune rabbit serum or with incubation buffer alone. After three washes of 15 min each with 0.1 M PBS containing 0.2% BSA, the tissues and sections were incubated for 1 h at room temperature, in darkness, with goat fluorescein isothiocyanate-conjugated antirabbit or anti-mouse IgG diluted at 1:200 in 0.1 M PBS containing 2% BSA. Cellular nuclei were counterstained with 0.1% propidium iodide or with Hoechst's dye (1:500 dilution in 0.1 M PBS, pH 7.2, containing 0.2% BSA) for sections. Specific fluorescence was examined by confocal microscope (Zeiss, Paris, France) and a microscope equipped with an epifluorescence illuminating system (Reichert, Paris, France). Microphotographs were taken on Fuji film (color Sensia 400 ASA; Fuji, Tokyo, Japan). The same exposure time was used for both control and experimental sections. Analysis of Metabolism of Arachidonic Acid by Embryos To determine the profile of arachidonic acid metabolites synthesized by conceptuses, twelve Day 14 embryos were incubated individually in 6 ml of Minimum Essential Medium containing 0.05% polyvinylpyrrolidone and 0.5 Ci/ml of [5,6,8,9,11,12,14,15(N)- 3H]arachidonic acid (specific activity: 200 Ci/mmol; Amersham). Incubations were carried out for 6 h at 38°C in an atmosphere of 5% CO 2 in air. Control culture medium containing radioactive arachidonic acid without an embryo was incubated under identical conditions to assess possible instability of arachidonic acid during the incubation. At the end of the incubation

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period, the embryonic tissues were removed and the culture media were centrifuged (10 000 x g, 15 min, 4°C). Supernatants were acidified to pH 3.5 with 100 .il/mlof 0.5 M citric acid and stored at -80°C until HPLC analysis. Samples were simultaneously extracted and analyzed in a single-step HPLC procedure as described by Powell [24] with some modifications. Arachidonic acid metabolites were extracted from the culture media using a precolumn containing octadecylsilyl silica (Nucleosil C18, 30 x 4.6 mm), which was connected on-line to a Zorbax C18 analytical column (250 x 4.6 mm) via an automated switching valve. Nucleosil was purchased from Touzart et Matignon (Les Ulis, France), and the Zorbax column was from Shandon (Cergy-Pontoise, France). The HPLC system was from Waters Associates (St-Quentin, France) and included three pumps (Model 510) coupled to a Model 680 gradient controller, a U6K injector, and an automatic 6-port switching valve. Two pumps were used to deliver the mobile phases to the separation column, and the third was used for sample loading and extraction onto the pre-column. Products were detected using a multi-wavelength UV detector (Waters Model 490) and a Packard radioactivity detector (Model Flo-on A500) equipped with a cell of 0.5 ml for liquid scintillation counting. The liquid scintillator was Flo-Scint III from Packard (Rungis, France) and was delivered at 3 ml/min. Samples were adjusted to 15% methanol by addition of pure methanol and were centrifuged (10 000 x g, 10 min, 4°C). Samples were then loaded in a 10-ml loading loop, and the loop was connected to the precolumn, which had been previously equilibrated with 15% methanol containing 0.01% acetic acid. The precolumn was washed for 8 min at a flow rate of 2 ml/mn and was then coupled to the separation column. Reverse-phase HPLC separation was conducted at 1 ml/mn using a gradient prepared from water: acetic acid (100:0.01) (solvent A) and acetonitrile:acetic acid (100:0.01) (solvent B) as follows: isocratic elution from 0 to 32 min at 36% B; 32 to 40 min, linear gradient to 50% B; 40 to 52 min, linear gradient to 55% B; 52 to 76 min, linear gradient to 60% B; 76 to 100 min, isocratic elution at 90% B. After completion of the chromatography, the precolumn was disconnected from the column flow line and washed with 85% methanol:acetic acid (100:0.01) before being equilibrated with the starting solvent. The column was washed with 100% B for 10 min and then conditioned for the next run at 36% B. Standards were obtained from Cayman Chemical (SpiBio). Prostaglandin B2 (PGB 2), prostaglandin D2 (PGD 2), prostaglandin E2 (PGE 2), prostaglandin F20 (PGF 2.), thromboxane-B 2 (TxB 2), 6-keto prostaglandin Fl, (6-ketoPGF,,), 13,14-dihydro-15-keto prostaglandin F 2. (DHKPGF 2.), 13,14-dihydro-15-keto prostaglandin E 2 (DHKPGE 2 ), 5(S)-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE), 1 l(S)-hydroxy-6,8,12,14-eicosatetraenoic acid (11-HETE), 12(S)-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE), 15(S)-hydroxy-5,8,11,13-eicosatetraenoic acid (15-HETE), and 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT) were obtained in individual vials and kept at -20°C under nitrogen. Peptide-leukotrienes were obtained as a mixture containing leukotriene C4, leukotriene D4, leukotriene E4, leukotriene F4, and N-acetyl-leukotriene E4. Statistical Analysis Comparison of the relative amounts of immunoreactive cyclooxygenases were made by one-way ANOVA followed

