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BOR - Papers in Press, published online ahead of print September 17, 2003.
Biol Reprod 2003, 10.1095/biolreprod.103.019083
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BIOLOGY OF REPRODUCTION 70, 191–197 (2004)
DOI: 10.1095/biolreprod.103.019083
© 2004 by the Society for the Study of Reproduction, Inc.

Female Reproductive Tract

Interferon-{tau} Blocks the Stimulatory Effect of Tumor Necrosis Factor-{alpha} on Prostaglandin F2{alpha} Synthesis by Bovine Endometrial Stromal Cells1

Kiyoshi Okuda2, Yuko Kasahara, Shuko Murakami, Hitomi Takahashi, Izabela Woclawek-Potocka, and Dariusz J. Skarzynski

Laboratory of Reproductive Endocrinology,3 Faculty of Agriculture, Okayama University, Okayama 700-8530, Japan Department of Animal Breeding and Reproduction,4 National Institute of Livestock and Grassland Science, Ibaraki 305-0901, Japan Division of Reproductive Endocrinology and Pathophysiology,5 Institute of Animal Reproduction and Food Research, Polish Academy of Sciences, Olsztyn 10-747, Poland


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Tumor necrosis factor-{alpha} (TNF{alpha}) has been shown to be a potent stimulator of prostaglandin (PG) F2{alpha} synthesis in bovine endometrial stromal cells. The aims of the present study were to determine the effect of interferon-{tau} (IFN{tau}) on TNF{alpha}-stimulated PGF2{alpha} synthesis and the intracellular mechanisms of TNF{alpha} and IFN{tau} action in the stromal cells. When cultured bovine stromal cells were exposed to TNF{alpha} (0.006–0.6 nM) for 24 h, the production of PGF2{alpha} and cyclooxygenase (COX)-2 gene expression were stimulated by TNF{alpha} (0.06–0.6 nM, P < 0.05). Moreover, a specific COX-2 inhibitor (NS-398; 5 nM) blocked the stimulatory effect of TNF{alpha} on PGF2{alpha} production (P < 0.05). Although IFN{tau} (0.03–30 ng/ml) did not stimulate basal PGF2{alpha} production in the stromal cells, it suppressed TNF{alpha} action in PGF2{alpha} production dose dependently (P < 0.05). Moreover, the stimulatory effect of TNF{alpha} (0.6 nM) on COX-2 gene expression was completely blocked by IFN{tau} (30 ng/ml; P < 0.05), although the gene expression of COX-2 was not influenced by IFN{tau}. The overall results indicate that the stimulatory effect of TNF{alpha} on PGF2{alpha} production is mediated by the up-regulation of COX-2 gene expression and suggest that one of the mechanisms of the inhibitory effect of IFN{tau} on luteolysis is the inhibition of TNF{alpha} action in PGF2{alpha} production in the stromal cells by the down-regulation of COX-2 gene expression stimulated by TNF{alpha}.

cytokines, female reproductive tract, mechanisms of hormone action, ovulatory cycle, uterus


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In nonpregnant cows, prostaglandin (PG) F2{alpha} is secreted from the uterus in a pulsatile manner to cause regression of the corpus luteum in the late luteal phase [1, 2]. In our previous studies, although both oxytocin (OT) and tumor necrosis factor-{alpha} (TNF{alpha}) affected endometrial PGF2{alpha} output at the follicular stage, PGF2{alpha} output was stimulated only by TNF{alpha} at the mid- and late luteal stages acting through specific binding sites present in bovine endometrium [3]. TNF{alpha} stimulates PGF2{alpha} synthesis in the stromal cells but not in the epithelial cells of bovine endometrium [4], suggesting that this cytokine locally induces autoamplification PGF2{alpha} cascade in bovine endometrium [5] and initiates a positive loop between hypophysial and ovarian OT and uterine PGF2{alpha} to complete luteolysis [2, 5–7]. Based on these findings, we assume that TNF{alpha} is a trigger for the initial output of PGF2{alpha} from stromal cells (subluteolytic PGF2{alpha}) that initiates luteolytic cascade in the epithelial cells of the endometrium at luteolysis in cattle [3, 4].

