Fresh semen was collected from three Danish Duroc boars and centrifuged at 2000 x g for 20 minutes at 4˚C. The supernatant was centrifuged again; the seminal plasma was pooled, collected to 15-ml tubes with caps and stored at −20˚C. The total volume of collected seminal plasma was approximately 150 ml.

Seminal plasma proteins were separated on column HiPrep 16/10 Heparin FF, 20 ml (GE Healthcare BioSciences AB, Uppsala, Sweden) by fast protein liquid chromatography (FPLC) according to Sanz, et al. [12] and Varilova, et al. [24] with some modifications. The column was used directly on ÄKTAdesign™ systems (Amersham Pharmacia Biotech, Uppsala, Sweden) with UNICORN™ software for data calculations on line. Samples of seminal plasma (0.5 ml) were injected through a valve with 0.5 ml sampling loop. The non-heparin-binding proteins, the first peak (F1) were eluted with 0.02 M Tris-HCl buffer containing 0.156 M NaCl, pH 7.5. The proteins adsorbed on heparin, the second peak (F2), were eluted using NaCl gradient (within 10 min) from 0.156 M to 1.5 M in 0.02 M Tris-HCl buffer, pH 7.5. The used flow rate was 1 ml/min. The yield of eluted proteins was evaluated by measurement of absorbance at 280 nm and 2 ml fractions were collected, lyophilized and stored at −20˚C.

In Experiments I-III, the uterus and cervix were collected from gilts slaughtered approximately one week after (gilt no. g 08) and before oestrus (gilt no. g11) in a local abattoir.

The cells were separated as described earlier [25-28] and recently modified by us [1]. Briefly, uterus and cervix were transported to the laboratory within 60 min after slaughter in ice-cold phosphate buffered saline (PBS), pH 7.4, saturated with O₂:CO₂ (95%:5%) and supplemented with antibiotics. After digestion of the uterine tissues with 0.48% dispase in Hank’s incomplete balanced salt solution (HIBSS) with antibiotics, the endometrial epithelial (pUE) cells were centrifuged and washed once with Medium 199. Afterwards, red blood cells lysing buffer (RBCLB) was used to remove erythrocytes. The cells were washed and plated in T-125 flasks for culture at 37˚C in a humidified atmosphere of 95% air: 5% CO₂. The culture medium Medium 199 containing 2% BSA, 10% foetal bovine serum (FBS), penicillin-streptomycin (100 IU/ml - 100 μg/ml), gentamycin (50 μg/ml) and amphotericin B (1 μg/ml) was changed every second day until cells were confluent (5 - 7 days). In order to obtain endometrial stromal (pUS) cells, the remaining tissue was first incubated with 0.25% trypsin solution for 1 hour at 37˚C, filtered (40-μm nylon sieve), centrifuged and washed with fresh medium. The undigested tissue fragments were further treated with 0.06% collagenase for 1.5 h at 37˚C. The cell suspensions obtained after trypsin and collagenase digestions were pooled together, washed, counted and plated in T-125 flasks for culture at 37˚C in humidified atmosphere of 95% air: 5% CO₂. The culture medium with additives as described for pUE above was changed every second day until the cells were confluent (5 - 7 days).

The cervix was treated with a solution of dispase and pancreatin (0.48% and 1.25%, respectively) in HIBSS solution containing antibiotics. After this treatment the remaining tissue samples were incubated with a 0.06% collagenase, 0.01% DNase, 0.06% trypsin in HIBSS solution containing antibiotics to obtain cervical stromal (pCS) cells. The undigested fragments were filtered through 200-μm and 40-μm pore-size stainless steel and nylon sieves. The pCS cells which passed through the sieves were incubated with the lysing buffer RBCLB, washed and plated in T-125 flasks for culture at 37˚C in a humidified atmosphere of 95% air: 5% CO₂. The culture medium Dulbecco’s Modified Eagle`s Medium Nutrient Mixture F-12 HAM (DMEM-F-12) containing 2% BSA, 10% FBS penicillin-streptomycin (100 IU/ml - 100 μg/ml), gentamycin (100 μg/ml) and amphotericin B (1 μg/ml) was changed every second day until the cells were confluent (5 - 7 days). Cryopreservation and thawing of separated cervical stromal cells from gilt no. g08 (pCS-80) was performed as described earlier in our work [1].