FIG. 1. Cox-2 immunodetection in ovine embryos 10-17 days old. Upper panel) Representative Western blot of embryo lysates. One microgram protein was probed with a mouse monoclonal anti-Cox-2 mAb 294 (3 Jxg/ml) (10-min film exposure). Lower panel) Densitometric analysis of the Cox-2 expression was determined by scanning the chemiluminescent signals obtained from 5 to 10 embryos by day of pregnancy. Values are means + SE of relative density.

by Student-Newman-Keuls test as a multiple comparison method. Concentrations of cyclooxygenases were determined from standard curves produced by analyzing the intensity of bands from known amounts of cyclooxygenase standards. Results were expressed as means ± standard errors. Radioactive metabolites of arachidonic acid were expressed as percentage of total radioactive derivatives. RESULTS Expression of the Cyclooxygenases in Ovine Embryos Western blots showed that ovine embryo tissues expressed a 70-kDa Cox-2 protein (Fig. 1). In order to evaluate the change in Cox-2 expression levels during the development, 1 g of protein extract from each embryo was subjected to Western blot. This amount corresponded to the whole protein content of a Day 10 embryo. Densitometric analysis of Cox-2 band obtained from 5 to 10 embryos per stage showed that the expression of Cox-2 was detected from Day 10 and was considerably increased on Day 14 (p < 0.001). The expression remained high on Day 15 and 16 and declined by Day 17 (p < 0.05). There was a 30-fold increase in Cox-2 protein levels per unit of conceptus proteins between Day 10 and Day 14 of pregnancy (p < 0.001). The main increase occurred between Day 13 and Day 14 (8-fold; p < 0.001). Taking into account that the protein content of embryo increased with embryo devel0.05 [xg on Day 10; 278 ± 0.112 Ig on Day opment (1 13; 1850 + 0.270 Rg on Day 14), there was a 50 000-fold increase in the Cox-2 content of the whole conceptus from Day 10 to Day 14. To determine the concentration of cyclooxygenases in embryonic tissue, both standard Cox-1 and standard Cox-2 proteins were immunoblotted with their respective antibod-

COX-1 AND COX-2 IN OVINE EMBRYOS

FIG. 2. Determination of Cox-2 amounts in embryonic cell lysates by Western blot. Lanes 1-5 contained variable concentrations of ovine Cox-2 standard protein. Lanes 6-9 were loaded with various amounts of proteins from a Day 14 embryo. Amounts of Cox-2 protein in embryo lysate were determined relative to chemiluminescent intensity of the standard Cox-2 (1-min film exposure).

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FIG. 3. Determination of Cox-1 amounts in embryonic cell lysates by Western blot. Lanes 1-5 and lane 7 were loaded with variable concentrations of ovine Cox-1 standard protein. Lane 6 was loaded with 500 ng standard ovine Cox-2 protein. Lanes 8-10 were loaded with variable amounts of proteins from a Day 14 embryo (1-min film exposure).

