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BOR - Papers in Press, published online ahead of print April 30, 2003.
Biol Reprod 2003, 10.1095/biolreprod.102.015057
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BIOLOGY OF REPRODUCTION 69, 780–787 (2003)
DOI: 10.1095/biolreprod.102.015057
© 2003 by the Society for the Study of Reproduction, Inc.

Female Reproductive Tract

Polyunsaturated Fatty Acids and Bovine Interferon-{tau} Modify Phorbol Ester-Induced Secretion of Prostaglandin F2{alpha} and Expression of Prostaglandin Endoperoxide Synthase-2 and Phospholipase-A2 in Bovine Endometrial Cells1

Ricardo Mattos, Aydin Guzeloglu, Lokenga Badinga, Charles R. Staples, and William W. Thatcher2

Department of Animal Sciences, University of Florida, Gainesville, Florida 32611-0910


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Embryonic mortality in cattle may occur because of inadequate inhibition of uterine secretion of prostaglandin (PG) F2{alpha} mediated by bovine interferon-{tau} (bIFN-{tau}). The objectives of the present study were to determine whether polyunsaturated fatty acids inhibit secretion of PGF2{alpha} from bovine endometrial cells induced by stimulating protein kinase C with phorbol 12,13 dibutyrate (PDBu) and to investigate possible mechanisms of action. Confluent cells were exposed for 24 h to 100 µM of linoleic, arachidonic (AA; C20:4, n-6), linolenic (LNA; C18:3, n-3), eicosapentaenoic (EPA; C20:5, n-3), or docosahexaenoic (DHA; C22:6, n-3) acid. After incubation, cells were washed and stimulated with PDBu. The EPA, DHA, and LNA attenuated secretion of PGF2{alpha} in response to PDBu. The EPA and DHA were more potent inhibitors than LNA. The EPA inhibited secretion of PGF2{alpha} at 6.25 µM. Secretion of PGF2{alpha} in response to PDBu decreased with increasing incubation time with EPA. Both bIFN-{tau} and EPA inhibited secretion of PGF2{alpha}, and their inhibitory effects were additive. The bIFN-{tau}, but not EPA, reduced the abundance of PG endoperoxide synthase-2 (PGHS-2) mRNA. Incubation with 100 µM EPA, DHA, or AA for 24 h followed by treatment with PDBu did not affect concentrations of PGHS-2 and phospholipase A2 proteins. The EPA and DHA inhibit secretion of PGF2{alpha} through a mechanism different from that of bIFN-{tau}. The effect of EPA on PGF2{alpha} secretion may be caused by competition with AA for PGHS-2 activity or reduction of PGHS-2 activity. The use of EPA and DHA to inhibit uterine secretion of PGF2{alpha} and to improve embryonic survival in cattle warrants further investigation.

conceptus, cytokines, female reproductive tract, implantation, uterus


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
A high proportion of embryonic loss occurs in cattle during the first 3 wk of pregnancy [1]. This high proportion of losses coincides with the period of embryonic inhibition of uterine prostaglandin (PG) F2{alpha} secretion and suggests that some loss may be occurring because certain embryos are unable to inhibit secretion of PGF2{alpha}. Therefore, strategies to further inhibit secretion of PGF2{alpha} may increase embryonic survival. In a recent report, increased PG synthesis induced by oxytocin during Days 5–8 of pregnancy reduced the pregnancy rates of beef cows at 30 days after artificial insemination from the 80% observed in the untreated controls to 30%. Treating cows concomitantly with flunixin meglumine, an inhibitor of prostanoid synthesis, restored pregnancy rates to 80% [2]. Another study [3] demonstrated that chronic treatment with PGF2{alpha} reduced the pregnancy rate when given between Days 5 and 8 of pregnancy and not on Days 10–13 and 15–18. These results demonstrate that increased PG secretion during early pregnancy causes embryonic loss. In addition, the results support the hypothesis that reducing PG synthesis during early pregnancy reduces embryonic loss and improves pregnancy rates.

Secretion of prostanoids, such as PGF2{alpha}, requires a cascade of intracellular events, including activation of the phospholipase (PL) C enzyme and activation of a calcium-dependent PLA2 through a multiple antigenic peptide kinase-dependent pathway [4]. The activated PLA2 translocates to the plasma membrane and cleaves fatty acids from the sn-2 position of membrane phospholipids, to which a high proportion of polyunsaturated fatty acids (PUFAs), such as arachidonic acid (AA), is esterified. Released AA is converted to PGH2 by the PG endoperoxide H synthase (PGHS) enzymes. Two PGHS enzymes, PGHS-1 and PGHS-2, with similar structures and functions but different expression patterns, have been described [5]. Prostaglandin H2 is subsequently reduced to PGF2{alpha} by PGF2{alpha} synthase or is isomerized to other prostanoids of the 2 series, such as PGE2 [6].

The PLA2 and PGHS enzymes also are capable of processing other fatty acids, such as eicosapentaenoic acid (EPA), which is the precursor for synthesis of prostanoids of the 3 series. Prostanoids of the 3 series, which include PGF3 and PGE3, have three double bonds in the carbon chain, as opposed to the two double bonds found in prostanoids of the 2 series. Increased availability of n-3 PUFAs, such as EPA and docosahexaenoic acid (DHA), in membrane phospholipids could inhibit PGF2{alpha} secretion through three mechanisms. First, EPA could displace AA, leading to increased synthesis of prostanoids of the 3 series at the expense of prostanoids of the 2 series, such as PGF2{alpha}. Second, EPA and another n-3 PUFA, DHA, may reduce expression of the PGHS enzymes [7], which could make these enzymes less available and further reduce prostanoid synthesis. Third, the conversion of EPA into prostanoids of the 3 series is less efficient than the conversion of AA into prostanoids of the 2 series [8]. A lower efficiency of catalysis may result in reduced total prostanoid synthesis because of insufficient converting capacity.

