Heparin-binding proteins from boar seminal plasma affecting the release of prostaglandins and interleukin-6 by porcine endometrial and cervical cells and bovine endometrial cells

Malgorzata Madej, Claus Hansen, Anders Johannisson, Andrzej Madej

Danish Agriculture and Food Council, Copenhagen, Denmark.
Department of Anatomy, Physiology and Biochemistry, Swedish University of Agricultural Sciences, Uppsala, Sweden.

DOI: 10.4236/ns.2013.57A004                

Abstract

The objectives of this study were to explore whether heparin-binding proteins, separated by fast protein liquid chromatography from boar seminal plasma influence the release of prostaglandins F_2α, (PGF_2α), E₂ (PGE₂) and interleukin-6 (IL-6) by porcine endometrial and cervical cells and even bovine endometrial cells. In Experiment I, we showed that release of PGF_2α by endometrial epithelial, endometrial stromal and cervical stromal cells to the medium was inhibited (p < 0.05) to 9.0% - 60.6% after 24 h incubation with 125 μg of heparin-binding proteins. Tumor necrosis factor α (TNFα) stimulated release of IL-6 by endometrial and cervical stromal cells after 24 h incubation, but in the presence of heparin-binding proteins, this stimulation was attenuated. Release of PGF_2α by cryopreserved (Experiment II) and primary (Experiment III) cervical stromal cells was significantly inhibited after 3 h incubation with 66 - 95.4 μg of heparin- binding proteins. A significant inhibition of PGE₂ release by cryopreserved and primary cervical stromal cells was already achieved after incubation with 16.5 - 23.9 μg of heparin-binding proteins. The release of IL-6 by cryopreserved cells was stimulated after 3 h incubation with heparin- binding proteins in a dose dependent manner in contrast to the release of IL-6 by freshly isolated cervical stromal cells. We also found (Experiment IV) that porcine heparin-binding seminal plasma proteins inhibited release of PGF_2α and stimulated release of IL-6 by bovine endometrial epithelial cells. In conclusion, a group of heparin-binding proteins separated by fast protein liquid chromatography from boar seminal plasma inhibit PGF_2α, PGE₂ and stimulate IL-6 release by porcine endometrial and cervical cells and even by bovine endometrial cells. Thus, these proteins have a similar effect as the entire seminal plasma.

Keywords

Seminal Plasma Proteins; Cell Culture; Porcine; Bovine; Prostaglandins, Interleukin-6

1. INTRODUCTION

The potent biological activity of boar seminal plasma to inhibit the release of prostaglandins and to stimulate IL-6 release by porcine endometrial, cervical and bovine endometrial cells has been demonstrated previously [1]. It is well known that prostaglandins F_2α and E₂, which are of endometrial origin, regulate a number of important physiological functions during the estrous cycle and pregnancy in pigs [2,3]. These two prostaglandins are also involved in acute and chronic inflammatory conditions [4,5]. Seminal plasma suppresses the migration of polymorphonuclear neutrophil granulocytes (PMNs) into the uterus, decreases PMNs infiltration of the endometrium and enhances the rate of disappearance of uterine inflammation in gilts following breeding [6,7]. Rozeboom, et al. [8] suggested that non-estrogenic factors in seminal plasma may protect spermatozoa from an inflamed uterine environment and consequently a suitable population of viable spermatozoa is able to reach the site of fertilization. Porcine SP does not activate neutrophil chemotaxis, but it might play a different role in the modulation of post breeding uterine neutrophil migration compared to spermatozoa in the sow [9]. There are also data that showing that SP induced the inflammatory response of the pig endometrium during mating or artificial insemination (AI) [10,11]. Boar SP contains two groups of proteins belonging to the spermadhesin family classified according to their affinity to heparin i.e. heparinbinding and non-heparin-binding [12-16]. RodriguezMartinez, et al. [17] studied the effects of these two groups of proteins on immunological events in the uterine lumen of sows. These authors found that only nonheparin binding spermadhesin PSP-I/PSP-II heterodimer induced migration of PMNs into the uterine cavity of anesthetized and conscious estrous sows, presumably in relation to specific cytokines. The presence of different groups of proteins in boar SP became a key issue for their relationship to fertility [18]. Novak, et al. [19] reported that non-heparin-binding spermadhesin PSP-I abundance was negatively correlated with both in vivo and in vitro fertility. Furthermore, heparin-binding spermadhesin AWN-1 seems to be of importance for future investigation as biological marker of fertility in boars [20]. Our previous observations that AI resulted in an increase in the prostaglandin F_2a metabolite (PGFM) concentrations in sows, whereas in mated sows no such elevation in PGFM occurred [21-23] led us to the hypothesis that seminal plasma might be responsible for the inhibition of prostaglandins. To test our hypothesis we have done a series of experiments in vitro [1] and demonstrated that boar seminal plasma inhibit prostaglandins (PGFM, PGF_2α, PGE₂) release and stimulate IL-6 release by not only porcine endometrial and cervical cells but also by bovine endometrial cells. Thus, the aim of the present study was to investigate the effect of heparin-binding proteins separated by fast protein liquid chromatography (FPLC) from boar SP on the release of PGF_2α, PGE₂ and IL-6 from porcine endometrial and cervical cells and bovine endometrial cells.