ies (Figs. 2 and 3). The first five lanes (1-5) in Figure 2 demonstrate the cross reaction of the anti-Cox-2 antibody mAb294 with standard ovine Cox-2 and made it possible to determine the linear regression curve between the intensity of the immunoreactive bands and protein concentration. The linearity ranged from 12 ng to 250 ng of Cox-2 (p = 0.006). The next four lanes (6-9) were loaded with various amounts of protein from a Day 14 embryo homogenate. Cox-2 was detected in this embryo with as little as 0.6 pIg of total protein. Making reference to the standard curve, the embryo content of Cox-2 was estimated to be 50 ng/Lg of total protein. Figure 3 shows a nitrocellulose membrane probed with anti-Cox-1 antibody L-855. The first five lanes and lane 7 were loaded with standard ovine Cox-1 protein. Lane 6 was loaded with 500 ng standard Cox-2 and displayed no immunoreactive band. However, it might be possible to detect a weak cross-reactivity of the anti-Cox-l antibody L-855 with Cox-2 protein at concentrations greater than 1 g when the film was exposed for an extended period (data not shown). On the basis of densitometric analysis, cross-immunoreactivity of the anti-Cox-l antibody L-855 with Cox-2 protein was estimated to be < 0.5%. Lanes 8, 9, and 10 were respectively loaded with 40, 20, and 10 jig of protein from the same Day 14 ovine embryo used for Cox-2 analysis. No immunoreactivity was detected. Moreover, no band was observed with an extended period of film exposure. Blots done with similar amounts of proteins from Day 15 and Day 16 embryos (data not shown) revealed no immunoreactive bands, suggesting that Cox-l was not expressed in ovine conceptuses during these stages.

Some trophoblastic areas were stained whereas others were not immunoreactive. On Day 17 and in later stages of pregnancy, the distribution of immunostaining was studied on sections of uterus containing the implanted embryos. On Day 17 the trophoblast cells were weakly and heterogenously stained (Fig. 4D). Moreover, a slight immunoreactivity was observed in the epithelial cells of the endometrium, confirming the expression of Cox-2 in the ovine uterus as reported in a previous study [21]. On Day 25 of pregnancy, the adherent trophoblast did not express Cox-2 whereas the uterine glands exhibited a positive immunostaining for Cox-2 (Fig. 4F). Immunoreactivity was sparsely distributed in the luminal epithelium in apposition to the trophoblast. Both trophoblast and uterine tissues were recognizable as seen in Figure 4, E and G, which presented the counterstaining with Hoechst's dye. On Day 130 of pregnancy, the trophoblast again exhibited high levels of Cox-2 expression in both the cotyledonary and intercotyledonary areas (Fig. 4, H and I). The endometrial epithelium adherent to trophoblast in the cotyledonary did not express Cox-2 (Fig. 4H). In contrast, in the intercotyledonary, Cox-2 was highly expressed in the glandular epithelium (Fig. 41). Immunofluorescence studies failed to detect Cox-1 in the nonimplanted Day 14 embryos. The intensity of the staining was not higher than that in the control (data not shown). However, Cox-l was observed in tissues from late pregnancy. Actually, trophoblastic cells as well as both epithelial and stromal cells from Day 130 pregnant ewes exhibited Cox-l (Fig. 4J).

Immunocytochemical Localization of Cyclooxygenases

Figure 5 shows the typical HPLC separation of arachidonic acid metabolites synthesized by Day 14 conceptuses. Radiolabeled arachidonic metabolites produced by embryos were identified by reference to the nonlabeled standards detected by UV absorption. The first part of the gradient, i.e., the isocratic elution consisting of 36% acetonitrile and 0.01% acetic acid, separated the cyclooxygenase products, 6-keto-PGFI,, thromboxane B 2, PGF 2., PGE 2, PGD 2, 13,14-dihydro- 15-keto-PGF 2 ,, 13,14-dihydro- 15 -ketoPGE 2, and PGB 2 at 5.8, 8.7, 10.9, 13.7, 16.6, 22.2, 25.3, and 37.3 min, respectively. Leukotrienes from the standard mixture were incompletely resolved. However, they eluted as a group of peaks between 40 and 55 min, after the cyclooxygenase metabolites and before the HETEs, hence avoiding any confusion with those products. The final part of the chromatography separated 5-, 11-, 12-, and 15-HE-

Immunohistofluorescence studies show that Cox-2 is localized in the trophoblastic cells of the conceptus (Fig. 4). The cells of the embryonic mass were not stained. No positive immunoreactivity was observed in controls (not shown). Confocal fluorescence scanning microscopy showed that Cox-2 was present in the cytoplasm and on the nuclear envelope. As previously observed for other cell types [25], the immunostaining was mainly concentrated in the nuclear envelope of the trophoblastic cells (Fig. 4B). Cox-2 was present in nonhatched blastocysts on Day 8 of development (Fig. 4A). All the trophoblastic cells were immunoreactive. A similar pattern of Cox-2 expression was observed in Day 10 embryos. However, in Day 14 embryos, a mosaic pattern of staining was observed (Fig. 4C).