Prostanoids of the 3 series are less bioactive [9], and their role in ruminant luteolysis is currently unknown. Prostaglandin F3{alpha} has only 25% affinity for the ovine luteal PGF2{alpha} receptor when compared to PGF2{alpha} [10]. In addition, DHA is not a substrate for the PGHS enzymes but is a strong competitive inhibitor of their activity [11].

The bovine interferon-{tau} (bIFN-{tau}) attenuates secretion of endometrial PGF2{alpha} both in vivo [12] and in vitro [13–16]. Experimental approaches used to test the effects of bIFN-{tau} have included explants [13], primary cultures of endometrial cells collected on Days 1–4 [15] or on Day 15 of the estrous cycle [14], and bovine immortalized endometrial (BEND) cells [16]. The BEND cells are a line of spontaneously replicating endometrial cells originating from Day 14 cycling cows [17]. Systems used to evaluate secretion of PGF2{alpha} from endometrial cells generally involve stimulation of cells with an activator of protein kinase C, such as phorbol 12-myristate 13-acetate and phorbol 12,13 dibutyrate (PDBu) [16]. These activators induce expression of the PGHS-2 gene and stimulate secretion of PGF2{alpha}. The bIFN-{tau} attenuates both of these effects [15, 16].

The objectives of the present study were to determine the effects of PUFAs on secretion of PGF2{alpha} from BEND cells stimulated with PDBu, to test the interaction of PUFAs with the inhibitory effect of bIFN-{tau}, and to explore possible mechanisms of action by determining the effects of PUFAs on steady-state concentrations of PGHS-2 mRNA and protein expression of PLA2 and PGHS-2.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials

Recombinant bIFN-{tau} (1.08 x 107 U of antiviral activity per milligram) was donated kindly by Dr. R. Michael Roberts (University of Missouri, Columbia, MO). Polystyrene tissue-culture Costar six-well plates and culture dishes (100 x 20 mm) were purchased from Corning (Corning Glass Works, Corning, NY). Polystyrene flasks for cell culture (175 cm2) were from Sarstedt, Inc. (Newton, NC). Acrylamide, N,N'-methylenebisacrylamide, and Nonidet 40 were from BDH Laboratory Supplies (Poole, U.K.).

The Ham F-12, Eagle minimum essential medium (MEM), antibiotic-antimycotic solution (AbAm), 3-(N-Morpholino) propanesulfonic acid (MOPS), and EDTA were from Sigma Chemical Co. (St. Louis, MO). The lysis buffer was from Promega (Reporter lysis buffer; Madison, WI). Isotopically labeled [5, 6, 8, 11, 12, 14, 15,-3H-PGF2{alpha}] (212 Ci/mol), and nitrocellulose membranes (Hybond-ECL) were from Amersham Pharmacia Biotech (Arlington Heights, IL). BioTrans Nylon membrane was from ICN Chemicals (Irvine, CA). Hanks balanced salt solution (HBSS) and TRIzol were from Life Technologies (Grand Island, NY). X-ray film was from Eastman Kodak Co. (X-Omat Blue XB-1; Rochester, NY). Enhanced chemiluminescence (ECL) kit (Renaissance Western Blot Chemiluminescence Reagent Plus) was from NEN Life Science Products (Boston, MA). Anti-PGHS-2, PGHS-2 electrophoresis standard, and the fatty acids EPA (C20:5, n-3), DHA (C22:6, n-3), AA (C20:4, n-6), linolenic (LNA; C18:3, n-3), linoleic (LA; C18:2, n-6), and oleic (OA; 18:1, n-9) were purchased from Cayman Chemical (Ann Arbor, MI). Cytosolic PLA2 polyclonal antibody was purchased from Santa Cruz Biotechnologies, Inc. (Santa Cruz, CA). The bovine PGHS-2 cDNA probe was cloned from an ovarian follicular cDNA library [18]. The probe was labeled by nick translation using a Nick Translation kit (N5000; Amersham Pharmacia Biotech, Little Chalfont, U.K.) and 32P-labeled deoxycytidine triphosphate (dCTP; ICN Chemicals, Costa Mesa, CA). The procedure used has been described by Badinga et al. [19].

Cell Culture and Experimental Designs

The BEND cell line is deposited and characterized by the American Type Culture Collection (ARCC no. CRL-2398; Manassas, VA). The BEND cells (16,22) were plated on 35-mm wells of six-well plates (1 x 105 cells/well) in culture medium (40% Ham F-12, 40% MEM, 10 ml/L of AbAm, 200 U/L of insulin, 0.343 g/L of D-valine, 10% fetal bovine serum, and 10% horse serum) for experiments 1–6 and 9 or on 100- x 20-mm Petri dishes (2.4 x 106 cell/dish) for experiments 7 and 8. The ratio of culture area to volume of culture medium was the same in both culture containers. Cells were grown to confluence at 37°C in an incubator (model MCO 17AI; Sanyo Electric Co., Ltd., Osaka, Japan) under a humidified atmosphere containing 95% air and 5% CO2. On reaching confluence, cells were washed twice with HBSS at 37°C and cultured for an additional 24 h in culture medium devoid of serum with or without fatty acid. The 24-h incubation period with fatty acids was chosen based on reported effects of AA on PGF2{alpha} secretion from bovine endometrial explants after incubation for 24 h [20]. At completion of this 24-h incubation period (designated as 0 h), cells were washed again with HBSS to remove fatty acids and exposed to serum-free medium mixed with appropriate treatments. In experiments 1–6, samples (250 µl) were collected immediately before cell wash (0 h) and at 3 and 6 h after addition of PDBu. At 3 h, the volume removed was replaced by adding 250 µl of medium devoid of serum with PDBu (100 ng/ml). In experiments 7–9, samples of medium were collected at 6 h only. After collection, all samples were stored at -20°C until analyzed for PGF2{alpha}.