The trypan blue exclusion method was applied for counting of the obtained cells. All types of cells were repeatedly controlled for growth and morphology using an inverted microscope (Telaval 31, Carl Zeiss Jena GmbH, Jena, Germany) until they attached and reached confluence. The viability of cells varied between 80 and 90%. The cells cultured in T-125 flasks were removed by treatment with trypsin—EDTA (0.5% - 0.2%) solution, counted and plated on 24-well plates (seeding density 2.5 x 10⁵ cells/well) and cultured at 37˚C in a humidified atmosphere of 95% air: 5% CO₂ until 80% - 90% confluence was reached and experiments could be started. The immunofluorescent staining for specific markers of epithelial cells (cytokeratin) and for stromal cells (vimentin) was applied as described by Blitek and Ziecik [28].

Bovine endometrial epithelial cells (commercial name —BEnEpC) derived from healthy bovine uterus includeing cell growth media and subculture reagent kit were purchased from Cell Applications, Inc., San Diego, USA, and cultured according to the recommendations of the manufacturer. These bovine cells shipped at 2nd passage were propagate to 4th passage and used in Experiment IV.

Porcine UE, US and CS cells were cultured for 3 and 24 h with heparin-binding F2 proteins (125 µg/well) added to the culture medium. The total incubation volume was 1.0 ml/well. Controls with or without these proteins, AA (20 μg/ml) and TNFα (1.05 ng/ml) were always in triplicate. The test was repeated 2 or 3 times. The collected media were analysed for the content of PGF_2α and IL-6.

Cryopreserved and thawed pCS-80 cells were cultured for 3 h with heparin-binding F2 proteins at different concentrations (0 - 132 µg/well) with 20.0 µg/ml of AA in triplicates. The test was repeated 2 or 3 times. The collected media were analyzed or the content of PGF_2α, PGE₂ and IL-6.

Freshly collected porcine CS cells from gilt no. g11 (pCS-g11) were cultured for 3 h with heparin-binding F2 proteins at different concentrations (0 - 190.8 µg/well) with 20.0 µg/ml of AA in 6 replicates. The collected media were analyzed for the content of PGF_2α, PGE₂ and IL-6.

Bovine cells were cultured for 3 and 24 h with heparin-binding F2 proteins (125 µg/well) added to the culture medium. The total incubation volume was 1.0 ml/well. Controls with or without these proteins, AA (20 μg/ml) and TNFα (1.05 ng/ml) were always in triplicate. The test was repeated 2 - 3 times. The collected media were analysed for the content of PGF_2α and IL-6.

When incubation time was complete, the medium from each well in all experiments was collected to tubes containing 10 µg/ml of indomethacin (Fluka, BioChemika, Switzerland) and stored at −20˚C. The remaining cells on the bottom of the well were then lysed with 0.1 N NaOH (0.5 ml/well), for the total cellular protein determination, and stored at −20˚C.

The quantitative determination of PGF_2α and PGE₂ in collected media was performed by competitive immunoassay kits, the Assay Designs’ Correlate-EIA™ Prostaglandin F_2α and the Assay Designs’ Correlate-EIA™ Prostaglandin E₂, respectively, according to the manufacturer’s recommendations (Assay Designs, Inc., Ann Arbor, MI, USA). The intra-assay coefficient of variation for PGF_2α was below 15% between 10.0 and 5000 pg/ml. The inter-assay coefficients of variations were 12.5% at 170.7 pg/ml, 7.2% at 1814 pg/ml and 10.2% at 17,573 pg/ml. The practical limit of the assay sensitivity for 0.05 ml of medium was approximately 7 pg⁄ml. The intraassay coefficient of variation for PGE₂ was below 10% between 120.1 and 4689 pg/ml. The inter-assay coefficient of variation was approximately 13% at 2330.2 pg/ml. The practical limit of the assay sensitivity for 0.05 ml of medium was approximately 13 pg⁄ml.

Porcine IL-6 (pIL-6) was measured by using solid phase ELISA (R&D Systems Europe, Ltd. Abingdon, UK) according to the manufacturer’s recommendations. No significant relative cross-reactivity was observed with TNFα. The inter-assay coefficient of variation was approximately 14.3% at 251.7 pg/ml. The practical limit of sensitivity for the assay analysing 0.1 ml of medium was approximately 10 pg⁄ml. Bovine IL-6 (bIL-6) was measured using the xMAP™ technology as applied by Dernfalk, et al. [29] and modified by Madej, et al. [1].

The total protein content of the cells in each well was determined with the Bio-Rad Protein Microassay kit (Bio-Rad Laboratories, Life Science Group, Sundbyberg, Sweden) according to the manufacturer’s recommendations.

All chemicals used in the present study were purchased from Sigma-Aldrich Sweden AB, Stockholm, Sweden, unless otherwise stated.