Arachidonic Acid Metabolism by Embryos

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FIG. 4. Immunohistochemical localization of Cox-2 (A-I) and Cox-1 () in ovine embryo. Green fluorescence indicates positive immunostaining revealed by fluorescein isothiocyanate. A-C) Confocal fluorescence scanning microscopy. Red color indicates nuclei counterstained with propidium iodide. A) Day 8 blastocyst. B) Day 10 trophoblastic cells. C) Day 14 conceptus showing a mosaic pattern of Cox-2-positive cells. D-J) Cryostat sections of embryos in uterine horns. D) Day 17 of pregnancy. E) Same field as in D, counterstained with Hoechst's dye. F) Day 25 of pregnancy. G) Same field as in F,counterstained with Hoechst's dye. H) Day 130 placental cotyledon. I, J) Day 130 intercotyledonary areas. e, Uterine epithelium; en, maternal endometrium; g, uterine gland; icm, inner cell mass; s, uterine stroma; t, trophoblast. Magnification is x600 for A and C, x1500 for B, x250 for D-J.

COX-I AND COX-2 IN OVINE EMBRYOS 0.2

1037 FIG. 5. Arachidonic acid metabolites synthesized by Day 14 ovine conceptuses. HPLC chromatogram of authentic standards (UV absorbance profile) and representative profile of [3H]arachidonic acid metabolites released in the culture medium by conceptuses (cpm profile). Embryo incubation medium and standards were chromatographied as described in Materials and Methods. UV detection of the unlabeled standards was monitored at their maximal absorption wavelength (X205 nm: 0-27 min; X280 nm: 27-47 min; X237 nm: 47-52 min; X280 nm: 52-61 min; X237 nm: 61-75 min; X205 nm: 75-100 min). [3H]Arachidonic acid metabolites were detected on-line with a Flo-on 3 counter and were identified by reference to authentic standards.

o 0.1

0.0 2000

0 0

20

40

60

80

100

Time (min)

TEs at 64.4, 66.8, 69.4, and 71.4 min, respectively. Arachidonic acid was observed as a sharp peak at the end of the separation, i.e., 81.7 min. HHT that could result from either cyclooxygenase or nonenzymatic free-radical oxygenation was also separated and had a retention time of 48 min. Day 14 ovine embryos mainly produced cyclooxygenase metabolites of arachidonic acid (Table 1). Cyclooxygenase derivatives counted for 80-85% of the overall radioactive arachidonic acid metabolites. The remaining 15-20% consisted of unresolved products in the region of leukotriene elution and of 15-, 11-, and 12-HETEs. The control involving incubation of arachidonic acid in culture medium without an embryo showed no radioactive peaks generated from possible instability of the substrate. The conceptuses synthesized the five principal cyclooxygenase derivatives: 6-keto-PGFI,, TxB 2, PGF 2 ., PGE 2, and PGD 2. Two unknown peaks eluting in the cyclooxygenase metabolites region were systematically detected. One eluted at 12 min of retention time between PGF2 . and PGE 2 and the other at 35.5 min. Neither DHK-F 2 nor DHK-E 2 was detected. It should be noted that the embryos displayed a high incorporation rate for arachidonic acid, because only a residual peak of [3 H]arachidonic acid was found in the media after the short-term cultures. DISCUSSION The present study is the first to demonstrate that the inducible isoform of prostaglandin synthase, Cox-2, was expressed in ovine embryos during the periimplantation period. We demonstrated that the expression of embryonic Cox-2 was developmentally regulated with a pattern of expression that coincided with a critical period of embryo development. Between Day 10 and Day 16, the ovine blastocyst undergoes a significant increase in size to reach a filamentous conceptus that is more than 10 cm long. During this elongation period, it should be noted that Cox-2 expression paralleled other molecular events that take place in the trophoblast. The pattern of Cox-2 expression was quite similar to the pattern of interferon-tau expression that

begins on Day 10 and stops on Day 21 [26-28]. A similar pattern of expression was observed in ovine trophoblast regarding fos protooncogenes [29]. The regulation of the corresponding genes in the trophoblast remains unknown. However, the elongation process that is the result of an intense proliferation of trophoblastic cells could be related to Cox-2 expression, since the relationships between Cox-2 expression and mitogenicity have been reported in a wide variety of cells and tissues. Mitogens such as serum, phorbol esters, platelet-derived growth factor, or epidermal growth factor [8, 10, 30, 31] have been shown to induce Cox-2 expression. Other differentiating agents, such as interleukin (IL)-13, that cause induction of Cox-2 in many cultured cells and tissues (reviewed in [7]) are putative candidates as regulators of the embryonic Cox-2, since some of them are abundantly expressed at the feto-maternal interface (reviewed in [32]). Actually, IL-1 3 has been shown to be implicated in implantation [33, 34] and exhibits a developmentally regulated expression [35] that has a temporal relationship to the Cox-2 expression reported in the present study. The termination of embryonic Cox-2 expression occurring at Day 17-18 coincides with the attachment of the TABLE 1. Distribution of arachidonic acid metabolites synthesized by Day 14 ovine conceptuses incubated with 0.5 jiCi/ml [3 Hlarachidonic acid for 6 h.* Arachidonic acid metabolites