Preparation of Fatty Acid Solutions

Stock solutions of polyunsaturated fatty acids were diluted in ethanol (50 mg/ml), aliquoted, and stored at -80°C. At preparation of culture medium with fatty acid, the ethanol was evaporated with nitrogen gas, and the remaining concentrated fatty acid solution was diluted to 1 mM in culture medium (50% MEM and 50% Ham F-12) containing 33 mg/ml of fatty acid-free BSA (fraction V, low endotoxin). This solution was incubated for 2 h in a water bath at 37°C to allow binding of the fatty acid to BSA and then further diluted in culture medium to final treatment concentrations. The BSA:fatty acid ratio was maintained constant at all fatty acid concentrations tested.

Experiment 1 The objective of this experiment was to test the effect of different PUFAs and BSA on secretion of PGF2{alpha} induced by PDBu. Cultures (3 ml of culture medium, six-well plates, n = 30) were assigned randomly to incubation for 24 h with medium devoid of serum with no fatty acid, medium devoid of serum with BSA (3.3 mg/ml), or medium devoid of serum with BSA and one of the following fatty acids (100 µM): EPA, DHA, AA, LNA, LA, or OA. Three 35-mm wells were assigned to each treatment. After the 24-h incubation, cells were washed with HBSS and exposed to culture medium containing PDBu (100 ng/ml). The control groups included cells incubated with BSA that subsequently were exposed or not exposed to PDBu and cells incubated for 24 h with culture medium alone (no BSA) and subsequently stimulated with PDBu.

Experiment 2 This experiment was conducted to test the effect of different concentrations of EPA, DHA, and LNA on PGF2{alpha} secretion from BEND cells stimulated with PDBu. Cultures (3 ml of culture medium, six-well plates, n = 54) were assigned in triplicate to incubation for 24 h in medium devoid of serum with treatments of a 3 x 6 factorial experiment. Factors were fatty acid (EPA, DHA, or LNA) and concentration (0, 20, 40, 60, 80, or 100 µM). At 0 h, cells were washed twice with HBSS and treated with medium devoid of serum containing PDBu (100 ng/ml).

Experiment 3 This experiment was conducted to test the effect of concentrations of EPA between 0 and 20 µM on PGF2{alpha} secretion from BEND cells stimulated with PDBu. Cultures (3 ml of culture medium, six-well plates, n = 12) were assigned in triplicate to incubation with medium devoid of serum with BSA and 0, 6.25, 12.5, or 20 µM EPA for 24 h and subsequently washed and stimulated with PDBu (100 ng/ml) dissolved in medium devoid of serum.

Experiment 4 This experiment was conducted to test the effect of different durations of incubation on PGF2{alpha} secretion from BEND cells stimulated with PDBu. Cultures (3 ml of culture medium, six-well plates, n = 18) were assigned in triplicate to incubation with medium devoid of serum with BSA and 100 µM EPA for 0, 1, 3, 6, 12, or 24 h. After incubation, cells were washed and treated with PDBu (100 ng/ml) dissolved in culture medium devoid of serum.

Experiment 5 This experiment was conducted to test the effect of concentrations of bIFN-{tau} between 0 and 25 ng/ml on PGF2{alpha} secretion from BEND cells stimulated with PDBu. Cultures (3 ml of culture medium, six-well plates, n = 27) were incubated for 24 h with medium devoid of serum without fatty acids. After incubation, cells were washed and assigned in triplicate to receive medium devoid of serum containing PDBu (100 ng/ml) in combination with 0, 0.01, 0.05, 1, 2, 3.125, 12.5, or 25 ng/ml of bIFN-{tau}.

Experiment 6 This experiment was conducted to test the effect of combinations of different doses of EPA and bIFN-{tau} on secretion of PGF2{alpha} from BEND cells stimulated with PDBu. Cultures (24 ml of culture medium, six-well plates, n = 27) were assigned in triplicate to a 3 x 3 factorial experiment. Factors were incubated for 24 h with medium devoid of serum containing EPA (0, 3, or 20 µM) and subsequent treatment with bIFN-{tau} (0, 50, or 100 pg/ml) given concurrently with PDBu (100 ng/ml). At the end of incubation with EPA, cells were washed twice with HBSS to remove EPA and BSA and treated with PDBu (100 ng/ml) dissolved in medium devoid of serum and containing the different concentrations of bIFN-{tau}. Whole-cell extracts (WCEs) were collected at 6 h to determine protein yields. This experiment was replicated once.

