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Biology of Reproduction 60, 656-663 (1999)
©Copyright 1999 Society for the Study of Reproduction, Inc.

Down-Regulation of Oxytocin-Induced Cyclooxygenase-2 and Prostaglandin F Synthase Expression by Interferon-{tau} in Bovine Endometrial Cells1

Chao Wu Xiaoa, Bruce D. Murphya, Jean Siroisa, and Alan K. Goff2,a

a Centre de recherche en reproduction animale, Faculté de médecine vétérinaire, Université de Montréal, St-Hyacinthe, Quebec, Canada J2S 7C6


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Oxytocin (OT) is responsible for the episodic release of luteolytic prostaglandin (PG) F2{alpha} from the uterus in ruminants. The attenuation of OT-stimulated uterine PGF2{alpha} secretion by interferon-{tau} (IFN-{tau}) is essential for prevention of luteolysis during pregnancy in cows. To better understand the mechanisms involved, the effect of recombinant bovine IFN-{tau} (rbIFN-{tau}) on OT-induced PG production and cyclooxygenase-2 (COX-2) and PGF synthase (PGFS) expression in cultured endometrial epithelial cells was investigated. Cells were obtained from cows at Days 1–3 of the estrous cycle and cultured to confluence in RPMI medium supplemented with 5% steroid-free fetal calf serum. The cells were then incubated in the presence or absence of either 100 ng/ml OT or OT+100 ng/ml rbIFN-{tau} for 3, 6, 12, and 24 h. OT significantly increased PGF2{alpha} and PGE2 secretion at all time points (p < 0.01), while rbIFN-{tau} inhibited the OT-induced PG production and reduced OT receptor binding in a time-dependent manner. OT increased the steady-state level of COX-2 mRNA, measured by Northern blot, which was maximal at 3 h (9-fold increase) and then decreased with time (p < 0.01). OT also caused an increase in COX-2 protein, which peaked at 12 h (11-fold increase), as measured by Western blot. Addition of rbIFN-{tau} suppressed the induction of COX-2 mRNA (89%, p < 0.01) and COX-2 protein (50%, p < 0.01) by OT. OT also increased PGFS mRNA, and this stimulation was attenuated by rbIFN-{tau} (p < 0.01). To ensure that the decrease in COX-2 was not solely due to down-regulation of the OT receptor, cells were stimulated with a phorbol ester (phorbol 12-myristate 13-acetate; PMA) in the presence and absence of rbIFN-{tau}. The results showed that rbIFN-{tau} also decreased PMA-stimulated PG production and COX-2 protein. It can be concluded that rbIFN-{tau} inhibition of OT-stimulated PG production is due to down-regulation of OT receptor, COX-2, and PGFS.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Prostaglandin (PG) F2{alpha}, primarily secreted from the surface epithelium of the uterus, is the luteolytic agent in ruminants [1–3], and oxytocin (OT) is responsible for its episodic release [4–7]. OT stimulates secretion of PGF2{alpha} from the endometrium [8–12], and the uterine OT receptor concentrations determine the sensitivity of endometrium to OT stimulation [6, 9, 13].

The attenuation of pulsatile secretion of PGF2{alpha} from the uterus is essential for the prevention of luteolysis during pregnancy in cows. The trophoblast secretes a protein, interferon-{tau} (IFN-{tau}), between Days 15 and 24 of gestation [14] that prevents luteolysis by suppressing endometrial PGF2{alpha} secretion. Conceptus secretory proteins suppress OT-induced PGF2{alpha} production [15, 16]; also, treatment with natural or recombinant bovine IFN-{tau} (rbIFN-{tau}) reduces the OT-induced secretion of PGF2{alpha} and PGE2 by bovine endometrial epithelial cells in vitro [8, 17, 18]. The suppression of OT-induced PGF2{alpha} by IFN-{tau} is generally thought to be due to a decrease in OT receptor number. It has been demonstrated that the endometrial OT receptor concentration on ovine uterine endometrium is reduced in pregnant animals [19–21] and that intrauterine infusion of rbIFN-{tau} attenuates OT binding to the endometrium in cattle [22] and sheep [23].