Mean

+ SEM

Cyclooxygenase derivatives 6-keto-PGF l TxB2 PGF2,, PGE2 PGD 2

18.2 22.5 21.0 14.5 2.7

+ 4.2 ±15.9 +11.0 + 7.4 + 2.6

Lipoxygenase derivatives Leukotrienes HETEs

12.6 4.9

+ 9.2 + 4.1

*Values are mean percentages - SEM of percentage of total converted

arachidonic acid in cpm within each metabolite identified by HPLC (number of conceptuses is 12).

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trophoblast to the uterus. The anti-inflammatory cytokine, transforming growth factor 3, which has been shown to down-regulate Cox-2 expression [36], has been demonstrated at the time of ovine embryo attachment [37], suggesting that it could be involved in the termination of Cox-2 expression. The hormonal control of embryonic Cox-2 could also be considered, since in a recent study we demonstrated that Cox-2 was highly expressed in ovine endometrium during the luteal phase of the estrous cycle, and progesterone is likely responsible for expression of ovine endometrial Cox-2 [21]. It could be suggested that the progestative uterine milieu acts as an inducer of Cox-2 expression in embryos. The temporal correlation between the patterns of expression of Cox-2 in both the endometrium and embryo supports this hypothesis. However, in a previous work Chakraborty et al. [38], who showed differential expression of Cox-1 and Cox-2 in the periimplantation mouse uterus, suggested that cox-2 gene was not inducible by ovarian steroids. The immunohistochemical analysis showed that Cox-2 was located in trophoblast cells and that the inner cell mass did not express Cox-2. All trophoblastic cells were stained in Day 8 and Day 10 embryos, whereas a mosaic pattern of staining was observed in the older conceptuses. It could be suggested that this mosaic pattern is linked to the first areas of interaction that take place between the embryo and endometrial epithelium, and that attachment might occur either in Cox-2-positive or Cox-2-negative domains of the trophoblast. A similar phenomenon was observed for trophoblastic interferon [27] and fos protooncogene [29] that were no longer expressed in cells that had established contact with the uterine epithelium, whereas they were still strongly expressed in nonimplanted areas. Our results indicated that Cox-2 was most concentrated in the nuclear membrane of the trophoblast cell. This pattern of immunostaining was previously reported to be characteristic of Cox-2 [25], suggesting that eicosanoids produced via Cox-2 were released, in part, in the nucleus and consequently should be involved in transcriptional events. In mice embryos [11, 12], the subcellular distribution of prostaglandin synthase differs from the intracellular location of Cox-2 in the ovine trophoblast. In mice embryos, prostaglandin synthase was reported to be predominantly located in the cytoplasm [11, 12], which is consistent with the known localization of Cox-1 to the endoplasmic reticulum [39, 40]. In these studies the antibodies for anti-prostaglandin synthase used did not discriminate between Cox-1 and Cox-2. Thus, the absence of staining in the nuclear membrane provided indirect evidence suggesting that Cox-2 was probably not expressed in the mouse embryo. Cox-2 seemed to be the only cyclooxygenase expressed in early ovine conceptus, since Cox-1 was undetectable. Undetectable levels of Cox- 1 have been previously reported in bovine [41-43] and avian granulosa cells [44], whereas Cox-2 was expressed in abundance in these studies. In our study, extremely low levels of Cox- are possible that could not have be detected by Western blot and immunocytofluorescence. More sensitive methods such as reverse transcription-polymerase chain reaction could be used to provide more definite data about the presence or absence of Cox-1 in the trophoblast cells of preimplanted embryos. Nevertheless, the high levels of Cox-2, which reached 5% of total embryo protein on Day 14, underlined the importance of the role of Cox-2 in embryonic development and reduced the significance of Cox- if it was expressed. However, the fact that Cox-1 was expressed in significant amounts on

