Cytokine 67 (2014) 85–91
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Ovarian steroid-dependent tumor necrosis factor-a production and its action on the equine endometrium in vitro Anna Z. Szóstek a, Marek Adamowski a, António M. Galvão a,b, Graça M. Ferreira-Dias b, Dariusz J. Skarzynski a,⇑ a b
Department of Reproductive Immunology and Pathology, Institute of Animal Reproduction and Food Research, Olsztyn, Poland C.I.I.S.A., Faculty of Veterinary Medicine, University of Lisbon, Lisbon, Portugal
a r t i c l e
i n f o
Article history: Received 18 July 2013 Received in revised form 6 February 2014 Accepted 12 February 2014 Available online 15 March 2014 Keywords: Tumor necrosis factor-a Prostaglandin Ovarian steroids Endometrium Mare
a b s t r a c t Tumor necrosis factor-a (TNF) is a cytokine that plays important roles in functions of the endometrium. The aims of this study were to determine whether (i) ovarian steroids modulate TNF production by endometrial cells (Experiment 1); (ii) TNF effects on prostaglandin (PG) production in cultured equine endometrial cells and tissue (Experiment 2). Epithelial and stromal cells were isolated from equine endometrium (Days 2–5 of the estrous cycle; n = 20) and treated after passage 1. In Experiment 1, epithelial and stromal cells were exposed to progesterone (P4; 107 M), 17-b estradiol (E2; 109 M) or P4+E2 (107/ 109 M) for 24 h. Then, TNF mRNA transcription was determined using Real-time PCR. Additionally, TNF protein production was investigated in response to ovarian steroids for 24 h using Enzyme-Linked Immunosorbent Spot (EliSpot). In Experiment 2, epithelial and stromal cells and endometrial explants (mid-luteal phase of the estrous cycle; n = 5) were exposed in vitro to TNF (10 ng/ml) and to oxytocin (OT; positive control; 107 M) for 24 h. The concentrations of PGE2 and PGF2a were determined using a direct enzyme immunoassay (EIA) method. The transcription of prostaglandin-endoperoxide synthase2 (PTGS-2), prostaglandin E2 synthase (PGES) and PGF2a synthase (PGFS) mRNAs in the endometrial explants was determined using Real-time PCR. Results showed that TNF is produced by two types of equine endometrial cells and its production is up-regulated by ovarian steroids (P < 0.05) in stromal cells and by P4 (P < 0.05) and E2 (P < 0.01) in epithelial cells. Epithelial and stromal cells can also produce PG in response to TNF. In endometrial explants, TNF stimulated PGE2 production to a large extent and PGF2a secretion to a lesser extent. These actions are mediated by up-regulation of PG synthases mRNA transcription. The study indicates that TNF production is closely related to ovarian steroid actions and that the interaction between TNF and PG regulates physiologic processes in the equine endometrium. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Tumor necrosis factor-a (TNF) is a non-glycosylated protein with a wide spectrum of bioactivities; most cells show at least some responsiveness to TNF. In general, this cytokine displays a functional duality, being involved in regeneration as well as destruction of tissues [1]. The distinctive effects of TNF are due to two types of TNF receptor which have different intracellular signaling pathways [2]. Tumor necrosis factor-a receptor type I (TNFRI) contains an intracellular death domain which is necessary for signaling pathways associated with apoptosis. This type of receptor is constitutively expressed in most tissues and seems to
⇑ Corresponding author. Tel./fax: +48 89 5393130. E-mail address: [email protected] (D.J. Skarzynski). http://dx.doi.org/10.1016/j.cyto.2014.02.005 1043-4666/Ó 2014 Elsevier Ltd. All rights reserved.
be a key mediator of TNF signaling [2]. In turn, TNFRII is strongly regulated and predominantly expressed in immune cells and its plays a major role in the lymphoid system. Signaling via TNFRII induce apoptosis but also support survival promoting tissue repair and angiogenesis [2]. Tumor necrosis factor-a is a cytokine that plays important autoparacrine roles in female reproduction [3–6]. It is well known that TNF is produced mainly by macrophages [7] and endothelial cells [8]. However, the ability of endometrial cells to produce TNF has been shown in several species [9–11]. Okuda et al. [9] revealed that TNF is co-located mainly in bovine luminal and glandular epithelial and endothelial cells in the estrous cycle. Furthermore, TNF was detected in stromal cell lysates and in conditioned cultured media [9]. In addition, it was confirmed that TNF is expressed to a larger extent in epithelial cells and, to a lesser extent, in stromal and lymphoid cells in human endometrium [10,11].