Although IFN-{tau} reduces OT receptor number in the endometrium, this is probably not the only effect the embryo has on PG secretion during early pregnancy. An inhibitor of PG synthesis is induced in the endometrium of pregnant cows [24–26]. This inhibitor has recently been identified as linoleic acid [27] and is thought to compete with arachidonic acid for the active site on the cyclooxygenase (COX) enzyme, which is the key rate-limiting enzyme responsible for the conversion of arachidonic acid to PGG2 and PGH2. These compounds are the precursors for PGF2{alpha}, PGE2, and other members of the PG family [28]. Two isoforms of COX (COX-1 and COX-2) have been identified in mammalian cells. COX-1 is a constitutively expressed enzyme; COX-2 is induced by various substances, such as phorbol esters, mitogens, cytokines, and serum [29–31], and by OT in the uterus [11, 18, 32].

The inhibitory effect of IFN-{tau} on OT-induced PGF2{alpha} is well established, but the molecular mechanisms involved have not yet been completely elucidated. One mechanism by which IFN inhibits the OT-induced PGF2{alpha} synthesis is by reducing estradiol receptor number and thus preventing an estrogen-induced increase in OT receptor number [33, 34]. However, other mechanisms such as the production of an inhibitor of PG synthesis [27, 35] may also be involved. Furthermore, since treatment of ovariectomized cows with progesterone alone results in an increase in response to OT [36, 37], some induction of OT receptor may be independent of estradiol action. If this is the case, and IFN suppresses estradiol-induced OT receptor, then IFN may also have other effects on PG synthesis to ensure that luteolysis does not occur. Since OT increases COX-2 mRNA in vivo [11] and in vitro [18], the objectives of the present study were to determine 1) the effect of IFN-{tau} on OT-induced COX-2 and 2) the effect of OT and IFN-{tau} on PGF synthase (PGFS), the enzyme that converts PGH2 to PGF2{alpha}. Since IFN-{tau} decreases OT receptor number in vivo, the effect of IFN-{tau} on phorbol 12-myristate 13-acetate (PMA)-stimulated PG synthesis was also examined to ensure that effects of IFN-{tau} were not solely due to changes in OT binding.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals and Reagents

Tissue culture medium (RPMI 1640), Hanks' buffered saline solution (HBSS; calcium and magnesium free), fetal calf serum (FCS), antibiotics, and trypan blue were purchased from Gibco (Grand Island, NY). Collagenase (type II), trypsin (type III, from bovine pancreas), DNase I (type I, from bovine pancreas), gentamicin, calf thymus DNA, Hoechst no. 33258, estradiol-17ß, progesterone, PMA, and E-TOXATE kit were purchased from Sigma Chemical Co. (St. Louis, MO). Matrigel was obtained from VWR Scientific (Mississauga, ON, Canada). OT was purchased from Vetroquinol (Joliette, PQ, Canada). Recombinant bovine INF-{tau} was generously provided by Dr. R.M. Roberts (University of Missouri, Columbia, MO) [38]. The polyclonal rabbit COX-2 antibody (MF243) was kindly provided by Drs. Jilly F. Evant and Stacia Kargman (Merck Frosst Center for Therapeutic Research).

Preparation and Culture of Cells

Uteri from cows at Days 1–3 of the estrous cycle were collected at the slaughterhouse and transported on ice to the laboratory. Days 1–3 were selected because the stage of the estrous cycle can be accurately determined from slaughterhouse material due to the presence of the corpus hemorrhagicum. The endometrial epithelial and stromal cells were separated as described previously [39, 40]. Briefly, the two horns of each uterus were washed with sterile HBSS containing 100 U penicillin, 100 µg streptomycin, and 0.25 µg amphotericin per milliliter. The myometrial layers were dissected away and the horns were then everted to expose the epithelium. The everted horns were digested for 2 h in HBSS with 0.3% trypsin at room temperature. At the end of incubation, the digested horns were scraped gently with forceps and then washed twice in HBSS. The scrapings and washings were combined with the digested cells, and then FCS was added to a final concentration of 10% to block the action of trypsin. The cell suspension was centrifuged at 60 x g for 5 min. The pellet was washed three more times with HBSS. Since most epithelial cells are in clumps after trypsin digestion, it is possible to separate them from completely dispersed stromal cells by this low-speed centrifugation (60 x g for 5 min). The pellet was then suspended in 20 ml RPMI medium containing 50 µg/ml gentamicin, plated onto 100 x 20-mm Nunclon Petri dishes (Gibco), and incubated at 37ÅC in 5% CO2:95% air for 3 h to allow for attachment of contaminating stromal cells. At the end of incubation, the unattached epithelial cells were collected. After cell counting and viability determination by trypan blue exclusion, cells were plated onto Matrigel (Collaborative Biochemicals, Bedford, MA)-coated 100 x 20-mm Nunclon Petri dishes. At the time of plating, the cell viability was greater than 95%.