We demonstrated that the stimulatory effect of TNF{alpha} on PGF2{alpha} production in the bovine endometrial cells is mediated via the activation of phospholipase A2 (PLA2) and arachidonic acid (AA) accumulation and suggested that TNF{alpha} acts farther down in the PG biosynthesis cascade including regulation of prostaglandin endoperoxide H synthase (cyclooxygenase [COX]) [4, 7]. Two isoforms of COX, which is the key rate-limiting enzyme responsible for the conversion of AA to the precursors for PGF2{alpha}, PGG2 and PGH2, have been identified in mammalian cells [8]. COX-1 is a constitutively expressed enzyme [9] in the uterus and other tissues. On the other hand, COX-2 is induced by various substances including OT and cytokines [8, 10–12]. TNF{alpha} has been shown to induce the gene expression of COX-2 in bovine endometrial stromal cells [13] and is involved in the regulation of luteotropic PGE2 production [13, 14] during pregnancy and the luteal phase. Therefore, it could be interesting to investigate whether TNF{alpha} induces initial PGF2{alpha} production during luteolysis by activating COX-2 gene expression in bovine endometrial stromal cells.

If the bovine endometrium possesses endogenous mechanisms for initiation of PGF2{alpha} secretion during luteolysis [4, 7], it is essential for the establishment of pregnancy to attenuate the stimulative effect of TNF{alpha} and other cytokines on luteolytic PGF2{alpha}. In ruminants, at the time of recognition of pregnancy, the conceptus produces a signal, first identified as trophoblastic protein-1 and now called trophoblastic interferon-{tau} (IFN{tau}) [15]. This cytokine is released at high levels by the conceptus trophectoderm between Days 10 and 25 of gestation and prevents luteolysis by suppressing endometrial PGF2{alpha} secretion [16–18]. Because TNF{alpha} has been suggested to play an important role in initiating PGF2{alpha} output [3] from the bovine endometrial stromal cells at luteolysis [4], we further hypothesized that IFN{tau} suppresses the stimulatory effect of TNF{alpha} on endometrial PGF2{alpha} production by reducing COX-2 gene expression. On the other hand, IFN{tau} has been generally thought to act in a paracrine manner on endometrial epithelial cells. However, there is increasing evidence that IFN{tau} acts not only on endometrial epithelial cells but also on the stromal cells [13, 19–21]. In the present study, we investigated the effects of IFN{tau} on TNF{alpha}-stimulated PGF2{alpha} synthesis and the intracellular mechanisms of IFN{tau} action on the TNF{alpha}-stimulated PGF2{alpha} synthesis in the bovine stromal cells, especially focusing on the COX-2 gene expression.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Isolation of Endometrial Stromal Cells

Uteri of Holstein cows were obtained from a local abattoir in accordance with protocols approved by the local institutional animal care and use committee. Uteri were obtained within 30 min after exsanguination and were transported to the laboratory within 1–1.5 h on ice. The stages of the estrous cycle were determined by macroscopic observation of the ovary and uterus as described previously [3]. In this study, uteri of the early estrous cycle (Days 2–5) were used. The stromal cells from the bovine endometrium were separated using a modification of procedures described previously [4]. The horn ipsilateral to the corpus luteum was used for culture. A polyvinyl catheter was inserted into the side of the oviduct, and the ends of the horn were tied shut to retain a trypsin solution for solubilizing the epithelial cells as described below. The uterine lumen was washed three times with 30–50 ml of sterile Ca2+- and Mg2+-free Hanks balanced salt solution (HBSS) supplemented with 100 IU/ml penicillin, 100 µg/ml streptomycin, and 0.1% (w/v) BSA (Roche Diagnostics GmbH, Mannheim, Germany; #735078). Thirty to fifty milliliters of sterile HBSS containing 0.3% (w/v) trypsin (Sigma Chemical Co., St. Louis, MO; #T4665) was then infused into the uterine lumen through the catheter. Epithelial cells were removed from the endometrial surface by incubation at 37°C for 60 min with gentle shaking.