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The action of TNF-a induced via prostaglandin (PG) stimulation in the corpus luteum (CL), oviduct and endometrium in many domestic species [3–6]. Prostaglandins play crucial roles in the regulation of several reproductive processes such as ovulation, luteolysis, uterine vascularization and maintenance of pregnancy [12]. These molecules are capable of a very broad spectrum of effects modulating a multitude of biological processes. In the PG production cascade, the prostaglandin-endoperoxide synthase (PTGS) enzyme converts arachidonic acid (AA) into prostaglandin (PG)H2 [13]. There are two isoforms of PTGS, designated PTGS-1, which is constitutively expressed, and PTGS-2 which is the inducible isoform. Prostaglandin F2a synthase (PGFS) and PGE2 synthases (PGES) are downstream enzymes which catalyze the conversion of PGH2 into PGF2a or PGE2, respectively. Although Grünig and Antczak [14] have previously demonstrated that TNF is expressed in gravid endometrium and trophoblast in the mare, the knowledge about the role of TNF in the equine endometrium is still insufficient. We have recently shown the immunolocalization of TNF in equine endometrial epithelial luminal and glandular cells as well as in stromal cells [15]. In above-mentioned study TNF protein expression was up-regulated in the mid-luteal and follicular phases of the estrous cycle suggesting sex steroids-dependent mechanisms regulating TNF expression in the equine endometrium [15]. Therefore, the present work attempts to explain whether: (i) ovarian steroids modulate TNF production, and (ii) TNF influences PG secretion and PG synthases mRNA transcription. 2. Materials and methods 2.1. Animals and endometrial tissue collection Uteri (n = 20) were collected post-mortem from cyclic mares at a local abattoir. All procedures for animal handling and tissue collection were approved by the Local Animal Care and Use Committee in Olsztyn, Poland (Agreement No. 51/2011). The mares were healthy as declared by official governmental veterinary inspection. The material was collected within 5 min of an animal’s death. Immediately before death, peripheral blood samples were collected into heparinized tubes (MonovettesÒ-Sarstedt, Numbrecht, Germany) for P4 and 17-b E2 analysis. The phases of the estrous cycle were identified based on P4 and E2 analysis of blood serum and macroscopic observation of the ovaries as described before [16]. A small piece of each endometrium was fixed in buffered 4% paraformaldehyde for histological analysis [17], and for characterization according to the classification system developed by Kenney [18] and extended by Kenney and Doig [19]. Only cells derived from endometria that were class I (no degenerative changes) according to the Kenney [18] classification were assigned to this study. For tissue and cell culture, the entire uterus was collected within 5 min of the animal’s death, placed in sterile, incomplete (Ca2+and Mg2+- free) Hank’s balanced salt solution (HBSS) supplemented with gentamicin (20 lg/mL; Sigma–Aldrich, #G1272) and 0.1% bovine serum albumin (BSA; Sigma–Aldrich, #A9056), kept on ice and transported quickly to the laboratory. 2.2. Epithelial and stromal cell isolation and culture A total of 15 uteri on days 2–5 of the estrous cycle were used. Equine epithelial and stromal cells were isolated following the methodology described previously [20]. The endometrial cells were cultured at 38.5 °C in a humidified atmosphere of 5% CO2 in air. The culture medium was Dulbecco’s modified Eagle’s medium/Nutrient Ham’s F-12 (DMEM/Ham’s F-12; Sigma–Aldrich;
D8900) supplemented with 10% fetal calf serum (FCS; Sigma– Aldrich, Madison, USA; #C6278) and 1% antibiotic and antimycotic solution (Sigma–Aldrich; #A5955); it was changed every 2–3 days. After reaching 90–95% confluence (Day 5 or 7 for epithelial or stromal cells, respectively), the cells were trypsinized as described previously [20]. Next, depending on the experiment, epithelial cells were seeded at a density of 5 105 viable cells/mL and stromal cells at a density of 2 105 viable cells/mL in 24- or 96-well plates. The viability of epithelial and stromal cells was 80% and 90%, respectively. The homogeneity of cell culture was evaluated using immunofluorescent staining for specific markers of epithelial cells (cytokeratin) and stromal cells (vimentin) as described previously [20]. 2.3. Tissue culture A total of 5 uteri at the mid-luteal phase of the estrous cycle were used. This phase of the estrous cycle was chosen based on a previous study [15] which showed that TNF, TNFRI and TNFRII expression was up-regulated at the mid-luteal phase of the estrous cycle. Endometrial explants were minced into small pieces, then 50 mg amounts were washed three times in PBS containing gentamicin (20 lg/lL) and placed into culture tubes, each of which contained 1 mL Dulbecco’s Modified Eagle’s medium without phenol red (Sigma–Aldrich; D#2960) with 0.1% BSA and antibiotic/ antimycotic solution as described above. Tissue explants were preincubated on a shaker inside a tissue culture incubator at 38.0 °C with 5% CO2 in air for 6 h, then the medium was replaced with fresh DMEM supplemented with 0.1% BSA and antibiotics and antimycotic. After this, endometrial tissue was further incubated for 24 h with TNF (10 ng/ml) and OT (107 M; positive control). Finally, viability of endometrial explant cells was confirmed using Alamar Blue according to the manufacturer’s instructions (Invitrogen; Burlington; Ontario, Canada; #DAL1025). 2.4. Experimental procedure 2.4.1. Experiment 1. In vitro production of TNF by endometrial cells 2.4.1.1. Experiment 1.1. Effect of ovarian steroids on TNF mRNA transcription in equine endometrial cells. Stromal (n = 5) and epithelial (n = 5) cells derived from passage 1 were placed in a 24-well plate in DMEM/Ham’s F-12 supplemented with 10% FCS and antibiotic and antimycotic solution. When the cells reached 90% confluence, the medium was replaced with fresh DMEM without phenol red, supplemented with 0.1% BSA and antibiotics and antimycotic. The most effective dose and the optimal treatment time for action of ovarian steroids were established in a preliminary study (data not shown). The epithelial and stromal cells were stimulated with P4 (107 M), E2 (109 M) or P4+E2 (107/109 M) for 24 h. Next, the culture medium was removed and to each well 250 ll of Fenozol was added in order to determine TNF mRNA transcription using Real-time PCR. 2.4.1.2. Experiment 1.2. Effect of ovarian steroids on TNF production by equine endometrial cells. To determine TNF production by endometrial cells, the equine ELISpot system was used (R&D Systems, Minneapolis, USA; #EL1814) following the manufacturer’s instructions. Stromal (n = 5) and epithelial (n = 5) cells derived from passage 1 were seeded at a density of 2 104 per well in a MultiScreen sterile 96-well plate with a PVDF membrane (Millipore) using fresh DMEM without phenol red supplemented with 0.1% BSA and antibiotics and antimycotic solution. The density of cell seeding was established in a preliminary experiment. Then, cells were incubated with vehicle, P4 (107 M), E2 (109 M) or P4+E2 (107/109 M) for 24 h. The following controls were used:
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positive control (TNF); negative control (unstimulated cells at the same density as stimulated cells); background control (culture medium alone) and Detection Antibody control (phosphate buffered saline substituted for the Detection Antibody). Spots were visualized using the BCIP/NBT substrate detection system according to the manufacturer’s instructions. The spots were analyzed using an Eli.Scan scanner and software (A.EL.VIS GmbH; Hannover; Germany). Data were expressed as spot number per 2 104 cells. 2.4.2. Experiment 2. TNF effect on PG production in vitro 2.4.2.1. Experiment 2.1. TNF effect on PG secretion by equine endometrial cells. Epithelial (n = 5) and stromal (n = 5) cells derived from passage 1 were placed in a 24-well plate in DMEM/Ham’s F-12 supplemented with 10% FCS and antibiotic and antimycotic solution. When the cells reached 90% confluence, the medium was replaced with fresh DMEM without phenol red supplemented with 0.1% BSA and antibiotics and antimycotic solution. The most effective dose and the optimal treatment time for TNF action was established in a preliminary study (data not shown). The epithelial and stromal cells were stimulated with TNF (10 ng/ml) for 24 h or with OT (107 M) as a positive control. Conditioned media were collected into tubes with 5 ll EDTA, 1% acetylsalicylic acid solution (Sigma–Aldrich; #A2093), and frozen at 20 °C until PG measurement. To collect cells from wells for single-step DNA isolation, 250 ll of TRI Reagent (Sigma–Aldrich; #T9424) was added to each well. Cells were then collected from 4 wells per each treatment, then DNA was isolated according to the TRI Reagent manufacturer’s procedure. The DNA content was used to standardize the results. 2.4.2.2. Experiment 2.2. TNF effect on PG secretion and PG synthase mRNA transcription by equine endometrial explants. Endometrial explants (n = 5) were exposed to TNF (10 ng/ml). The most effective dose and the optimal treatment time for TNF action was established in a preliminary study (data not shown); OT (107 M) was used as a positive control. After incubation, conditioned culture medium was collected in tubes with 5% EDTA, 1% acetylsalicylic acid solution and kept frozen at 20 °C until PG determination. In order to normalize results, the concentration of PGE2 and PGF2a was assessed per 1 g of tissue. Additionally, after incubation, endometrial strips were placed in RNAlater (Invitrogen, #AM7021) for determination of PTGS-2, PGES, PGFS mRNAs transcription using Real-time PCR. 2.5. Methods 2.5.1. Total RNA isolation and cDNA synthesis Total RNA was extracted using the Total RNA Prep Plus Kit (A&A Biotechnology, Gdansk, Poland; #031-50) according to the manufacturer’s instructions. RNA samples were stored at 80 °C. Before use, RNA concentration and quality were determined spectrophotometrically and in agarose gel electrophoresis. The ratio of absorbance at 260 nm and 280 nm (A260/280) was approximately 2. Then, 1 lg RNA was reverse-transcribed into cDNA using a ThermoScript™ RT-PCR System (Qiagen; #205311) according to the manufacturer’s instructions. The cDNA was stored at 20 °C until Real time PCR was carried out. 2.5.2. Real time PCR Real time PCR was performed with an ABI Prism 7300 sequence detection system using SYBR Green PCR master mix (Applied Biosystems, Foster City, CA, USA). The sequences for PTGS-2, PGES, PGFS and ACTB primers [21] and for TNF [22] were determined recently. After a preliminary study, ACTB was chosen as the best housekeeping gene. All primers were synthesized by GenoMed (Warsaw, Poland). Total reaction volume was 20 ll containing:
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1 ll cDNA (20 ng/ll), 2 ll forward and reverse primers each (250nM) and 10 ll SYBR Green PCR master mix. Real time PCR was carried out as follows: initial denaturation (10 min at 95 °C), followed by 40 cycles of denaturation (15 s at 95 °C) and annealing (1 min at 60 °C). After each PCR reaction, melting curves were obtained by stepwise increases in temperature from 60 °C to 95 °C to ensure single product amplification. Specificity of the product was confirmed by electrophoresis on 2% agarose gel. Data were analyzed using the method described by Zhao and Fernald [23]. 2.5.3. PG and ovarian steroid determination The concentration of PGE2 in the conditioned medium was determined using a Prostaglandin E2 EIA kit (Cayman Chemical Company, Ann Arbor, USA; #514010) according to the manufacturer’s instructions. The concentration of PGF2a was determined using the direct enzyme immunoassay (EIA) method described previously by Uenoyama et al. [24] with modification. The standard curve for PGE2 ranged from 16.5 pg/mL to 1000 pg/mL. The intra- and inter-assay coefficients of variation (CV) were 2.0% and 3.2%, respectively. The standard curve for PGF2a ranged from 0.19 ng/mL to 50 ng/mL and CV were 4.2% and 3.1%, respectively. The concentration of P4 in blood plasma was determined using EIA as described previously [25]. The standard curve for P4 ranged from 0.0925 ng/mL to 25 ng/mL and CV were 4.5% and 5.3%, respectively. The antibody (Anti-P4, code SO/91/4; kindly donated by Dr. S. Okrasa, Warmia-Mazury University, Olsztyn, Poland) was characterized previously [26]. The concentrations of E2 in blood plasma were assayed by radioimmunoassay (RIA) after extraction with diethyl ether (extraction efficiency: 89%) as described before [27]. The antibody (Anti-E2, code BS/88/754; gift from Dr. B. Szafranska, Warmia-Mazury University, Olsztyn, Poland) was characterized previously [28]. The intra- and inter-assay CV averaged 5.1% and 6.2%, respectively. The E2 standard curve ranged from 0.5 to 80 pg/mL, and the effective dose for 50% inhibition (ED50) of the assay was 1.98 pg/mL. The intra- and inter-assay CV averaged 5.3% and 6.6%, respectively. 2.6. Statistical analysis Data are shown as the mean±SEM of values obtained in separate experiments, each performed in quadruplicate. The statistical analyses of TNF mRNA transcription in response to ovarian steroid exposure (Experiment 1.1), and TNF protein production in response to ovarian steroid exposure (Experiment 1.2), were determined by nonparametric one-way ANOVA Kruskal–Wallis followed by Dunn’s test (GraphPad Software version 5, San Diego, USA). The influence of TNF on PG secretion by epithelial and stromal cells (Experiment 2.1), and by endometrial explants (Experiment 2.2), was statistically analyzed by Student’s t-test. The statistical analysis of TNF influence on PTGS-2, PGES and PGFS mRNAs transcription by endometrial explants was determined by the nonparametric Mann–Whitney U test. All results were considered significantly different when P < 0.05.
3. Results 3.1. Experiment 1. In vitro production of TNF by endometrial cells 3.1.1. Experiment 1.1. Ovarian steroid effects on TNF mRNA transcription in equine endometrial cells During 24-h stimulation, P4 up-regulated TNF mRNA transcription in epithelial cells (Fig. 1A; P < 0.01). Ovarian steroids did not affect TNF mRNA transcription in stromal cells when compared to the control (Fig. 1B; P > 0.05).