Treatment of Cells

After reaching confluence, usually at about 7 days, cells were incubated in RPMI medium supplemented with 5% dextran-charcoal-treated FCS (DC-FCS) in presence or absence of 100 ng/ml rbIFN-{tau}, 100 ng/ml OT, or OT+rbIFN-{tau} for 3, 6, 12, and 24 h. In a second experiment, cells were incubated in the presence or absence of PMA (100 ng/ml) or PMA+rbIFN-{tau} (100 ng/ml) for 12 h. At the end of the culture, medium was collected for PG measurement. The cells were either lysed with guanidinium isothiocyanate and stored at -70ÅC for RNA isolation or scraped from the dish and suspended in 5 ml HBSS and then pelleted by centrifugation at 1000 x g for 5 min at 4ÅC. The cell pellets were stored at -70ÅC for Western blotting. For the OT-binding experiment, the confluent epithelial cells were incubated in RPMI medium supplemented with 5% DC-FCS in the presence or absence of 100 ng/ml rbIFN-{tau} for 3, 6, 12, and 24 h. The doses of OT and IFN-{tau} used in these experiments were those that gave the maximum response in preliminary dose-response studies (data not shown). The concentration of IFN-{tau} in the uterine lumen is not known, and the dose of IFN-{tau} used is in the same range as in previously published studies [8]. DNA content was determined by the bisbenzimide fluorescent dye method of Labarca and Paigen [41].

Isolation of Total RNA and Northern Blot Analysis

Total RNA was isolated from the cultured cells by centrifugation through a density gradient of 5.7 M cesium chloride. Twenty-five micrograms of total RNA was denatured at 56ÅC for 15 min, electrophoresed in 1.2% agarose gel, and passively transferred to Hybond (Amersham) nylon membranes by capillary blotting. The nylon membranes were UV-cross-linked for 30 sec at 150 mJ in a UV chamber (Bio-Rad GS Gene Linker; Bio-Rad Labs, Richmond, CA) and prehybridized for 4–6 h in hybridization buffer at 55ÅC. Blots were hybridized with the appropriate 32P-labeled probes (1 x 106 cpm/ml) for 16 h at 55ÅC, washed in double-strength saline-sodium citrate (SSC; single-strength SSC is 0.15 M sodium chloride and 0.015 M sodium citrate) + 0.1% SDS at 60ÅC for 15–20 min, and then successively washed in double-strength SSC + 0.1% SDS at 55ÅC for 15–20 min twice. Autoradiography was performed with Kodak XAR-5 (Mandel Scientific Company Ltd., Guelph, ON, Canada) and double intensifying screen at -70ÅC for various exposure times. For rehybridization with a different probe, blots were boiled for 3 min in diethyl pyrocarbonate-treated H2O containing 0.1% SDS and exposed to film overnight to check for completeness of the removal of the probe. Autoradiographic bands were scanned using Foto/Analyst (Fotodyne Inc., New Berlin, WI), and the intensity of the bands was quantitated by the NIH-Image (Bethesda, MD) program. The amount of mRNA loaded was normalized using 28S mRNA.

Probes

Mouse COX-2, bovine PGFS, and human 28S cDNAs were used as probes to detect COX-2, PGFS, and 28S gene expression in cultured bovine endometrial epithelial cells. A mouse COX-2 cDNA [42] had been previously validated for use with bovine tissues [43]. A bovine PGFS cDNA probe was generated by reverse transcription-polymerase chain reaction (RT-PCR). Five micrograms of RNA extracted from granulosa cells of bovine preovulatory follicles [43] was reversed-transcribed using AMV (avian myeloblastosis virus) reverse transcriptase (Pharmacia Biotech, Montreal, PQ, Canada) and oligodeoxythymidine primers. For the PCR reaction, homologous primers were designed from the published bovine PGFS sequence lung form [44]. The sense 25-mer primer 5'-TTAATGATGGCCACTTCATTCCTGT-3' corresponded to the region from +29 to +53 base pairs (bp) from the start codon, and the antisense 25-mer primer 5'-GAGTCAGTTCAAAGTCAAACACCTG-3' was from +841 to +865 bp of the bovine PGFS lung form [44]. The expected 837-bp PCR product was subcloned into the pCR 2.1 vector (Invitrogen, Carlsbad, CA), and its identity was confirmed by DNA sequencing using the T7 Sequencing Kit (Pharmacia), which employs the dideoxynucleotide chain termination method [45].