Experiment 7 This experiment was conducted to compare the effect of three concentrations bIFN-{tau} and three concentrations of EPA on PGF2{alpha} secretion and steady-state concentrations of PGHS-2 mRNA in BEND cells stimulated with PDBu. Cultures (24 ml of culture medium, 100-mm dishes, n = 12) were assigned in duplicate to incubation for 24 h with medium devoid of serum containing 0, 20, or 100 µM EPA followed by treatment with 100 ng/ml of PDBu in serum-free medium without fatty acids or incubation for 24 h with serum-free medium without fatty acids followed by treatment at 0 h with 100 ng/ml of PDBu in combination with 0, 0.5, or 50 ng/ml of bIFN-{tau}. Total RNA extraction was conducted at 6 h. This experiment was replicated once.

Experiment 8 This experiment was conducted to test the effect of different concentrations of EPA, DHA, and AA on secretion of PGF2{alpha} and intracellular concentrations of PGHS-2 and cPLA2 protein in BEND cells stimulated with PDBu. Cultures (24 ml of culture medium, 100-mm dishes, n = 12) were assigned in duplicate to incubation for 24 h with medium devoid of serum containing 100 µM EPA, DHA, or AA. After incubation, cells were washed twice with HBSS and treated with 100 ng/ml of PDBu dissolved in media devoid of serum. The WCEs were collected at 6 h after addition of PDBu for PGHS-2 and PLA2 immunoblotting. This experiment was replicated once.

Experiment 9 This experiment was conducted to test the effect of combinations of EPA and AA concentrations on secretion of PGF2{alpha} from BEND cells stimulated with PDBu. Cultures (3 ml of culture medium, six-well plates, n = 27) were assigned in triplicate to a 3 x 3 factorial experiment. Cells were incubated for 24 h with medium devoid of serum containing all factorial combinations of EPA (0, 25, and 100 µM) and AA (0, 25, and 100 µM). After incubation, cells were washed twice with HBSS and treated with 100 ng/ml of PDBu dissolved in medium devoid of serum. Medium samples were at 6 h after PDBu.

Preparation of Cell Extracts and Immunoblotting Analysis

The WCEs were harvested immediately after collection of the last medium sample. Cells were washed twice with HBSS at 4°C and incubated for 15 min with 1 ml of lysis buffer. Dishes were then scraped using a rubber policeman to collect cell fragments attached to the dish. Lysates and cell fragments were transferred to 1.5-ml conical tubes and agitated for 5 min using a rotating device (Roto-torque; Cole-Parmer Instrument Co., Chicago, IL) to allow further cell lysis. Cell debris was separated by centrifugation using a microcentrifuge (13 000 x g) for 2 min. Supernatants were transferred to 1.5-ml tubes and stored at -20°C. Concentrations of protein in WCEs were determined using the Bradford method [21]. A sample of each WCE was diluted 1:25 (40 µl of sample in 960 µl of ddH2O) and used for determination of protein concentration. Twenty-five microgram of protein were loaded onto duplicate 7.5% acrylamide gels. A PGHS-2 eletrophoresis standard (50 ng) also was loaded onto one lane of the gel and used as a positive control. Proteins were separated by one-dimensional SDS-PAGE (150 V, 100 mA, 25 min). Proteins were transferred to nitrocellulose membranes as previously described [16]. After transfer, membranes were blocked for 2 h in 5% nonfat dried milk and subsequently incubated for 2 h with a polyclonal antibody against PGHS-2 (1:500 dilution in 5% nonfat dry milk solids in Tris-buffered saline [TBS]). After incubation with the primary antibody, membranes were washed once with TBS containing 0.1% Tween 20 (TBST) for 15 min and thrice for 5 min. Membranes were then incubated for 1 h with anti-rabbit immunoglobulin G horseradish peroxidase-linked whole antibody from sheep (1:5000 dilution in TBST). Antibody complexes were detected using ECL, with exposure times of 1 min for PGHS-2 and 2 min for PLA2. Protein bands were quantified by densitometry as described by Binelli et al. [16].

RNA Isolation and Northern Blot Analysis

At 6 h, culture medium was aspirated and cells washed twice using HBSS at 4°C. Cells were lysed using 3 ml of Trizol at 4°C for 5 min. Total RNA was extracted following the manufacturer's instructions. The precipitated RNA was resuspended using 25 µl of RNase-free water. Concentration of RNA was determined using a spectrophotometer set at OD260. Total cellular RNA (30 µg/lane) was fractionated in a 1% agarose-formaldehyde gel, stained with ethidium bromide, blotted to a nylon membrane, and hybridized with a homologous bovine PGHS-2 cDNA probe labeled with [32P]dCTP. The PGHS-2 mRNA transcript was identified by autoradiography, and hybridization signals were analyzed by densitometry.

Radioimmunoassay

Concentrations of PGF2{alpha} were measured in medium samples collected at 0 and 6 h after treatment of cells with PDBu. Twenty-five microliters of each sample were further diluted in 75 µl of 50 mM Tris-HCl (pH 7.5, Tris buffer). Therefore, the total volume of diluted samples for assay was 100 µl. Danet-Desnoyers et al. [14] have described the RIA procedure. To prepare standard curves, known amounts of nonradioactive PGF2{alpha} (1.25–1000 pg/tube) were diluted in Tris buffer (100 µl). An anti-PGF2{alpha} antiserum [22] was diluted 1:5000 (Tris buffer, 100 µl/tube). Final assay volume was 400 µl (100 µl of Tris buffer, 100 µl of diluted sample, 100 µl of antibody solution, and 100 µl of radiolabeled PGF2{alpha} solution). The minimum detectable concentration of PGF2{alpha} was 3.32 pg/tube. Inter- and intraassay coefficients of variation were 11.2% and 13.3%, respectively. Assay was validated for serum-free medium by adding 25 pg of PGF2{alpha} per 0.1 ml to medium. Average recovered PGF2{alpha} was 25.1 ± 1.42 pg per 0.1 ml, which yielded a calculated recovery of 100.25%. A test using known amounts of PGF3{alpha} resulted in a cross-reactivity of 39.8%. Cross-reactivity with PGE3 was less than 1%. Concentrations of PGF3{alpha} used in the test of cross-reactivity were 0.625, 1.25, 2.5, 5, 10, and 20 ng/ml. Concentrations of PGE3 tested were 20, 40, 80, and 100 ng/ml. Concentrations of PGF2{alpha} are reported as picograms per milliliter of culture medium because treatments did not affect cell numbers, as determined by protein yield (experiments 6 and 8).