After removal of the epithelial cells, the uterine lumen was washed with sterile HBSS supplemented with antibiotics and 0.1% BSA. The horn was then cut transversely into several segments with scissors, and then the segments were slit to expose the endometrial surface. Intercaruncular endometrial strips were dissected from the myometrial layer with a scalpel and washed once in 50 ml of sterile HBSS containing antibiotics. The endometrial strips were then cut into small pieces (1 mm3). The minced tissues (approximately 5 g) were digested by stirring for 60 min in 50 ml of sterile HBSS containing 0.05% (w/v) collagenase (Sigma; #C0130), 0.005% (w/v) deoxyribonuclease I (Sigma; #D5025), and 0.1% BSA. The dissociated cells were filtered through metal meshes (100 µm and 80 µm) to remove undissociated tissue fragments. The filtrate was washed three times by centrifugation (10 min at 100 x g) with Dulbecco Modified Eagle Medium (DMEM; Sigma; #D1152) supplemented with antibiotics and 0.1% BSA. After the washes, the cells were counted with a hemocytometer. The cell viability was higher than 85% as assessed by 0.5% (w/v) trypan blue dye exclusion. The homogeneity of the stromal cells and contamination of the cells with epithelial cells were evaluated by the method of Malayer and Woods [22], using the immunofluorescent staining for specific markers of epithelial cells (cytokeratin) and stromal cells (vimentin). The cells obtained consisted of more than 99% of stromal cells and only a few glandular epithelial cells.

Culture of Endometrial Stromal Cells

The final pellet of the stromal cells was resuspended in culture medium (DMEM/Ham F-12; 1:1 (v/v); Sigma; #D8900) supplemented with 10% (v/v) calf serum (Sigma; #C6278), 20 µg/ml gentamicin (Invitrogen Co., Carlsbad, CA; #15750–060), and 2 µg/ml amphotericin B (Sigma; #A9528). Stromal cells were separately seeded at a density of 1 x 105 viable cells/ml in 24-well plates (Costar, Cambridge, MA) and cultured at 37.5°C in a humidified atmosphere of 5% CO2 in air. To purify the stromal preparation, the medium was changed 2 h after plating, by which time selective attachment of stromal cells had occurred [4, 23]. The medium was changed every 2 days until confluency was reached. When the cells were confluent (6–7 days after the start of the culture), the medium was then replaced with fresh DMEM/Ham F-12 supplemented with 0.1% BSA, 5 ng/ml sodium selenite (Sigma; #S5261), 0.5 mM ascorbic acid (Wako Pure Chemical Industries, Ltd., Osaka, Japan, #013–12061), 5 µg/ml transferrin (Sigma; #T3400), 2 µg/ml insulin (Sigma; #I-4011), and 20 µg/ml gentamicin. The cells were then exposed to various stimulants for the following experiments.

Experiment 1 To determine the dose-dependent effect of TNF{alpha} (Dainippon Pharmaceutical Co. Ltd., Osaka, Japan) on COX-2 gene expression in the stromal cells, the cells were exposed to TNF{alpha} (0.006–0.6 nM) for 24 h, disrupted with 1 ml of TRIZOL Reagent (Invitrogen; #15596), and frozen at -80°C until reverse transcription–polymerase chain reaction (RT-PCR).

Experiment 2 To determine the effect of a selective COX-2 inhibitor (NS-398; BIOMOL, Plymouth Meeting, PA; #EI-261; 5 nM) on TNF{alpha}-stimulated PGF2{alpha} production in the stromal cells, the cells were exposed to NS-398, TNF{alpha} (0.06 nM), or both for 24 h.

Experiment 3 To determine the dose-dependent effect of recombinant bovine IFN{tau} (classified into the rb-1a group) produced by an Autographa californica nuclear polyhedrosis virus expression system [24] on PGF2{alpha} production in the stromal cells, the cells were exposed to IFN{tau} (0.03–30 ng/ml) for 24 h.