Day 130 of pregnancy suggests that a differentiating event occurs in trophoblasts cells near term. Moreover, the expression of both Cox-1 and Cox-2 indicated that the two cyclooxygenases are needed for the prostaglandin synthesis occurring at term. Following the report of Wimsatt et al. [17], who found Cox-2 in ovine placentae at term, histological studies were developed to identify the tissues and cells expressing Cox-2 at parturition [18, 19]. Our results, which indicated the expression of Cox-2 in trophoblastic cells, agreed with the previous data. However, conflicting results remain on the cellular localization of maternal Cox-2, which was found in either the stromal cells [18] or epithelial cells [19]. Unlike Gibb et al. [18], we did not observe any immunoreactivity in the endometrial stroma on Day 130. We found that Cox-2 expression was restricted to the uterine glands of the intercotyledonary areas, in contrast with Zhang et al. [19], who observed Cox-2 in both the luminal and glandular epithelium. Our study also evaluated the production of arachidonic acid metabolites by ovine conceptuses. The high production rates of cyclooxygenase metabolites as compared to low levels of lipoxygenase derivatives reflected the high level of Cox-2 expression in the trophoblast. Nevertheless, for the first time, the present data indicate the formation of lipoxygenase products by ovine embryos during the preimplantation period. Only few studies have reported lipoxygenase derivatives being synthesized by human or ovine placental tissues [45, 46]. However, in spite of the fact that HETEs might be considered to be mediators in immunological processes at the feto-maternal interface, their exact role in pregnancy remains unknown. We reported that Day 14 ovine embryos produced the five primary cyclooxygenase derivatives of arachidonic acid. In particular we identified the formation of PGD2 and thromboxane-A ,2 determined by its stable metabolite thromboxane-B 2. These two metabolites have not been identified in previous studies [5, 14, 47], probably as a result of the use of less accurate analytical methods. We used on-line radioactive detection because it was more relevant than the collection of fractions as used by others [5, 14]. However, it must be pointed out that eight unknown radioactive peaks were detected by Sayre and Lewis [5] in the culture medium of ovine embryos, and the retention time of some of them indicates that they could be thromboxane-B 2 and PGD.2 Unlike those authors, we detected neither DHK-F2,, nor PGB 2. There is abundant evidence from various species that uterine prostaglandins are involved in the implantation process by controlling the vascular reactivity (reviewed in [1, 2]). However, the function of prostaglandins originating from the embryo is not established. The fact that ovine trophoblast Cox-2 was highly expressed during the time of the implantation window suggested an important role for the prostaglandins released by the embryo in mediating interactions with the uterus. We suggest two hypotheses concerning the putative biological roles of trophoblast Cox-2 and related products. First, at implantation, the previously nonadhesive apical surface of the trophoblast cells becomes adhesive. These phenotypic changes could be related to the concomitant high level of Cox-2 expression. It was recently shown that adhesion to cellular matrix protein was enhanced in intestinal cells that are programmed to overexpress Cox-2 [48]. It was also reported that changes in cyclooxygenase content occurred along with human trophoblast differentiation [49]. Secondly, according to species, the degree of penetration of the trophoblast in the endometrium is controlled by de-

COX-I AND COX-2 IN OVINE EMBRYOS

gradative enzymes, matrix metalloproteinases, and their inhibitor counterparts-the tissue inhibitor of metalloproteinases (TIMP) [50]. In ruminants, the trophoblast cells are noninvasive and remain in close apposition with the intact uterine epithelium. Consequently, in these species the production and the activity of degradative enzymes must be prevented at the endometrium-trophoblast interface [51]. In ovine trophoblast, Cox-2 expression and the associated prostaglandin production could be the way in which the cells control the invasion of the trophoblast. It was effectively reported that cyclooxygenase end-products suppressed the production of metalloproteinases [52, 53] and enhanced the expression of their inhibitors [54-56]. However, in other models, prostaglandins were also found to promote the synthesis of proteolytic enzymes [57-59]. In fact, data from numerous studies suggest that the balance between eicosanoids may be more relevant than absolute levels to induce specific changes in functional activity. Thus, our data indicating a simultaneous production of the five primary prostaglandins by the trophoblast reinforce this concept. ACKNOWLEDGMENTS We thank Elinor Thompson for revision of the manuscript, Eric Thompson and Pierre Adenot for confocal microscopy, and Francis Fort for the photographic work.

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