Statistical Analyses

Data were analyzed by least-squares ANOVA using the general linear models procedure of SAS [23]. Data for each experiment were analyzed separately. The PGF2{alpha} data were analyzed as a split-plot design. Models included the effects of treatment well (or dish) within treatment, time, interaction between time and treatment, and residual error. The effect of well (or dish) within treatment was used as the error term to test for the effects of treatment. Preplanned orthogonal contrasts and pairwise comparisons were used to identify differences between means. Concentrations of PGF2{alpha} in medium at 6 h after addition of PDBu were greater than at 3 h (P < 0.01), and the pattern of responses among treatments were comparable at 3 and 6 h. The 6-h responses for all experiments are reported. For abundance of PGHS-2 and cPLA2, the models included the effect of treatment only. Values of PGHS-2 mRNA abundance in experiment 7 were adjusted for variability of loading using the abundance of 18s rRNA as a covariate. Preplanned pairwise comparisons were used to determine differences between means.

The test of homogeneity of polynomial regression [24] was used in experiment 2 to compare the inhibition curves obtained using increasing concentrations of EPA, DHA, and LNA.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of Polyunsaturated Fatty Acids on Secretion of PGF2{alpha}

Incubation for 24 h with 100 µM of the omega-3 fatty acids EPA, DHA, and LNA significantly attenuated secretion of PGF2{alpha} (P < 0.05) in response to PDBu when compared with incubation without fatty acid (Fig. 1). Arachidonic acid (100 µM) had the opposite effect and tended to increase secretion of PGF2{alpha} in response to PDBu (P = 0.1). The other fatty acids tested (100 µM), OA and LA, did not affect production of PGF2{alpha} (P > 0.05). Incubation with BSA without fatty acid did not affect secretion of PGF2{alpha} in response to treatment with PDBu. Cells incubated with BSA and not stimulated with PDBu did not secrete detectable amounts of PGF2{alpha}.



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  		FIG. 1. Experiment 1. Least-square means and SEM of concentrations of PGF2{alpha} in medium conditioned by BEND cells 6 h after treatment with PDBu (100 ng/ml). Cells were incubated with no fatty acid (Control) or with 100 µM EPA, DHA, AA, LA, LNA, or OA for 24 h before treatment with PDBu. A second control group was included to test the effect of BSA used to solubilized the fatty acids. The BSA did not affect PGF2{alpha} secretion. Differences between different fatty acids and the Control: aP < 0.10, *P < 0.05; and **P < 0.01

In experiment 2, incubation with increasing concentrations (dose) of n-3 PUFAs resulted in a dose-responsive reduction in secretion of PGF2{alpha} (P < 0.001). Treatment with 20 µM of any of the three fatty acids resulted in lower secretion of PGF2{alpha} in comparison to the control without fatty acid (P < 0.02). Treatment with EPA [Y = 6078 - (225 x dose) + (3.4 x dose2) - (0.016 x dose3); R2 = 0.92] and DHA [Y = 5930 - (212 x dose) + (3.1 x dose2) - (0.015 x dose3); R2 = 0.95] resulted in clear dose responses and greater inhibition of PGF2{alpha} secretion than with LNA [Y = 6067 - (96.6 x dose) + (1.52 x dose2) - (0.008 x dose3); R2 = 0.95; P < 0.01] (Fig. 2). Treatment with 20 µM LNA, DHA, or EPA resulted in a 22% (4835 pg/ml, P = 0.04), 60% (2464 pg/ml, P < 0.01), and 61% (2423 pg/ml, P < 0.01) inhibition of PGF2{alpha} secretion at 6 h in comparison to the control without fatty acid (6203 pg/ml, SEM = 381), respectively. Results from experiment 2 confirm the findings of experiment 1 for EPA, DHA, and LNA. When tested at a lower dose range (experiment 3), incubation for 24 h with EPA reduced secretion of PGF2{alpha} in response to PDBu in a dose-dependent manner (P < 0.01). Treatment with 6.25, 12.5, and 20 µM EPA resulted in an inhibition of PGF2{alpha} secretion at 6 h of 24.5% (3667 pg/ml, P = 0.02), 22.3% (3733 pg/ml, P = 0.03), and 46.7% (2590 pg/ml, P < 0.01), respectively, when compared to the control without fatty acid (4859 pg/ml, SEM = 299).



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  		FIG. 2. Experiment 2. Prostaglandin F2{alpha} response curves for increasing concentrations (0, 20, 40, 60, 80, or 100 µM) of the fatty acids EPA, DHA, or LNA. Concentrations were measured in medium conditioned by BEND cells 6 h after treatment with PDBu (100 ng/ml). Cells were incubated for 24 h before treatment with PDBu in a 3 x 6 factorial design. Both EPA and DHA were more potent inhibitors than LNA (P < 0.01)

The inhibitory effect of EPA on secretion of PGF2{alpha} induced by PDBu increased with increasing time of exposure of the cells to the medium containing 100 µM EPA (P < 0.01, experiment 4; Fig. 3).