2. MATERIALS AND METHODS

2.1. Separation of Proteins from Boar Seminal Plasma

2.1.1. Seminal Plasma

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.

2.1.2. Affinity Liquid Chromatography of Seminal Plasma

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.

2.2. Cell Culture

2.2.1. Animals

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.

2.2.2. Isolation, Culture and Cryopreservation of Porcine Endometrial and Cervical Cells

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].

2.2.3. Bovine Endometrial Epithelial Cells

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.

2.3. Experiments

2.3.1. Experiment I—Effect of SP Proteins on Release of PGF_2α and IL-6 by Primary Porcine Cells from Gilt No. g08

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.

2.3.2. Experiment II—Effect of SP Proteins on Release of PGF_2α, PGE₂ and IL-6 by Cryopreserved and Thawed Porcine Cervical Stromal Cells from Gilt No. g08 (pCS-80)

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.

2.3.3. Experiment III—Effect of SP Proteins on Release of PGF_2α, PGE₂ and IL-6 By Primary Porcine Cervical Stromal Cells from Gilt No. g11

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.

2.3.4. Experiment IV—Effect of SP Proteins on Release of PGF_2α and IL-6 by Bovine Cells (BEnEpC)

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.

2.4. Biochemical Measurements

2.4.1. Prostaglandin F_2α and Prostaglandin E₂

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.

2.4.2. Porcine and Bovine IL-6

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].

2.4.3. Protein Analysis

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.

2.5. Statistical Analysis

In Experiment I-II and IV, F2 (125 µg/well), controls with or without AA and TNFα were in triplicate and tests were repeated 2 or 3 times. In Experiment III, F2 (0 - 190.8 µg/well) and controls with or without AA were either in triplicates or six replicates. Prostaglandin F_2α, E₂ and IL-6 output data in 3 and 24 h post-treatment media were expressed per μg of cellular proteins. Data were log transformed and analysed with a one-way ANOVA followed by Newman-Keuls Multiple Comparison Test for multiple groups to compare the differences between treatment groups according to the GraphPad Prism version 5.02 for Windows (GraphPad Software, San Diego, CA, USA). Data are presented as a mean ± SEM.

3. RESULTS

3.1. Affinity Liquid Chromatography

Figure 1 depicts a typical chromatogram of boar SP using an affinity stationary phase with immobilized heparin on the column HiPrep 16/10 Heparin FF. Based on the total protein content (16.4 mg/ml) and recovery from FPLC (63.9%) we estimated that each ml of this pooled seminal plasma consists of 13.0 mg non-heparinbinding proteins (F1) and 3.4 mg heparin-binding proteins (F2). In total, 196 mg of F1 and 54 mg of F2 was obtained.

Conflicts of Interest

The authors declare no conflicts of interest.

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