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Fig. 1. Effect of P4 (107 M), E2 (109 M) and P4+E2 (107/109 M) on TNF mRNA transcription (A, B) and TNF protein expression (C, D) by epithelial and stromal cells. All values are presented as n-fold increase relative to the control. Asterisks indicate significant differences (⁄P < 0.05; ⁄⁄P < 0.01) from the respective control, as determined by nonparametric one-way ANOVA Kruskal–Wallis followed by Dunn’s test.
3.1.2. Experiment 1.2. Ovarian steroid effects on TNF production by equine endometrial cells Production of TNF by epithelial cells was increased by P4 and E2 compared to the control group (Fig. 1C; P < 0.05; P < 0.01). Progesterone+E2, E2 and P4 increased TNF production by stromal cells compared to the control group (Fig. 2D; P < 0.01; P < 0.05; P < 0.05). 3.2. Experiment 2. TNF effect on PG production in vitro 3.2.1. Experiment 2.1. TNF effects on PG secretion by equine endometrial cells Basal in vitro PGE2 and PGF2a secretion by epithelial cells was 2.03 ± 0.312 ng/lg DNA and 2.54 ± 0.220 ng/lg DNA, respectively. Basic PGE2 and PGF2a secretion by stromal cells was 1.97 ± 0.361 ng/lg DNA and 2.71 ± 0.236 lg DNA, respectively. Oxytocin increased PGE2 and PGF2a secretion by epithelial and stromal cells compared to the control group (Fig. 2; P < 0.05). Tumor necrosis factor-a increased PGE2 secretion by epithelial cells compared to the control group (Fig. 2A; P < 0.01), while in stromal cells it increased not only PGE2 but also PGF2a (Fig. 2C and D; P < 0.001). 3.2.2. Experiment 2.2. TNF effect on PG secretion and PG synthase mRNA transcription by equine endometrial explants Basal PGE2 and PGF2a production by endometrial tissue incubated in vitro for 24 h was 568.0 ± 82.1 ng/g tissue and 659.3 ± 69.31 ng/g tissue, respectively (Fig. 3A and B). Oxytocin increased PGE2 and PGF2a secretion by endometrial explants compared to the control group (Fig. 3A and B; P < 0.001; P < 0.01, respectively). Oxytocin increased transcription of PTGS-2, PGES and PGFS mRNAs in endometrial explants compared to the control group (Fig. 4A–C; P < 0.01; P < 0.05; P < 0.001, respectively).
The data indicate that the tissue was reactive during the experiment and maintained its physiological properties. Tumor necrosis factor-a stimulated PGE2 as well as PGF2a production in the endometrium after 24-h incubation, compared to the control group (Fig. 3A; P < 0.001; P < 0.05, respectively). Tumor necrosis factor-a also increased transcription of PTGS-2, PGES and but PGFS mRNAs in endometrial explants compared to the control group (Fig. 4A–C; P < 0.05).
4. Discussion The first issue addressed in the study was to determine the ability of epithelial and stromal cells to produce TNF in response to E2 and P4 in the mare endometrium. It was shown that expression of TNF is regulated by ovarian steroids and by itself in human and bovine endometrial cells [9,11]. The fact that endometrial cells can produce TNF helps elucidate the auto-/paracrine modulation processes occurring in the endometrium, especially considering the ability of TNF to induce its own production [9,11]. Besides the effect of P4 on TNF mRNA transcription in epithelial cells, there was a lack of effect of ovarian steroids on TNF mRNA transcription, but they up-regulated TNF protein secretion during 24 h of incubation. 17b-estradiol and P4 increased production of TNF by epithelial cells. In turn P4, E2 and P4+E2 increased TNF production by stromal cells. The reason for differences between mRNA transcription and protein expression is not clear. Vogel and Marcotte [29] demonstrated that production and maintenance of cellular protein requires a vast series of linked processes, from transcription, processing and degradation of mRNAs to translocation, localization, modification and programmed destruction of the proteins themselves. It was shown that about 40% of the
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Fig. 2. Effect of TNF (10 ng/ml) on PGE2 (A, C) and PGF2a (B, D) in vitro secretion by epithelial and stromal cells. Oxytocin (OT; 107 M) was used as a positive control. All values are expressed as n-fold change from control. Asterisks indicate significant differences (⁄P < 0.05; ⁄⁄P < 0.01; ⁄⁄⁄P < 0.001) from the respective control, as determined by a two-tailed Student’s t-test.
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Fig. 3. Influence of TNFa (10 ng/ml) on (A) PGE2 and (B) PGF2a secretion from mid-luteal phase endometrial explants. All values are expressed as n-fold change from the control. Asterisks indicate significant differences (⁄P < 0.05; ⁄⁄P < 0.01; ⁄⁄⁄P < 0.001) from the respective control, as determined by a two-tailed Student’s t-test.
variation in protein concentration can be explained by differences in mRNA transcription [30,31]. This may result from the combination of different post-transcriptional regulation processes [29] and might be the explanation for differences in TNF mRNA transcription and protein production in response to ovarian steroids. In the mare, TNF expression and its influence on PG expression have most recently become subjects of interest [14,15]. Nevertheless, the action of endometrial TNF has been studied in domestic animals for at least a decade [4–6,32,33]. In the mare, knowledge concerning this issue is sparse. In the cow, endometrial TNF is known as a luteolytic agent, as well as being luteotropic depending on its concentration, which was established by several in vivo studies [4,25]. Our present and previous [15] findings suggest that TNF, produced by and acting on the equine endometrium, acts as an
autocrine-/paracrine factor, activating potential luteotropic mechanisms that induce PGE2 production in mid-luteal phase of the estrous cycle. Firstly, it was shown that endometrial expression of TNF, TNFRI and TNFRII was up-regulated at the mid-luteal phase of the estrous cycle [15]. Secondly, the present findings show that TNF stimulated PGE2 secretion in endometrial cells and tissues. The in vivo antiluteolytic action of PGE2 was confirmed by Vanderwall et al. [34]. Nonpregnant mares were continuously infused with PGE2, on Days 10–16 postestrus. This intrauterine administration of PGE2 prolonged CL function [34]. Additionally, Lukasik et al. [35] showed that PGE2 stimulated P4 secretion from equine luteal steroidogenic cells in vitro. In the present study, the results from Experiment 1 indicated that stromal cell production of TNF increased in response to P4, the level of which is highest at the mid-luteal phase of the estrous cycle. Even though our present
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porcine luminal epithelial cells during early pregnancy, PTGS-2, PGES-1 and PGES-2 mRNA transcription was up-regulated in response to TNF [5]. In summary, our study provides better understanding of TNF production and action, as well as interactions among TNF, PG and ovarian steroids, in the equine endometrium. Our results indicated that TNF is produced by epithelial and stromal cells of the equine endometrium and that its secretion is up-regulated by ovarian steroids. Furthermore, it was confirmed that epithelial as well as stromal cells are able to produce PG in response to TNF exposure. Additionally, it was shown that TNF stimulated PG secretion via up-regulation of PG synthases mRNA transcription. Taking into consideration all these findings, it is suggested that local production of TNF is closely related to ovarian steroid action and the interaction between TNF and PG regulates physiologic processes in the equine endometrium. Thus, TNF plays auto-/paracrine roles in the equine endometrium during the estrous cycle.