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  		FIG. 3. Experiment 4. Least-square means and SEM of concentrations of PGF2{alpha} in medium conditioned by BEND cells 6 h after treatment with PDBu (100 ng/ml). Cells were incubated with the fatty acid EPA (100 µM) for 0 (Control), 1, 3, 6, 12, or 24 h before treatment with PDBu. Secretion of PGF2{alpha} in response to PDBu decreased with increasing incubation time (P < 0.01). Differences between different incubation times and the Control: aP < 0.1, *P < 0.01, and **P < 0.001

Treatment of cells with PDBu in combination with increasing concentrations of bIFN-{tau} (experiment 5) resulted in a dose-dependent decrease in concentrations of PGF2{alpha} (P < 0.001). Concentrations of PGF2{alpha} in the medium of cells treated with bIFN-{tau} at 0.01, 0.1, 0.5, 1, 2, 3.125, 12.5, or 20 ng/ml were 6570, 4485, 3314, 2388, 2628, 1793, 966, and 1131 pg/ml (SEM = 398), respectively. Inhibition of PGF2{alpha} secretion was significant at all concentrations of bIFN-{tau} tested when compared to the control without bIFN-{tau} (P < 0.01).

In Experiment 6, both incubation for 24 h with EPA (P < 0.002) and subsequent treatment with bIFN-{tau} (P < 0.001) inhibited secretion of PGF2{alpha} in response to PDBu (Fig. 4). No interaction between bIFN-{tau} and EPA was detected, indicating that EPA and bIFN-{tau} acted in an additive manner. Treatments did not affect concentrations of protein measured in WCEs collected at 6 h.



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  		FIG. 4. Experiment 6. Least-square means and SEM of concentrations of PGF2{alpha} in medium conditioned by BEND cells 6 h after treatment with PDBu (100 ng/ml). Cells were assigned to a 3 x 3 factorial experiment. Factors were incubation for 24 h with 0, 3, or 20 µM EPA and treatment with PDBu in combination with 0, 50 or 100 pg/ml of bIFN-{tau}. Both EPA and bIFN-{tau} attenuated the secretion of PGF2{alpha} in response to PDBu. Both EPA and bIFN-{tau} inhibited secretion of PGF2{alpha} (P < 0.01). The interaction between EPA and bIFN-{tau} was not significant

Effects of Polyunsaturated Fatty Acids and bIFN-{tau} on Steady-State Concentrations of PGHS-2 mRNA: PGHS-2 Northern Blot Analysis

Incubation of cells with EPA for 24 h did not affect PGHS-2 mRNA concentrations (P > 0.1). However, bIFN-{tau} reduced PGHS-2 mRNA concentrations in a dose-responsive manner (P < 0.01, experiment 7; Fig. 5). Reduction of PGHS-2 mRNA abundance relative to the control not treated with bIFN-{tau} was 26.5% and 90.7% for 0.5 and 50 ng/ml of bIFN-{tau}, respectively. Both EPA and bIFN-{tau} attenuated the secretion of PGF2{alpha} at 6 h after addition of PDBu (P < 0.01). Concentrations of PGF2{alpha} in medium from controls (no EPA and no bIFN-{tau}) were greater (5504.3 ± 217 pg/ml, P < 0.01) than those in medium from cells treated with 20 µM EPA (3100 ± 307 pg/ml), 100 µM EPA (1777 ± 307 pg/ml), 0.5 ng/ml of bIFN-{tau} (2240 ± 307 pg/ml), or 50 ng/ml of bIFN-{tau} (334 ± 307 pg/ml).



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  		FIG. 5. Experiment 7. Northern blot analysis of PGHS-2 mRNA in BEND cells 6 h after treatment with PDBu (100 ng/ml). Cells were incubated with EPA (20 or 100 µM) for 24 h before treatment with PDBu or were treated with PDBu in combination with bIFN-{tau} (0.5 or 50 ng/ml). Controls were treated with PDBu alone. Top) Autoradiographic exposure of abundance of PGHS-2 mRNA (upper row) and ethidium bromide staining of 18S ribosomal RNA (lower row). Bottom) Least-square means and SEM of abundance of PGHS-2 mRNA arbitrary densitometric units. Details of statistical differences are described in Results. The bIFN-{tau}, but not the EPA, attenuated the increase in abundance of PGHS-2 mRNA induced by PDBu (P < 0.01)

Effects of Polyunsaturated Fatty Acids on PGHS-2 and cPLA2 Protein Expression: PGHS-2 and PLA2 Immunoblotting

Concentrations of PGF2{alpha} in medium at 6 h after PDBu were increased by incubation for 24 h with AA (1991 ± 84.2 pg/ml, P < 0.01) and decreased (P < 0.01) by EPA (567 ± 84.2 pg/ml) and DHA (756 ± 84.2 pg/ml) relative to the control with PDBu alone (1454 ± 84.2 pg/ml). Concentrations of PGHS-2 were not affected by any of the fatty acids tested (P > 0.05). Treatment with DHA tended to reduce the abundance of PLA2 (P = 0.08, experiment 8; Fig. 6). Treatments did not affect concentrations of protein measured in WCEs collected at 6 h.