Experiment 4 To determine the effect of IFN{tau} on TNF{alpha}-stimulated PGF2{alpha} production and COX-2 gene expression in the stromal cells, the cells were exposed to TNF{alpha} (0.06 nM), IFN{tau} (30 ng/ml), or both for 24 h, disrupted with 1 ml of TRIZOL reagent, and frozen at -80°C until RT-PCR.

After culture, the conditioned media were collected in tubes with 5 µl 0.3 M EDTA, 1% aspirin (Sigma; #A2093) solution (pH 7.3), and frozen at -30°C until the PGF2{alpha} assay. The DNA content, estimated by a spectrophotometric method of Labarca and Paign [25], was used to standardize the results.

PGF2{alpha} Determination

The concentration of PGF2{alpha} in the culture medium was determined with an enzyme immunoassay as described previously [26]. The PGF2{alpha} standard curve ranged from 0.016 ng/ml to 4 ng/ml, and the ED50 of the assay was 0.25 ng/ml. The intra- and interassay coefficients of variation were on average 7.1% and 11.3%, respectively.

Semiquantitative RT-PCR

Total RNA was prepared from cultured cells using TRIZOL reagent according to the manufacturer's directions. One microgram of each total RNA was reverse transcribed using a T-primed first-strand kit (Amersham Pharmacia Biotech, Piscataway, NJ; 27–9263–01), and one tenth of the reaction mixture was used in each PCR using specific primers for bovine COX-2, or ß-actin. Semiquantitative RT-PCR was carried out using the housekeeping gene, ß-actin, as an internal standard. The sequence of COX-2 primers, which were based on a report by Asselin et al. [19], were 5'-TCC AGA TCA CAT TTG ATT GAC A-3' (5' primer, 22 mer) and 5'-TCT TTG ACT GTG GGA GGA TAC A-3' (3' primer, 22 mer). The primers for ß-actin, which were designed as described by Asselin and Fortier [27], were 5'-GAG GAT CTT CAT GAG GTA GTC TGT CAG GTC-3' (5' primer, 30 mer) and 5'-CAA CTG GGA CGA CAT GGA GAA GAT CTG GCA-3' (3' primer, 30 mer). The PCR process has previously been described [28]. The PCRs were carried out using an AmpliTaq Gold DNA polymerase (Perkin Elmer, Foster City, CA; #N888-0240) and a thermal cycler (TP240; Takara, Otsu, Shiga, Japan). The conditions for the PCRs were as follows: after activation of the DNA polymerase by incubating for 7 min at 94°C, COX-2 was amplified for 34 cycles, and ß-actin was amplified for 29 cycles consisting of denaturation for 1 min at 94°C, annealing for 1 min at 54°C, and extension for 2 min at 72°C, followed by an additional extension for 5 min at 72°C. The ß-actin primers were added after five amplification cycles of COX-2 (primer-dropping method [29]). Two-fifths aliquot of each reaction mixture was electrophoresed on a 1.5% agarose gel containing ethidium bromide with a known standard (100-bp ladder, New England BioLabs Inc., Beverly, MA; #N3231S), and photographed under ultraviolet illumination. The amplified cDNA fragments were sequenced directly and/or after being subcloned into pGEM3Zf(+). Dideoxynucleotide sequencing was performed using fluorescent primers and an automated DNA sequencer (373A; Applied Biosystems, Foster City, CA). Sequence analysis was carried out using GENETYX software and the Blast program (National Center for Biotechnology Information, National Institutes of Health, Bethesda, MD; http://www.ncbi.nlm.nih.gov/).