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Fig. 4. Influence of TNFa (10 ng/ml) on (A) PTGS-2, (B) PGES and (C) PGFS mRNAs transcription in mid-luteal phase endometrial explants incubated in vitro. All values are presented as n-fold increase relative to the control. Asterisks indicate significant differences (⁄P < 0.05; ⁄⁄P < 0.01; ⁄⁄⁄P < 0.001) from the respective control as determined by nonparametric Mann–Whitney U test.
findings support the hypothesis that TNF acts as a luteotrophic agent, nevertheless, it requires confirmation by in vivo studies. Considering that TNF stimulates PG in other species, it should be pointed out that one of the purposes of this study was to determine which cell type responds to TNF exposure [32,33]. Our results indicated that equine epithelial cells are able to produce PGE2 and PGF2a in response to TNF. Nevertheless, PGE2 is only produced by epithelial cells in response to this cytokine. It is very interesting and worth notice that this response seems to be different depending on the species [32,33]. In the cow, Murakami et al. [32] showed that TNF strongly stimulated PGF2a secretion by stromal cells but did not affect epithelial cells. Our findings indicate that TNF-stimulated PG secretion by equine endometrium occurs via PG synthases mRNA transcription modulation. The up-regulation of PG synthases mRNA transcription by TNF in porcine endometrial cells was confirmed by others [5]. Also in the sow, on Days 12,13 and 15,16 of the estrous cycle, TNF up-regulated endometrial PGFS mRNA transcription [36]. In
Supported by Grant MAESTRO of National Science Centerdedicated to DJS (No 2011/02/A/NZ5/00338). DJS and GF-D were supported by joint Polish-Portugal project under the Agreement of MSHE (Poland) and FCT (Portugal). Presented at part on XIth International Symposium on Equine Reproduction (ISER XI), 26th–31st January 2014, Hamilton, New Zealand. References [1] Wajant H, Pfizenmaier K, Scheurich P. Tumor necrosis factor signaling. Cell Death Differ 2003;10:45–65. [2] Tartaglia LA, Goeddel DV. Two TNF receptors. Immunol Today 1992;13:151–3. [3] Townson DH, Pate JL. Mechanism of action of TNF-alpha-stimulated prostaglandin production in cultured bovine luteal cells. Prostaglandins 1996;52:361–73. [4] Skarzynski DJ, Bah MM, Woclawek-Potocka I, Deptula KM, Korzekwa A, Schibaya M, et al. Role of tumor necrosis factor-a in the regulation of the estrus cycle in cattle. Biol Reprod 2003;68:1674–81. [5] Waclawik A, Blitek A, Ziecik AJ. Oxytocin and tumor necrosis factor alpha stimulate expression of prostaglandin E2 synthase and secretion of prostaglandin E2 by luminal epithelial cells of the porcine endometrium during early pregnancy. Reproduction 2010;140:613–22. [6] Szóstek AZ, Siemieniuch MJ, Deptula K, Woclawek-Potocka I, Majewska M, Okuda K, et al. Ovarian steroids modulate tumor necrosis factor-a and nitric oxide-regulated prostaglandin secretion by cultured bovine oviductal epithelial cells. Domest Anim Endocrin 2011;41:14–23. [7] Carswell EA, Old LJ, Kassel RL, Green S, Fiore N, Williamson B. An endotoxininduced serum factor that causes necrosis of tumor. Proc Natl Acad Sci USA 1975;72:3666–70. [8] Hehnke–Vagnoni KE, Clark CL, Taylor MJ, Ford SP. Presence and locatization of tumor necrosis factor a in the corpus luteum of nonpregnant and pregnant pigs. Biol Reprod 1995;53:1339–44. [9] Okuda K, Sakumoto R, Okamoto N, Acosta TJ, Abe H, Okada H, et al. Cellular localization of genes and proteins for tumor necrosis factor-a (TNF) TNF receptor types I and II in bovine endometrium. Mol Cell Endocrinol 2010;330:41–8. [10] Hunt JS, Chen H-L, Tabibzadeh S. Tumor necrosis factor-alpha messenger ribonucleic acid and protein in human endometrium. Biol Reprod 1992;47:141–7. [11] Tabibzadeh S, Satyaswaroop PG, von Wolff M, Strowitzki T. Regulation of TNFalpha mRNA expression in endometrial cells by TNF-alpha and by oestrogen withdrawal. Mol Hum Reprod 1999;5:1141–9. [12] Weems CW, Weems YS, Randel RD. Prostaglandins and reproduction in female farm animals. Vet J 2006;171:206–28. [13] Simmons DL, Botting RM, Hla T. Cyclooxygenase isozymes: the biology of prostaglandin synthesis and inhibition. Pharmacol Rev 2004;56:387–437. [14] Grünig G, Antczak DF. Horse trophoblasts produce tumor necrosis factor alpha but not interleukin 2, interleukin 4, or interferon gamma. Biol Reprod 1995;52:531–9. [15] Galvão A, Valente L, Skarzynski DJ, Szóstek A, Piotrowska-Tomala K, Rebordão MR, et al. Effect of cytokines and ovarian steroids on equine endometrial function: an in vitro study. Reprod Fertil Dev 2013;25:985–97. [16] Roberto da Costa RP, Serrão PM, Monteiro S, Pessa P, Silva JR, Ferreira-Dias G. Caspase-3-mediated apoptosis and cell proliferation in the equine endometrium during the oestrous cycle. Reprod Fertil Dev 2007;19:925–32. [17] Roberto da Costa RP, Costa AS, Korzekwa AJ, Platek R, Siemieniuch M, Galvão A, et al. Actions of a nitric oxide donor on prostaglandin production and