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  		FIG. 6. Experiment 8. Immunoblot analysis of PGHS-2 and PLA2 in whole-cell extracts collected from BEND cells 6 h after treatment with PDBu (100 ng/ml). Cells were incubated for 24 h before PDBu with the fatty acids EPA (100 µM), DHA (100 µM), or AA (100 µM). a) Representative-enhanced ECL exposure of abundance of total PGHS-2 (top) and least-square means ± SEM of abundance of total PGHS-2 arbitrary densitometric units (ADU; bottom). b) Representative-enhanced ECL exposure of abundance of total PLA2 (top) and least-square means ± SEM of abundance of total PGHS-2 ADU (bottom). Incubation with DHA tended to decrease abundance of PLA2. Std, Standard. *P = 0.08

Effect of Coincubation with AA and EPA on PGF2{alpha} Secretion

Coincubation of BEND cells with AA and EPA for 24 h (experiment 9) demonstrated the competing effects of the two fatty acids. Arachidonic acid increased (P < 0.01) secretion of PGF2{alpha} in response to PDBu, whereas EPA was inhibitory (P < 0.01; Fig. 7). The response to incubation with 25 µM AA tended to decrease with increasing concentrations of EPA (P = 0.09, EPA x AA interaction). Incubation with 100 µM EPA resulted in a 72.4% (2198 pg/ml) inhibition of PGF2{alpha} secretion relative to the untreated control (7960 pg/ml), but this inhibitory effect was reversed with coincubation of EPA (100 µM) with AA (100 µM), causing the concentrations of PGF2{alpha} in the medium to be similar to that of the control (6715 pg/ml, P > 0.05).



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  		FIG. 7. Experiment 9. Least-squares means and SEM of concentrations of PGF2{alpha} in medium conditioned by BEND cells 6 h after treatment with PDBu (100 ng/ml). Cells were assigned to a 3 x 3 factorial experiment. Factors were incubated for 24 h with 0, 25, or 100 µM EPA or AA. Secretion of PGF2{alpha} in response to PDBu was attenuated by EPA (P < 0.01) and increased by AA (P < 0.01). The interaction between EPA and AA was not significant


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The experiments described indicate that PUFAs can modify the secretion of PGF2{alpha} in a model in which BEND cells are stimulated with PDBu. Binelli et al. [16] reported that PDBu (100 ng/ml) increases steady-state concentrations of PGHS-2 mRNA and protein and induces secretion of PGF2{alpha}. Moreover, it was determined that bIFN-{tau} (50 ng/ml) suppresses PGF2{alpha}, probably through reduction of PGHS-2 mRNA concentrations, protein expression, and enzymatic activity of PGHS-2 and PLA2. A control without PDBu was run only in experiment 1, because that experiment demonstrated that incubation of cells with PUFA did not induce secretion of PGF2{alpha}. Concentrations of PGF2{alpha} were undetectable in that group. In a previous report [16], maximal induction of PGF2{alpha} secretion was detected between 3 and 6 h after treatment of cells with PDBu and coincided temporally with an increase in the intracellular mass of PGHS-2 protein. Based on these results, the present experiments were designed to determine the effects of fatty acids on secretion of PGF2{alpha} and enzyme expression (PGHS-2 and PLA2) at 6 h after treatment with PDBu.

The antibody used to determine concentrations of PGF2{alpha} also had a significant cross-reactivity (39.8%) for PGF3{alpha}. Although we were unable to separate the contribution of PGF3{alpha} to the PGF2{alpha} measurements, any increased production of PGF3{alpha} as a result of incubation with EPA or other n-3 fatty acids would have reduced the apparent differences of PGF2{alpha} concentrations measured by RIA between cells treated with PDBu and EPA and cells treated with PDBu alone. In other words, if EPA or other n-3 fatty acids indeed increased production of PGF3{alpha}, then the real differences in production of PGF2{alpha} between controls treated with PDBu alone and cells incubated with EPA or other n-3 fatty acids were greater than those detected by the RIA.

Incubation of cells with culture medium containing a fatty acid bound to BSA increases the concentration of that particular fatty acid in the plasma membrane [25]. Experiment 4 demonstrated that the inhibitory effect of EPA on secretion of PGF2{alpha} increased with longer exposure of cells to EPA. Although incorporation of fatty acids to the plasma membrane was not measured in the present study, the mass of EPA incorporated to the plasma membrane likely increased with increasing incubation time, and this greater incorporation likely resulted in greater inhibition of PGF2{alpha} secretion. It is possible that longer incubation periods would result in a greater inhibition of PGF2{alpha} secretion. Therefore, it is also possible that the effects described in experiments 1–3 and 6–9 could have been attained using lower doses and longer incubation times.

In experiments 1 and 2, incubation of BEND cells with the n-3 PUFAs EPA, DHA, and LNA caused a dose-dependent reduction of PGF2{alpha} secretion in response to stimulation with PDBu. Incubation for 24 h with EPA was significant even at the lowest concentration tested (6.25 µM, experiment 3). These findings are in agreement with reports indicating that PUFAs of the n-3 and n-6 families can inhibit the secretion of prostanoids in several cell types cultured in vitro [7, 26]. Other studies have demonstrated reduced prostanoid synthesis when fatty acids of the n-3 and n-6 families are fed in the diet [27]. It is not clear why LNA was less inhibitory than DHA and EPA. Linolenic acid is the precursor for synthesis of DHA and EPA and can be converted to them in a process that relies on activities of desaturase and elongase enzymes. It is possible that some conversion of LNA to EPA and DHA occurred during the period of incubation, leading to the observed reduction in PGF2{alpha} secretion. The fact that LA did not affect secretion of PGF2{alpha} in response to PDBu somewhat contradicts previous reports indicating an inhibitory activity of this fatty acid [28, 29]. Moreover, LA has been considered to be a potential mediator of reduced endometrial PGF2{alpha} secretion in the pregnant cow [1]. Conversely, because LA is the most abundant precursor for synthesis of AA and PGF2{alpha}, it could be hypothesized that LA would increase secretion of PGF2{alpha} through increased precursor availability. This did not occur in the BEND cell system. Possible reasons could involve lack of an efficient system for conversion of LA to AA, which involves two steps of desaturation and one step of elongation. The fact that incubation with 100 µM AA increased production of PGF2{alpha} (experiments 1 and 8) supports the concept that availability of AA limits secretion of PGF2{alpha} and that conversion of LA to AA does not occur efficiently in the cell culture system used. Oleic acid was included as a fatty acid control that is not a substrate for the PGHS enzymes and does not interfere with synthesis of prostanoids [7]. Oleic acid did not affect secretion of PGF2{alpha} in response to stimulation with PDBu.