Statistical Analysis

The data are shown as the mean ± SEM of values obtained in three to four separate experiments, each performed in triplicate. Level of PGF2{alpha} production was standardized on DNA concentrations per well. The statistical significance of differences between controls and treated groups was assessed by one-way ANOVA followed by Bonferroni multiple comparison tests (GraphPad PRISM version 4; GraphPad Software, Inc., San Diego, CA).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Dose-Dependent Effects of TNF{alpha} on COX-2 Gene Expression and PGF2{alpha} Production in Stromal Cells

An RT-PCR analysis of bovine COX-2 gene expression in endometrial stromal cells identified the appropriate 449-bp band (Fig. 1A). ß-Actin was used as an internal control, and a 349-bp band was observed in all samples and no variation was observed. At the concentrations of 0.06 and 0.6 nM, TNF{alpha} promoted COX-2 gene expression (P < 0.05; Fig. 1B). Moreover, at concentrations of 0.06 and 0.6 nM, TNF{alpha} dose dependently and significantly increased PGF2{alpha} production (P < 0.05; Fig. 1C).



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  		FIG. 1. COX-2 mRNA expression and PGF2{alpha} production following treatment with increasing doses of TNF{alpha} in cultured bovine stromal cells. TNF{alpha} (0.006–0.6 nM) was added 24 h before the end of culture. A) The RT-PCR products of COX-2 (34 cycles) and ß-actin (29 cycles) are shown after electrophoresis. B) Ratios of COX-2:ß-actin signals (mean ± SEM, n = 3) determined by image analysis. C) PGF2{alpha} production (mean ± SEM, n = 3) measured by enzyme immunoassay. Different superscript letters indicate significant differences (P < 0.05), as determined by ANOVA followed by Bonferroni multiple comparison test

Effect of COX-2 Inhibitor on TNF{alpha}-Induced PGF2{alpha} Production in Stromal Cells

TNF{alpha}-stimulated PGF2{alpha} was blocked by a specific inhibitor (NS-398) of COX-2 activity, whereas NS-398 showed no significant effect on basal PGF2{alpha} production (Fig. 2).



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  		FIG. 2. Effect of a COX-2 inhibitor (NS-398) on TNF{alpha}-stimulated PGF2{alpha} production by cultured bovine stromal cells (mean ± SEM, n = 3). NS-398 (5 nM) and TNF{alpha} (0.06 nM) were added 24 h before the end of culture. Different superscript letters indicate significant differences (P < 0.05), as determined by ANOVA followed by Bonferroni multiple comparison test

Dose-Dependent Effect of IFN{tau} on PGF2{alpha} Production in Stromal Cells Treated With or Without TNF{alpha}

Figure 3 shows PGF2{alpha} production by cultured bovine endometrial stromal cells treated with or without TNF{alpha} and IFN{tau} (0.03–30 ng/ml) for 24 h. In a dose-dependent fashion, addition of IFN{tau} inhibited the TNF{alpha}-stimulated PGF2{alpha} production (Fig. 3A). IFN{tau} alone did not affect the PGF2{alpha} production of stromal cells (Fig. 3B).



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  		FIG. 3. Effect of IFN{tau} on basal or TNF{alpha}-stimulated PGF2{alpha} production by cultured bovine stromal cells (mean ± SEM, n = 3). IFN{tau} (0.03–30 ng/ml) with or without TNF{alpha} (0.06 nM) was added 24 h before the end of culture. Different superscript letters indicate significant differences (P < 0.05), as determined by ANOVA followed by Bonferroni multiple comparison test

Effects of IFN{tau} on TNF{alpha}-Induced COX-2 Gene Expression and PGF2{alpha} Production in Stromal Cells

Figure 4 shows COX-2 gene expression and PGF2{alpha} production by cultured bovine endometrial stromal cells in response to exposure to TNF{alpha}, IFN{tau}, or both for 24 h. As shown in Figure 1, an RT-PCR analysis of bovine COX-2 gene expression in stromal cells identified the appropriate 449-bp band, and 349 bp of ß-actin used as an internal control was observed in all samples (Fig. 4A). As illustrated in Figure 4B, IFN{tau} had no significant effect on COX-2 mRNA as compared with the control. However, addition of IFN{tau} reduced COX-2 mRNA levels stimulated by TNF{alpha} (Fig. 4B). When the stromal cells were exposed to IFN{tau}, IFN{tau} did not affect the basal production of PGF2{alpha} (Fig. 4C). However, when IFN{tau} and TNF{alpha} were added simultaneously, IFN{tau} reduced PGF2{alpha} production stimulated by TNF{alpha} (P < 0.05; Fig. 4C).