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Cytokine journal homepage: www.journals.elsevier.com/cytokine
Ovarian steroid-dependent tumor necrosis factor-a production and its action on the equine endometrium in vitro Anna Z. Szóstek a, Marek Adamowski a, António M. Galvão a,b, Graça M. Ferreira-Dias b, Dariusz J. Skarzynski a,⇑ a b
Department of Reproductive Immunology and Pathology, Institute of Animal Reproduction and Food Research, Olsztyn, Poland C.I.I.S.A., Faculty of Veterinary Medicine, University of Lisbon, Lisbon, Portugal
a r t i c l e
i n f o
Article history: Received 18 July 2013 Received in revised form 6 February 2014 Accepted 12 February 2014 Available online 15 March 2014 Keywords: Tumor necrosis factor-a Prostaglandin Ovarian steroids Endometrium Mare
a b s t r a c t Tumor necrosis factor-a (TNF) is a cytokine that plays important roles in functions of the endometrium. The aims of this study were to determine whether (i) ovarian steroids modulate TNF production by endometrial cells (Experiment 1); (ii) TNF effects on prostaglandin (PG) production in cultured equine endometrial cells and tissue (Experiment 2). Epithelial and stromal cells were isolated from equine endometrium (Days 2–5 of the estrous cycle; n = 20) and treated after passage 1. In Experiment 1, epithelial and stromal cells were exposed to progesterone (P4; 107 M), 17-b estradiol (E2; 109 M) or P4+E2 (107/ 109 M) for 24 h. Then, TNF mRNA transcription was determined using Real-time PCR. Additionally, TNF protein production was investigated in response to ovarian steroids for 24 h using Enzyme-Linked Immunosorbent Spot (EliSpot). In Experiment 2, epithelial and stromal cells and endometrial explants (mid-luteal phase of the estrous cycle; n = 5) were exposed in vitro to TNF (10 ng/ml) and to oxytocin (OT; positive control; 107 M) for 24 h. The concentrations of PGE2 and PGF2a were determined using a direct enzyme immunoassay (EIA) method. The transcription of prostaglandin-endoperoxide synthase2 (PTGS-2), prostaglandin E2 synthase (PGES) and PGF2a synthase (PGFS) mRNAs in the endometrial explants was determined using Real-time PCR. Results showed that TNF is produced by two types of equine endometrial cells and its production is up-regulated by ovarian steroids (P < 0.05) in stromal cells and by P4 (P < 0.05) and E2 (P < 0.01) in epithelial cells. Epithelial and stromal cells can also produce PG in response to TNF. In endometrial explants, TNF stimulated PGE2 production to a large extent and PGF2a secretion to a lesser extent. These actions are mediated by up-regulation of PG synthases mRNA transcription. The study indicates that TNF production is closely related to ovarian steroid actions and that the interaction between TNF and PG regulates physiologic processes in the equine endometrium. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Tumor necrosis factor-a (TNF) is a non-glycosylated protein with a wide spectrum of bioactivities; most cells show at least some responsiveness to TNF. In general, this cytokine displays a functional duality, being involved in regeneration as well as destruction of tissues [1]. The distinctive effects of TNF are due to two types of TNF receptor which have different intracellular signaling pathways [2]. Tumor necrosis factor-a receptor type I (TNFRI) contains an intracellular death domain which is necessary for signaling pathways associated with apoptosis. This type of receptor is constitutively expressed in most tissues and seems to
⇑ Corresponding author. Tel./fax: +48 89 5393130. E-mail address: [email protected] (D.J. Skarzynski). http://dx.doi.org/10.1016/j.cyto.2014.02.005 1043-4666/Ó 2014 Elsevier Ltd. All rights reserved.
be a key mediator of TNF signaling [2]. In turn, TNFRII is strongly regulated and predominantly expressed in immune cells and its plays a major role in the lymphoid system. Signaling via TNFRII induce apoptosis but also support survival promoting tissue repair and angiogenesis [2]. Tumor necrosis factor-a is a cytokine that plays important autoparacrine roles in female reproduction [3–6]. It is well known that TNF is produced mainly by macrophages [7] and endothelial cells [8]. However, the ability of endometrial cells to produce TNF has been shown in several species [9–11]. Okuda et al. [9] revealed that TNF is co-located mainly in bovine luminal and glandular epithelial and endothelial cells in the estrous cycle. Furthermore, TNF was detected in stromal cell lysates and in conditioned cultured media [9]. In addition, it was confirmed that TNF is expressed to a larger extent in epithelial cells and, to a lesser extent, in stromal and lymphoid cells in human endometrium [10,11].