Cell Extracts and Western Analysis of COX-2 Protein

Solubilized cell extracts were prepared as previously described [43]. Briefly, cells were homogenized on ice in 500 µl TED homogenization buffer (50 mM Tris, 10 mM EDTA, 1 mM diethyldithiocarbamic acid [DEDTC], pH 8.0) supplemented with 2 mM octyl glucoside and centrifuged at 30 000 x g for 1 h at 4ÅC. The crude pellets containing membranes, nuclei, and mitochondria were sonicated (5 sec/cycle; 4 cycles) in 200 µl TED sonication buffer (20 mM Tris, 50 mM EDTA, 0.1 mM DEDTC, pH 8.0) containing 32 mM octyl glucoside. The sonicates were centrifuged at 13 000 x g for 25 min at 4ÅC. The supernatants (solubilized cell extracts) were stored at -70ÅC until Western analysis. The protein concentration was determined using the Bio-Rad Protein Assay.

Fifty micrograms of cell extracts was separated by 10% one-dimensional SDS-PAGE and electrophoretically transferred onto nitrocellulose membrane. The membranes were incubated with COX-2 antibody MF243, and 125I-labeled protein A was used to visualize immunopositive proteins as described previously [43]. Autoradiographic bands were scanned by Foto/Analyst, and the intensity of the bands was quantitated by the NIH-Image program.

OT Receptor and Binding Assay

OT binding was measured using whole cells. At the end of the culture, the medium was aspirated, and 100 µl of 10 nM 3H-OT, either with (nonspecific binding) or without (total binding) a 200-fold excess of unlabeled OT in binding buffer (25 mM Tris-HCl, pH 7.6; 0.1% BSA (w:v); 1 mM MnCl2) [46], was added to each of three wells for each treatment. For saturation analysis of OT receptor, epithelial cells were incubated with 0.15–15 nM 3H-OT. The binding affinity of 3H-OT for cell supernatants was determined using the least-squares curve fitting program LIGAND [47]. Plates were incubated for 20 min at room temperature and then washed three times with 1 ml of saline. Solubilization solution (250 µl, 0.5% Triton X-100, 1 M NaOH) was added into each well, and incubation was performed overnight at 37ÅC. The solubilized cells were neutralized with 62.5 µl of 4 M HCl and then counted in 4 ml of scintillant liquid. Specific binding (total minus nonspecific binding) values were expressed as cpm/µg DNA.

RIA of PGF2{alpha} and PGE2

Concentrations of PGF2{alpha} were measured in 100-µl aliquots of culture medium after 10-fold or 100-fold dilution with assay buffer. Serial dilutions of medium samples (n = 3) were parallel to the standard curve. The antibody was purchased from Cayman Chemical Co. (Ann Arbor, MI); its cross-reactivities against 13,14-dihydro-15-keto-PGF2{alpha} (PGFM), 6-keto-PGF1{alpha}, PGD2, PGE2, and arachidonic acid were 0.07%, 6.1%, 0.6%, 0.2%, and 0.002%, respectively, at 50% displacement. The sensitivity of the assay was 62.5 pg/ml, and the intra- and interassay coefficients of variation were 9.2% and 12.3%, respectively.

Concentrations of PGE2 were measured directly in 10- or 100-µl aliquots of culture medium. The antiserum was purchased from Assay Designs Inc. (Ann Arbor, MI); its cross-reactivity against PGE1, PGF1{alpha}, PGF2{alpha}, and 6-keto PGF1{alpha} was 70%, 1.4%, 0.7%, and 0.6%, respectively. The sensitivity of the assay was 40 pg/ml, and the intra- and interassay coefficients of variation were 6.3% and 8.6%, respectively.

Endotoxin Assay

The Limulus amebocyte lysate assay was used to measure the endotoxin concentration in all reagents used in this experiment, according to the protocol provided by Sigma. The endotoxin content of all reagents, including rbIFN-{tau}, was lower than the minimum detectable level by this method (< 0.1 ng/ml).