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  		FIG. 4. TNF{alpha}-induced COX-2 mRNA expression and PGF2{alpha} production in cultured bovine stromal cells in response to IFN{tau}. TNF{alpha} (0.06 nM) and/or IFN{tau} (30 ng/ml) was added 24 h before the end of culture. A) The RT-PCR products of COX-2 (34 cycles) and ß-actin (29 cycles) are shown after electrophoresis. B) Ratios of COX-2:ß-actin signals (mean ± SEM, n = 3) determined by image analysis. C) PGF2{alpha} production (mean ± SEM, n = 3) measured by enzyme immunoassay. Different superscript letters indicate significant differences (P < 0.05), as determined by ANOVA followed by Bonferroni multiple comparison test


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The stimulatory effect of TNF{alpha} on PGF2{alpha} synthesis has been shown to be mediated by the activation of PLA2 in cultured bovine endometrial stromal cells [4]. It is generally accepted that PLA2 stimulates intracellular AA accumulation. Moreover, we have shown that TNF{alpha} promoted conversion of AA into PGF2{alpha} in cultured bovine endometrial stromal cells, suggesting that TNF{alpha} acts directly on the metabolism of AA downstream of the PLA2 action in the PG biosynthesis cascade [4]. In the present study, we demonstrated that TNF{alpha} stimulates COX-2 gene expression, resulting in promotion of PGF2{alpha} production in bovine endometrial stromal cells (Fig. 1). COX-2 is an inducible key rate-limiting enzyme for converting AA to the unstable form PGG2/PGH2, which is the first step in the synthesis of PGF2{alpha}, PGE2, and other members of the PG family [8, 9]. Moreover, a specific inhibitor of COX-2 (NS-398) blocked TNF{alpha}-stimulated PGF2{alpha} synthesis in the stromal cells (Fig. 2). These findings suggest that the increase of PGF2{alpha} production induced by TNF{alpha} is caused by increasing COX-2 mRNA expression and activity of this enzyme.

Recently, Parent et al. [13] demonstrated that TNF{alpha} induces PGE2 by increasing PGE synthase and COX-2 in cultured bovine endometrial stromal cells. The results of our previous study suggest that TNF{alpha} stimulates the secretion of both PGF2{alpha} and PGE2 from bovine endometrium throughout the estrous cycle, and that TNF{alpha} is involved in the initiation of luteolysis by decreasing the PGE2:PGF2{alpha} ratio at the late luteal stage [14]. The up-regulation of COX-2 mRNA by TNF{alpha} could explain the increase of PG synthesis. The cause of the change in the ratio of PG production is not clear, but it may be due to modulation of the activities of PGF synthase, PGE synthase, or PGE2-9-ketoreductase, which converts PGE2 into PGF2{alpha} [27, 30]. However, Madore at al. [31] recently found that AKR1C family members (to which all the currently known PGF synthases belong) are not expressed in the bovine endometrium. Alternatively, an aldose reductase known for its 20{alpha}-hydroxysteroid dehydrogenase (20{alpha}-HSD) activity, AKR1B5, is a likely candidate enzyme for controlling the sufficient and timely production of PGF2{alpha} in the bovine endometrium [31]. AKR1B5 has also been suggested to inactivate progesterone by transforming it into 20{alpha}-hydroxyprogesterone [32, 33]. In turn, progesterone and a glucocorticoid (dexamethasone) caused a dose-related decrease in 20{alpha}-HSD (AKR1B5) mRNA [34]. In addition, glucocorticoids have been demonstrated to antagonize functions of TNF{alpha} by inhibiting activity of the transcription factor nuclear factor kappa B (NF-{kappa}B) [35, 36] and by the induction of inhibitory protein (I-{kappa}B) that finally inhibits NF-{kappa}B [37, 38]. Therefore, it could be assumed that NF-{kappa}B is a key regulator of COX-2 expression in TNF{alpha}-induced PG production [39–41].