Experiment 6 demonstrated that EPA can amplify the inhibitory effects of bIFN-{tau} on secretion of PGF2{alpha} in an additive manner. The concentrations of EPA and bIFN-{tau} included in experiment 6 were based on the results of experiments 3 and 5, which determined concentrations of EPA and bIFN-{tau} that partially inhibit secretion of PGF2{alpha}. Utilizing these partially inhibitory doses, a significant amplification in PGF2{alpha} inhibition was detected in experiment 6 by addition of an equivalent dose of the other inhibitor. The implication of this finding is that supplementation with inhibitory fatty acids, such as EPA, during early pregnancy by dietary or parenteral means may further enhance the suppression of PGF2{alpha} secretion in concert with the action of embryonic bIFN-{tau}. Because a significant proportion of bovine embryos is thought to be lost because of inadequate inhibition of uterine PGF2{alpha} secretion, further inhibition by exogenous means may result in increased embryo survival. This hypothesis is supported by the study of Burke et al. [30], in which feeding a source of EPA and DHA, fish meal, to lactating dairy cows resulted in increased pregnancy rates.

Results from experiments 7 and 8 indicated that EPA and bIFN-{tau} inhibit secretion of PGF2{alpha} through different mechanisms. Bovine interferon-{tau}, but not EPA, reduced steady-state concentrations of PGHS-2 mRNA. In experiment 7, the degree of inhibition relative to the control without bIFN-{tau} attained after treating cells with 0.5 ng/ml of bIFN-{tau} was greater for concentration of PGF2{alpha} in the medium (60%) than for abundance of PGHS-2 mRNA (26.5%). The evidence that bIFN-{tau} reduces PGF2{alpha} secretion through regulation of steady-state concentrations of PGHS-2 mRNA does not preclude other possible mechanisms. Inhibition of PGF2{alpha} secretion by bIFN-{tau} is consistent with a previous report using similar concentrations of bIFN-{tau} (50 ng/ml) and PDBu (100 ng/ml) [16]. Conversely, PGHS-2 mRNA concentrations were up-regulated by bIFN-{tau} at high doses (1–20 µg/ml) [31]. This differential effect likely is related to the different doses of bIFN-{tau}, indicating that bIFN-{tau} exerts a complex regulation of PGHS-2 gene expression and activity to modulate PGF2{alpha} production. Incubation for 24 h with EPA did not affect steady-state concentrations of PGHS-2 mRNA or intracellular abundance of PGHS-2 and PLA2 proteins. Similarly, concentrations of PGHS-2 were unaffected by AA and those of PLA2 only slightly decreased by DHA. Collectively, these findings suggest that the effects of EPA and AA on secretion of PGF2{alpha} are mediated by mechanisms that involve competition of precursors for processing by the PGHS enzymes and regulation of enzyme activity.

Experiment 9 demonstrated that incubation of BEND cells with EPA or with AA has opposite effects on secretion of PGF2{alpha} and supported the concept of competition for processing by the enzymes involved in prostanoid synthesis. The reduced secretion of PGF2{alpha} observed in cells incubated with EPA likely is the result of a shift of the PGHS pathway from synthesis of prostanoids from the 2 series to synthesis of prostanoids from the 3 series. The cyclooxygenase reaction has unique characteristics in its absolute requirement for hydroperoxide to stimulate its activity and in its suicide reaction that causes self-inactivation after approximately 1400 catalytic turnovers [32]. The PGHS enzymes differ in their ability to process different substrates. Eicosapentaenoic acid has been shown to be a poor substrate for PGHS-2, probably because the cyclooxygenase activity occurs in an inefficient manner, causing insufficient self-activation of the cyclooxygenase domain [8]. This mechanism of enzyme inhibition could be another factor causing reduced secretion of PGF2{alpha}.


   ACKNOWLEDGMENTS
 
The authors would like to thank Dr. R. Michael Roberts for providing the recombinant bIFN-{tau} and Dr. Kevin Fritsche for providing the method of fatty acid enrichment of cells.


   FOOTNOTES
 
1 This research was supported in part by the NRI Competitive Grant Program/USDA, grant 98-35203-6367. This is Florida Agricultural Experimental Station Journal Series R-09552. Back

2 Correspondence: William W. Thatcher, Department of Animal Sciences, University of Florida. P.O. Box 11910, Gainesville, Florida 32611-0910. FAX: 352 392 5595; thatcher{at}animal.ufl.edu Back

Received: 30 December 2002.

First decision: 19 January 2003.

Accepted: 22 April 2003.


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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