Statistical Analysis

Each experiment was carried out using the cells from one uterus and was repeated with 4 different uteri collected at different times from the slaughterhouse. Effects of treatment on PGF2{alpha} and PGE2 production, OT receptor number, and COX-2 and PGFS expression in uterine cells were evaluated by least-squares ANOVA. The data for PGF2{alpha} concentrations were transformed to logarithms to eliminate heterogeneity of variance. The effect of treatment was analyzed using a 2-way factorial design, which included the main effects of hormone treatment (control, OT, and OT+rbIFN-{tau}) and incubation time, and the treatment x time interaction. Since uterus was nested within an experiment, it was included as a random variable in the F-test for the effect of experiment. If a significant treatment or treatment x time interaction was found, further analysis was performed using OT and OT+IFN-{tau} as treatments to isolate effects of IFN. Simple effect comparisons were performed to determine differences between individual means. A probability of p < 0.05 was considered to be statistically significant. The data were analyzed using the computer program JMP (SAS Institute Inc., Cary, NC).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of IFN-{tau} on OT-Induced PG Secretion

The basal secretion of PGF2{alpha} and PGE2 by untreated epithelial cells did not change with time (treatment x time interaction, p < 0.05). OT stimulated secretion of both PGF2{alpha} and PGE2, an effect that increased with time (p < 0.01) (Fig. 1, a and b). The increase in PGE2 was maximum at 12 h, whereas PGF2{alpha} continued to increase at 24 h. Addition of rbIFN-{tau} inhibited the OT-induced increase in PGF2{alpha} (p = 0.02) and PGE2 (p < 0.01). OT and OT+rbIFN-{tau} decreased the PGE2/PGF2{alpha} ratio at 24 h (p < 0.05), but not before (Fig. 1c).



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  		FIG. 1. Effect of OT and rbIFN-{tau} on PG production in epithelial cells. Confluent epithelial cells were incubated in RPMI medium supplemented with 5% DC-FCS (control, CTL; open bar), with 100 ng/ml OT (solid bar), or with OT+100 ng/ml rbIFN-{tau} (hatched bar) for 3, 6, 12, and 24 h. The culture medium was collected for PGF2{alpha} (a) and PGE2 (b) measurement by RIA. Data are normalized to the DNA content of the respective wells. The ratio of PGE2 to PGF2{alpha} was calculated (c). The data are presented as the least-square means ± SEM (n = 4). There was a significant inhibitory effect of rbIFN-{tau} on the OT-induced increase in both PGF2{alpha} (p = 0.02) and PGE2 (p < 0.01). OT and OT+rbIFN-{tau} decreased the PGE2/PGF2{alpha} ratio at 24 h (p < 0.05) but not before.

Effect of IFN-{tau} on OT-Induced COX-2

Unstimulated levels of COX-2 mRNA did not change with time (treatment x time interaction, p < 0.01). OT significantly up-regulated COX-2 mRNA (Fig. 2, p < 0.001) at all time points measured. The OT-induced COX-2 mRNA steady-state level was highest at 3 h and then decreased with time. Recombinant bovine IFN-{tau} dramatically reduced the OT-induced COX-2 mRNA levels at 3 and 6 h (p < 0.01) but had no significant effect at 12 and 24 h.



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  		FIG. 2. Northern blot analysis of COX-2 mRNA in epithelial cells. RNA was isolated from epithelial cells. A representative sample is shown (a); 25 µg of total RNA per lane was loaded and blots were hybridized with 32P-labeled mouse COX-2 cDNA probe. The stripped blots were rehybridized with human 28S cDNA probe as a loading control. Autoradiographic bands were scanned by a densitometer and normalized to their 28S values (b). Open bars, solid bars, and hatched bars represent Control, OT, and OT+rbINF-{tau}, respectively. Data are expressed as the least-square means ± SEM (n = 4). Recombinant bovine IFN-{tau} reduced the OT-induced COX-2 mRNA levels at 3 and 6 h (p < 0.001) but not later.

The specific COX-2 antibody used bound to two COX-2 protein bands with molecular weights of approximately74 000 Mr and 62 000 Mr, respectively (Fig. 3a). The smaller molecular weight band has previously been observed in rats [48], sheep [49], and cattle [43] and is believed to be a proteolytic fragment [43]. The concentration of COX-2 did not change with time in the untreated cells. OT markedly increased COX-2 protein at all time points (3.8-, 6.2-, 10.2-, and 5.2-fold at 3, 6, 12, and 24 h, respectively; Fig. 3, p < 0.001), with the maximum stimulation at 12 h. Addition of rbIFN-{tau} suppressed the induction of COX-2 protein by OT (p < 0.001) at all times, reducing its abundance by 42%, 56%, 52%, and 47% at 3, 6, 12, and 24 h, respectively.