Regression of the corpus luteum is essential for normal cyclicity because it allows the development of a new ovulatory follicle, whereas prevention of luteolysis is necessary to establish and maintain pregnancy [2]. In ruminants, IFN{tau} produced by the trophoblast tissue at the time of recognition of pregnancy acts on maintaining the corpus luteum [42–44]. In the present study, IFN{tau} had no effect on basal PGF2{alpha} production but attenuated TNF{alpha}-stimulated PGF2{alpha} production in a dose-dependent manner in bovine endometrial stromal cells. Because PGF2{alpha} is considered to be a luteolytic agent [2], the reduction of TNF{alpha}-stimulated PGF2{alpha} production by IFN{tau} in stromal cells seems to play a role in the prevention of luteolysis. Furthermore, it is interesting to note that IFN{tau} inhibited TNF{alpha}-stimulated COX-2 gene expression and PGF2{alpha} production in stromal cells, whereas both basal production of PGF2{alpha} and COX-2 gene expression were not affected by IFN{tau} (Fig. 4). Therefore, the decrease of TNF{alpha}-induced PGF2{alpha} secretion by IFN{tau} in the current study appears to be due to the reduction of COX-2 gene expression.

Although the mechanisms of IFN{tau} action on endometrial epithelial cells have been studied intensively [13, 17, 19, 20, 27, 31, 44–49], there is limited information on the action of IFN{tau} on bovine endometrial stromal cells [13, 20, 21, 27, 49]. Moreover, IFN{tau} has been generally thought to act in a paracrine manner primarily on the endometrial epithelial cells [50, 51]. In fact, IFN{tau} does not become detectable in the systemic blood circulation of early pregnant ewes [15, 18, 50]. It has been recently shown that the primary site of IFN{tau} actions is the uterine epithelium in sheep [51]. Therefore, it is assumed that IFN{tau} prevents luteolysis in ewes by inhibiting OT-induced PGF2{alpha} secretion in the uterine epithelium by reducing the number of estrogen receptors and thus preventing the estrogen-induced increase of OT receptor [2, 21, 46, 52]. However, in cattle, IFN{tau} inhibits OT-induced PGF2{alpha} secretion from the endometrium not simply by the down-regulation of the OT receptor but by decreasing the expression of COX-2 and PGF synthase via a mechanism independent of changes in the OT receptor [7, 47, 48]. Moreover, because OT appears to play a supplementary role rather than a mandatory role during luteolysis in cattle [3–7], there should be other mechanisms for preventing luteolysis, independent of the OT-stimulated PGF2{alpha} secretion from the endometrium, including cross-talk between the conceptus, endometrial epithelial and stromal cells and maternal immune cells [6, 11–14, 19–21, 53–56]. However, our and others' in vitro data [13, 20, 21, 27, 49, 53–56] may not represent the in situ situation. Moreover, INF-{tau} has been suggested to exert its action on the stromal cells via a product of the epithelial cells [51]. Therefore, the direct action of IFN{tau} across the epithelium on the stromal cells of the bovine endometrium remains speculative. Further in vitro and in situ studies are needed to clarify these points.

It is also not clear how IFN{tau} blocks the mechanisms by which TNF{alpha} promotes COX-2 gene expression. In bovine endometrial epithelial cells, IFN{tau} inhibited several intracellular mechanisms responsible for PGF2{alpha} production, and this could be transcriptional action through both cytosolic [47, 48] and nuclear receptors [48]. NF-{kappa}B induces inflammation, suppression of apoptosis and is involved in cell proliferation [57, 58]. The NF-{kappa}B heterodimer is typically localized to the cytoplasm by an inhibitory protein, I-{kappa}B. On stimulation of the cell, such as with TNF{alpha}, I-{kappa}B is phosphorylated, ubiquitinated, and degraded. This allows the free NF-{kappa}B to accumulate in the nucleus in which it can activate transcription. In addition, NF-{kappa}B has been shown to control transcription of the COX-2 gene [39–41]. Therefore, it is possible that the inhibitory effect of IFN{tau} on COX-2 gene expression is due to down-regulation of TNF{alpha}-activated NF-{kappa}B in bovine stromal cells. In addition, recombinant bovine IFN{tau} has been shown to inhibit OT-induced PGF2{alpha} secretion from bovine endometrial epithelial cells by decreasing the expression of COX-2 and OT receptor [46]. Thus, IFN{tau} may also decrease the number of TNF{alpha} receptors in the stromal cells. This hypothesis is now under investigation.

On the other hand, there are some reports that IFN{tau} stimulates COX-2 gene expression in bovine-cultured endometrial cells [13, 19]. As suggested by Parent et al. [13], this discrepancy might be due to differences in the doses, isoforms, or both of IFN{tau} used by the different studies. It has been demonstrated that different doses of INF-{tau} could produce biphasic effects [10, 13, 19, 20, 27, 49]. Low doses of INF-{tau} (nanogram level, as shown in our study) had either an inhibitory effect or no effect on basal PGF2{alpha} or PGE2 production or COX-2 mRNA expression [10, 13, 19]. However, at high (microgram level) doses, INF{tau} stimulated both PGs and induced expression of COX-2 [10, 13, 19, 20, 49]. Moreover, it has been recently shown that two isoforms of bovine IFN{tau} (rb-2b and rb-3b) either inhibited or had no effect on PG production at all concentrations tested [49]. However, another isoform of bovine IFN{tau} (rb-1a) inhibited PG synthesis at low doses and stimulated PG synthesis concomitantly with COX-2 induction at high concentrations [49]. We recently reported that rbIFN{tau} used in the present study is classified into the rb-1a group [24], based on phylogenetic analysis of nucleotide and amino acid differences. These findings suggest that the conceptus has the capacity for local modulation of the production of PGs in uterus during early pregnancy [13, 19, 49].

In conclusion, the overall results indicate that the stimulatory effect of TNF{alpha} on PGF2{alpha} production is mediated via not only the activation of PLA2 and AA conversion [4] but also the induction of COX-2 expression. The present results support the hypothesis made by our previous studies [3, 4, 14] that TNF{alpha} directly induces the output of subluteolytic PGF2{alpha} from the stromal cells, initiating the positive feedback loop between the epithelial PGF2{alpha} and the luteal OT to complete luteolysis in cattle. In addition, we demonstrated that TNF{alpha}-induced COX-2 gene expression was decreased by IFN{tau}. These findings imply that IFN{tau} inhibits TNF{alpha}-induced PGF2{alpha} secretion by down-regulating COX-2 mRNA expression, resulting in the maintenance of the corpus luteum during early pregnancy in cattle.


   ACKNOWLEDGMENTS
 
We thank Dr. Yoko Miyamoto for valuable suggestion, the Dainippon Pharmaceutical Co. Ltd. (Osaka, Japan) for the gift of recombinant human TNF-{alpha}, and Dr. Seiji Ito of Kansai Medical University, Osaka, Japan, for providing anti-PGF2{alpha} serum.


   FOOTNOTES
 
1 This research was supported by the Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (JSPS: (B)14360168); the Japanese Ministry of Agriculture, Forestry, and Fisheries (RCP2002-4210); and Polish State Committee for Scientific Research (KBN 5P06K 003 21), and Japanese-Polish Joint Research Project under the agreement between JSPS and the Polish Academy of Sciences. Back

2 Correspondence: Kiyoshi Okuda, Laboratory of Reproductive Endocrinology, Faculty of Agriculture, Okayama University, Okayama 700-8530, Japan. FAX: 81 86 251 8388; kokuda{at}cc.okayama-u.ac.jp Back

Received: 9 May 2003.

First decision: 29 May 2003.

Accepted: 9 September 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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