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  		FIG. 3. Western blot analysis of COX-2 in epithelial cells. Solubilized cell extracts were prepared from epithelial cells after OT and rbIFN-{tau} treatment as described for Figure 1. Fifty micrograms of protein per lane was loaded; the membranes were incubated with COX-2 antibody MF243, and 125I-labeled protein A was used to visualize immunopositive proteins (a). Autoradiographic bands were scanned by a densitometer and quantified (b). Open bars, solid bars, and hatched bars represent Control, OT, and OT+rbINF-{tau}, respectively. Data are expressed as the least-square means ± SEM (n = 4). Recombinant bovine IFN-{tau} suppressed the induction of COX-2 protein by OT at all time points (p < 0.001).

Effect of IFN-{tau} on OT-Induced PGFS mRNA Levels

A 1.4-kilobase transcript hybridized with the bovine PGFS cDNA probe (Fig. 4). The basal and stimulated levels of PGFS decreased with time (p < 0.05). OT up-regulated PGFS mRNA (p < 0.001) at all times, and rbIFN-{tau} significantly inhibited this induction (p < 0.01), reducing PGFS mRNA by 32%, 29.5%, 12.7%, and 24.1% at 3, 6, 12, and 24 h, respectively.



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  		FIG. 4. Northern blot analysis of PGFS in epithelial cells; the same blots as shown in Figure 2 for COX-2 hybridization were stripped and hybridized with 32P-labeled PGFS probe (a). The densitometric values of PGFS were normalized to their 28S values (b). Open bars, solid bars, and hatched bars represent Control, OT, and OT+rbINF-{tau}, respectively. Data are expressed as the least-square means ± SEM (n = 4). Recombinant bovine IFN-{tau} inhibited the OT-induced PGFS mRNA at all time points (p < 0.01).

Effect of IFN-{tau} on OT Binding

Results shown in Figure 5 indicated that specific binding of 3H-OT by epithelial cells was saturable and exhibited high affinity. The disassociation constant (Kd) was 4.9 nM. To determine whether rbIFN-{tau} altered OT binding to the cells in vitro, confluent epithelial cells were treated with 100 ng/ml rbIFN-{tau} for 3, 6, 12, and 24 h, and OT binding was measured using 3H-OT. The results show that rbIFN-{tau} significantly decreased OT binding to the cells as early as 3 h after treatment (p < 0.01, Fig. 6).



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  		FIG. 5. Saturation analysis of specifically bound 3H-OT to primary cultures of bovine endometrial epithelial cells. Cells were cultured to confluence in RPMI 1640 containing 5% DC-FCS. The cells were then incubated with 0.15–15 nM 3H-OT with or without 200-fold molar excess of unlabeled OT. a) Total binding, nonspecific binding, and specific binding by epithelial cells are presented as saturation curves. b) The corresponding Scatchard plot of specific binding is presented. B/F, bound-to-free ratio.



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  		FIG. 6. Effect of rbIFN-{tau} on OT receptor number in epithelial cells. Confluent epithelial cells were incubated in RPMI medium supplemented with 5% DC-FCS in presence or absence of 100 ng/ml rbIFN-{tau} for 3, 6, 12, and 24 h. At the end of the culture, the whole cells were incubated with 100 µl of 10 nM 3H-OT in the presence or absence of 200-fold molar excess of unlabeled OT in the binding buffer to measure the total and nonspecific binding. Data are presented as specific binding, normalized to the respective DNA content. Data are expressed as the least-square means ± SEM (n = 4); ** represents significant difference between medium control and rbIFN-{tau} treatment within the same time point (p < 0.01).

Effect of IFN-{tau} on PMA-Stimulated PGF2{alpha} Production

The effect of rbIFN-{tau} on PMA-stimulated PG synthesis was examined to determine whether the observed decrease in OT-induced PG secretion by rbIFN-{tau} was due solely to a decrease in OT binding to the cells. PMA stimulated PGF2{alpha} production by the epithelial cells, and this stimulation was reduced by rbIFN-{tau} (p < 0.01) (Fig. 7). Recombinant bovine IFN-{tau} also inhibited the PMA stimulation of COX-2 protein in the epithelial cells (p < 0.01) (Fig. 8).



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  		FIG. 7. Effect of rbIFN-{tau} on PMA-induced PGF2{alpha} in epithelial cells. Confluent epithelial cells were incubated in RPMI medium supplemented with 5% DC-FCS in absence (CTL) or presence of 100 ng/ml PMA or PMA+rbIFN-{tau} for 12 h. The culture medium was collected for PGF2{alpha} measurement by RIA. Data are normalized to the DNA contents of the respective wells. Data are presented as the least-square means ± SEM (n = 4). Recombinant bovine IFN-{tau} decreased the PMA-stimulated PGF2{alpha} production (p < 0.01).



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  		FIG. 8. Effect of rbIFN-{tau} on PMA-induced COX-2 protein. Solubilized cell extracts were prepared from epithelial cells after treatment for 12 h with PMA and rbIFN-{tau}. Fifty micrograms of protein per lane was loaded; the membranes were incubated with COX-2 antibody MF243, and 125I-labeled protein A was used to visualize immunopositive proteins (a). Autoradiographic bands were scanned by a densitometer and quantified (b). Data are expressed as the least-square means ± SEM (n = 3). Recombinant bovine IFN-{tau} suppressed the induction of COX-2 protein by PMA (p < 0.01).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Although it is well known that IFN-{tau} from the embryo inhibits OT-induced secretion of PGF2{alpha} from the endometrium, the exact mechanisms involved are not completely understood. It is widely accepted that IFN-{tau} decreases OT receptor number in the endometrium, resulting in a decrease in the response to OT in pregnant animals. In the cow there is also an increase in an inhibitor of PG synthesis in pregnant animals [35], which suggests that IFN-{tau} has actions on the endometrium other than decreasing OT receptor. In this study we investigated the possibility that IFN-{tau} inhibits the OT-stimulated COX-2. Since it is difficult to dissociate an effect of IFN-{tau} on OT receptor-mediated events and postreceptor effects in vivo, an in vitro model was used to examine the effect of rbIFN-{tau} on the induction of PG secretion, COX-2 mRNA and protein, and PGFS mRNA by OT. In the epithelial cell preparation used, OT significantly stimulated PG production, an effect that was attenuated by rbIFN-{tau}. This is in agreement with previous reports [8, 15, 17, 18, 50] and reflects the events that occur in vivo. The cells were prepared from uteri taken at Day 3 of the cycle, a time when the uterus is still responsive to OT and thus should respond in a similar fashion to the endometrium in vivo at the time of luteolysis. It is possible that the cells differentiate during the 7-day culture, but since these cells respond to OT and IFN-{tau} in a manner similar to that observed in vivo, we believe the results are representative of the situation in vivo.

The in vitro stimulation of PGF2{alpha} in bovine endometrial epithelial cells by OT is associated with an increase in COX-2 mRNA at 24 h after OT treatment [18], but changes in COX-2 mRNA, COX-2 protein, and PGFS over time have not been previously documented. Our results show a rapid induction in COX-2 mRNA by OT, which was maximal by 3 h and then decreased over time. The maximal OT-induced COX-2 protein appeared later than the mRNA, with the lag period of 3–9 h suggesting that the latter is the product of translation of the former. OT also up-regulated PGFS expression, which could explain, at least in part, the difference in the production of PGF2{alpha} and PGE2. Production of PGE2 by the epithelial cells plateaued at 12 h, resulting in a significant decrease in the ratio of PGE2 to PGF2{alpha} at 24 h. An increase in PGFS would be expected to result in an increase in the conversion of the precursor, PGH2, to PGF2{alpha} at the expense of PGE2.

In vivo studies in sheep also show that OT induces a rapid increase in both serum PGFM and endometrial COX-2 mRNA, peaking at 25 min postinjection [11]. Thus, the in vivo and in vitro data show that both COX-2 mRNA and protein are rapidly and transiently expressed in endometrial tissue. This is consistent with COX-2's being an immediate early gene in the endometrial epithelium, as in many other tissues [51]. In cattle, pulses of PGF2{alpha} occur every 6–8 h during luteolysis [52]. Since COX-2 protein concentration peaks about 12 h after administration of OT, it is possible that OT is not only responsible for the immediate stimulation of PGF2{alpha} during a luteolytic episode but that it may also increase COX-2 and PGFS and, thereby, the capacity of the uterus to synthesize PGF2{alpha} as luteolysis proceeds. There is an increase in COX around the time of luteolysis in sheep [53], but whether or not OT is involved in its induction remains to be established. Charpigny et al. [53] did not detect any difference between COX-2 levels in cycling and pregnant sheep, suggesting that OT is not involved. However, in their study, COX-2 protein was measured in whole endometrium. Therefore, since IFN-{tau} stimulates COX-2 in the stromal cells but inhibits COX-2 in the epithelial cells [40], it is possible that changes in COX-2 in the epithelial cells were masked.

INF-{tau} is responsible for inhibiting PGF2{alpha} secretion in pregnant ruminants, and one of its actions is to decrease OT receptor number in the endometrium. Rates of OT receptor gene transcription are 2-fold lower in the endometrium of Day 15 cyclic ewes receiving intrauterine injections of recombinant ovine IFN-{tau} than in control ewes [34]. Intrauterine infusion of rbIFN-{tau} also attenuates OT binding to the endometrium in the cow [22] and sheep [23]. Suppression of the estradiol up-regulation of OT receptor may not, however, be the only mechanism involved. OT is able to induce release of PGF2{alpha} in pregnant cows and ewes [54], albeit at a level much lower than that observed in the cyclic animals [50]. This suggests that functional OT receptors are present in the pregnant uterus. It is likely, therefore, that IFN-{tau} decreases PGF2{alpha} secretion in the endometrium of the pregnant cow by actions other than the reduction of OT receptors. One such mechanism is the production of an inhibitor of PG synthesis, as occurs in the endometrium of the pregnant cow [35]. This inhibitor has recently been identified as linoleic acid, which competes with arachidonic acid for the active site of the COX enzyme [27]. Our results show that rbIFN-{tau} also inhibits the OT-induced increase in COX-2 and PGFS mRNA. Together the data suggest that rbIFN-{tau} has several effects on the endometrium that result in decreased PGF2{alpha} secretion in the pregnant animal and thus the maintenance of the corpus luteum.

Since OT and IFN-{tau} were added at the same time, it is unlikely that the observed effect of IFN-{tau} was due to a change in OT receptor. However, rbIFN-{tau} did decrease OT binding by the epithelial cells. To ensure that the rbIFN-{tau} inhibition of the OT-induced COX-2 was not solely due to a decrease in OT receptor, the effect of rbIFN-{tau} on PMA-induced PGF2{alpha} secretion was examined. OT acts via the protein kinase C intracellular pathway. Thus, activating protein kinase C with PMA bypasses the interaction of OT with its receptor. PMA markedly stimulated PGF2{alpha} secretion by epithelial cells, a finding that is consistent with previous reports in cows [55, 56] and pigs [57]. PMA stimulated PG synthesis in the endometrial epithelial cells by increasing COX-2 protein, consistent with its effect in other cell types. The effect of PMA on PGF2{alpha} synthesis and COX-2 protein was significantly reduced when the cells were treated with rbIFN-{tau}. These results indicate that the inhibitory effect of IFN-{tau} on OT-induced PGF2{alpha} secretion is attributable not only to the decrease in OT receptor but also to down-regulation of COX-2. This is consistent with our previous demonstration that rbIFN-{tau} decreases basal PGF2{alpha} secretion by down-regulation of both COX-2 and PGFS mRNA [40].

In conclusion, the present study shows that OT stimulates PGFS as well as COX-2 in endometrial epithelial cells and that rbIFN-{tau} inhibits the OT-induced COX-2 and PGFS. These data suggest that during pregnancy, rbIFN-{tau} inhibits OT-induced PGF2{alpha} secretion from the endometrium not only by down-regulating OT receptor but also by decreasing COX-2 and PGFS via a mechanism independent of changes in OT receptor.


   ACKNOWLEDGMENTS
 
We thank Dr. R.M. Roberts for the generous gift of rbIFN-{tau}; Dr. D.L. Simmons (Brigham Young University) for the mouse COX-2 cDNA; Dr. G. Schultz for the 28S probe; Drs. Jilly F. Evant and Stacia Kargman (Merck Frosst Center for Therapeutic Research) for providing COX-2 antibody (MF243); and D. Rannou for technical assistance.


   FOOTNOTES
 
1 This work was supported by grants from the Natural Sciences and Engineering Research Council (to A.K.G.), Fonds pour la Formation de Chercheurs et l'Aide à la recherche (to B.D.M. and A.K.G.), and Medical Research Council of Canada (to J.S.). Back

2 Correspondence: Alan K. Goff, Centre de recherche en reproduction animale, Faculté de médecine vétérinaire, Université de Montréal, 3200 rue Sicotte, St-Hyacinthe, PQ, Canada J2S 7C6. FAX: 450 778 8103; goffak{at}medvet.umontreal.ca Back

Accepted: October 20, 1998.

Received: August 10, 1998.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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