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The action of TNF-a induced via prostaglandin (PG) stimulation in the corpus luteum (CL), oviduct and endometrium in many domestic species [3–6]. Prostaglandins play crucial roles in the regulation of several reproductive processes such as ovulation, luteolysis, uterine vascularization and maintenance of pregnancy [12]. These molecules are capable of a very broad spectrum of effects modulating a multitude of biological processes. In the PG production cascade, the prostaglandin-endoperoxide synthase (PTGS) enzyme converts arachidonic acid (AA) into prostaglandin (PG)H2 [13]. There are two isoforms of PTGS, designated PTGS-1, which is constitutively expressed, and PTGS-2 which is the inducible isoform. Prostaglandin F2a synthase (PGFS) and PGE2 synthases (PGES) are downstream enzymes which catalyze the conversion of PGH2 into PGF2a or PGE2, respectively. Although Grünig and Antczak [14] have previously demonstrated that TNF is expressed in gravid endometrium and trophoblast in the mare, the knowledge about the role of TNF in the equine endometrium is still insufficient. We have recently shown the immunolocalization of TNF in equine endometrial epithelial luminal and glandular cells as well as in stromal cells [15]. In above-mentioned study TNF protein expression was up-regulated in the mid-luteal and follicular phases of the estrous cycle suggesting sex steroids-dependent mechanisms regulating TNF expression in the equine endometrium [15]. Therefore, the present work attempts to explain whether: (i) ovarian steroids modulate TNF production, and (ii) TNF influences PG secretion and PG synthases mRNA transcription. 2. Materials and methods 2.1. Animals and endometrial tissue collection Uteri (n = 20) were collected post-mortem from cyclic mares at a local abattoir. All procedures for animal handling and tissue collection were approved by the Local Animal Care and Use Committee in Olsztyn, Poland (Agreement No. 51/2011). The mares were healthy as declared by official governmental veterinary inspection. The material was collected within 5 min of an animal’s death. Immediately before death, peripheral blood samples were collected into heparinized tubes (MonovettesÒ-Sarstedt, Numbrecht, Germany) for P4 and 17-b E2 analysis. The phases of the estrous cycle were identified based on P4 and E2 analysis of blood serum and macroscopic observation of the ovaries as described before [16]. A small piece of each endometrium was fixed in buffered 4% paraformaldehyde for histological analysis [17], and for characterization according to the classification system developed by Kenney [18] and extended by Kenney and Doig [19]. Only cells derived from endometria that were class I (no degenerative changes) according to the Kenney [18] classification were assigned to this study. For tissue and cell culture, the entire uterus was collected within 5 min of the animal’s death, placed in sterile, incomplete (Ca2+and Mg2+- free) Hank’s balanced salt solution (HBSS) supplemented with gentamicin (20 lg/mL; Sigma–Aldrich, #G1272) and 0.1% bovine serum albumin (BSA; Sigma–Aldrich, #A9056), kept on ice and transported quickly to the laboratory. 2.2. Epithelial and stromal cell isolation and culture A total of 15 uteri on days 2–5 of the estrous cycle were used. Equine epithelial and stromal cells were isolated following the methodology described previously [20]. The endometrial cells were cultured at 38.5 °C in a humidified atmosphere of 5% CO2 in air. The culture medium was Dulbecco’s modified Eagle’s medium/Nutrient Ham’s F-12 (DMEM/Ham’s F-12; Sigma–Aldrich;
D8900) supplemented with 10% fetal calf serum (FCS; Sigma– Aldrich, Madison, USA; #C6278) and 1% antibiotic and antimycotic solution (Sigma–Aldrich; #A5955); it was changed every 2–3 days. After reaching 90–95% confluence (Day 5 or 7 for epithelial or stromal cells, respectively), the cells were trypsinized as described previously [20]. Next, depending on the experiment, epithelial cells were seeded at a density of 5 105 viable cells/mL and stromal cells at a density of 2 105 viable cells/mL in 24- or 96-well plates. The viability of epithelial and stromal cells was 80% and 90%, respectively. The homogeneity of cell culture was evaluated using immunofluorescent staining for specific markers of epithelial cells (cytokeratin) and stromal cells (vimentin) as described previously [20]. 2.3. Tissue culture A total of 5 uteri at the mid-luteal phase of the estrous cycle were used. This phase of the estrous cycle was chosen based on a previous study [15] which showed that TNF, TNFRI and TNFRII expression was up-regulated at the mid-luteal phase of the estrous cycle. Endometrial explants were minced into small pieces, then 50 mg amounts were washed three times in PBS containing gentamicin (20 lg/lL) and placed into culture tubes, each of which contained 1 mL Dulbecco’s Modified Eagle’s medium without phenol red (Sigma–Aldrich; D#2960) with 0.1% BSA and antibiotic/ antimycotic solution as described above. Tissue explants were preincubated on a shaker inside a tissue culture incubator at 38.0 °C with 5% CO2 in air for 6 h, then the medium was replaced with fresh DMEM supplemented with 0.1% BSA and antibiotics and antimycotic. After this, endometrial tissue was further incubated for 24 h with TNF (10 ng/ml) and OT (107 M; positive control). Finally, viability of endometrial explant cells was confirmed using Alamar Blue according to the manufacturer’s instructions (Invitrogen; Burlington; Ontario, Canada; #DAL1025). 2.4. Experimental procedure 2.4.1. Experiment 1. In vitro production of TNF by endometrial cells 2.4.1.1. Experiment 1.1. Effect of ovarian steroids on TNF mRNA transcription in equine endometrial cells. Stromal (n = 5) and epithelial (n = 5) cells derived from passage 1 were placed in a 24-well plate in DMEM/Ham’s F-12 supplemented with 10% FCS and antibiotic and antimycotic solution. When the cells reached 90% confluence, the medium was replaced with fresh DMEM without phenol red, supplemented with 0.1% BSA and antibiotics and antimycotic. The most effective dose and the optimal treatment time for action of ovarian steroids were established in a preliminary study (data not shown). The epithelial and stromal cells were stimulated with P4 (107 M), E2 (109 M) or P4+E2 (107/109 M) for 24 h. Next, the culture medium was removed and to each well 250 ll of Fenozol was added in order to determine TNF mRNA transcription using Real-time PCR. 2.4.1.2. Experiment 1.2. Effect of ovarian steroids on TNF production by equine endometrial cells. To determine TNF production by endometrial cells, the equine ELISpot system was used (R&D Systems, Minneapolis, USA; #EL1814) following the manufacturer’s instructions. Stromal (n = 5) and epithelial (n = 5) cells derived from passage 1 were seeded at a density of 2 104 per well in a MultiScreen sterile 96-well plate with a PVDF membrane (Millipore) using fresh DMEM without phenol red supplemented with 0.1% BSA and antibiotics and antimycotic solution. The density of cell seeding was established in a preliminary experiment. Then, cells were incubated with vehicle, P4 (107 M), E2 (109 M) or P4+E2 (107/109 M) for 24 h. The following controls were used:
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positive control (TNF); negative control (unstimulated cells at the same density as stimulated cells); background control (culture medium alone) and Detection Antibody control (phosphate buffered saline substituted for the Detection Antibody). Spots were visualized using the BCIP/NBT substrate detection system according to the manufacturer’s instructions. The spots were analyzed using an Eli.Scan scanner and software (A.EL.VIS GmbH; Hannover; Germany). Data were expressed as spot number per 2 104 cells. 2.4.2. Experiment 2. TNF effect on PG production in vitro 2.4.2.1. Experiment 2.1. TNF effect on PG secretion by equine endometrial cells. Epithelial (n = 5) and stromal (n = 5) cells derived from passage 1 were placed in a 24-well plate in DMEM/Ham’s F-12 supplemented with 10% FCS and antibiotic and antimycotic solution. When the cells reached 90% confluence, the medium was replaced with fresh DMEM without phenol red supplemented with 0.1% BSA and antibiotics and antimycotic solution. The most effective dose and the optimal treatment time for TNF action was established in a preliminary study (data not shown). The epithelial and stromal cells were stimulated with TNF (10 ng/ml) for 24 h or with OT (107 M) as a positive control. Conditioned media were collected into tubes with 5 ll EDTA, 1% acetylsalicylic acid solution (Sigma–Aldrich; #A2093), and frozen at 20 °C until PG measurement. To collect cells from wells for single-step DNA isolation, 250 ll of TRI Reagent (Sigma–Aldrich; #T9424) was added to each well. Cells were then collected from 4 wells per each treatment, then DNA was isolated according to the TRI Reagent manufacturer’s procedure. The DNA content was used to standardize the results. 2.4.2.2. Experiment 2.2. TNF effect on PG secretion and PG synthase mRNA transcription by equine endometrial explants. Endometrial explants (n = 5) were exposed to TNF (10 ng/ml). The most effective dose and the optimal treatment time for TNF action was established in a preliminary study (data not shown); OT (107 M) was used as a positive control. After incubation, conditioned culture medium was collected in tubes with 5% EDTA, 1% acetylsalicylic acid solution and kept frozen at 20 °C until PG determination. In order to normalize results, the concentration of PGE2 and PGF2a was assessed per 1 g of tissue. Additionally, after incubation, endometrial strips were placed in RNAlater (Invitrogen, #AM7021) for determination of PTGS-2, PGES, PGFS mRNAs transcription using Real-time PCR. 2.5. Methods 2.5.1. Total RNA isolation and cDNA synthesis Total RNA was extracted using the Total RNA Prep Plus Kit (A&A Biotechnology, Gdansk, Poland; #031-50) according to the manufacturer’s instructions. RNA samples were stored at 80 °C. Before use, RNA concentration and quality were determined spectrophotometrically and in agarose gel electrophoresis. The ratio of absorbance at 260 nm and 280 nm (A260/280) was approximately 2. Then, 1 lg RNA was reverse-transcribed into cDNA using a ThermoScript™ RT-PCR System (Qiagen; #205311) according to the manufacturer’s instructions. The cDNA was stored at 20 °C until Real time PCR was carried out. 2.5.2. Real time PCR Real time PCR was performed with an ABI Prism 7300 sequence detection system using SYBR Green PCR master mix (Applied Biosystems, Foster City, CA, USA). The sequences for PTGS-2, PGES, PGFS and ACTB primers [21] and for TNF [22] were determined recently. After a preliminary study, ACTB was chosen as the best housekeeping gene. All primers were synthesized by GenoMed (Warsaw, Poland). Total reaction volume was 20 ll containing:
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1 ll cDNA (20 ng/ll), 2 ll forward and reverse primers each (250nM) and 10 ll SYBR Green PCR master mix. Real time PCR was carried out as follows: initial denaturation (10 min at 95 °C), followed by 40 cycles of denaturation (15 s at 95 °C) and annealing (1 min at 60 °C). After each PCR reaction, melting curves were obtained by stepwise increases in temperature from 60 °C to 95 °C to ensure single product amplification. Specificity of the product was confirmed by electrophoresis on 2% agarose gel. Data were analyzed using the method described by Zhao and Fernald [23]. 2.5.3. PG and ovarian steroid determination The concentration of PGE2 in the conditioned medium was determined using a Prostaglandin E2 EIA kit (Cayman Chemical Company, Ann Arbor, USA; #514010) according to the manufacturer’s instructions. The concentration of PGF2a was determined using the direct enzyme immunoassay (EIA) method described previously by Uenoyama et al. [24] with modification. The standard curve for PGE2 ranged from 16.5 pg/mL to 1000 pg/mL. The intra- and inter-assay coefficients of variation (CV) were 2.0% and 3.2%, respectively. The standard curve for PGF2a ranged from 0.19 ng/mL to 50 ng/mL and CV were 4.2% and 3.1%, respectively. The concentration of P4 in blood plasma was determined using EIA as described previously [25]. The standard curve for P4 ranged from 0.0925 ng/mL to 25 ng/mL and CV were 4.5% and 5.3%, respectively. The antibody (Anti-P4, code SO/91/4; kindly donated by Dr. S. Okrasa, Warmia-Mazury University, Olsztyn, Poland) was characterized previously [26]. The concentrations of E2 in blood plasma were assayed by radioimmunoassay (RIA) after extraction with diethyl ether (extraction efficiency: 89%) as described before [27]. The antibody (Anti-E2, code BS/88/754; gift from Dr. B. Szafranska, Warmia-Mazury University, Olsztyn, Poland) was characterized previously [28]. The intra- and inter-assay CV averaged 5.1% and 6.2%, respectively. The E2 standard curve ranged from 0.5 to 80 pg/mL, and the effective dose for 50% inhibition (ED50) of the assay was 1.98 pg/mL. The intra- and inter-assay CV averaged 5.3% and 6.6%, respectively. 2.6. Statistical analysis Data are shown as the mean±SEM of values obtained in separate experiments, each performed in quadruplicate. The statistical analyses of TNF mRNA transcription in response to ovarian steroid exposure (Experiment 1.1), and TNF protein production in response to ovarian steroid exposure (Experiment 1.2), were determined by nonparametric one-way ANOVA Kruskal–Wallis followed by Dunn’s test (GraphPad Software version 5, San Diego, USA). The influence of TNF on PG secretion by epithelial and stromal cells (Experiment 2.1), and by endometrial explants (Experiment 2.2), was statistically analyzed by Student’s t-test. The statistical analysis of TNF influence on PTGS-2, PGES and PGFS mRNAs transcription by endometrial explants was determined by the nonparametric Mann–Whitney U test. All results were considered significantly different when P < 0.05.
3. Results 3.1. Experiment 1. In vitro production of TNF by endometrial cells 3.1.1. Experiment 1.1. Ovarian steroid effects on TNF mRNA transcription in equine endometrial cells During 24-h stimulation, P4 up-regulated TNF mRNA transcription in epithelial cells (Fig. 1A; P < 0.01). Ovarian steroids did not affect TNF mRNA transcription in stromal cells when compared to the control (Fig. 1B; P > 0.05).
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Fig. 1. Effect of P4 (107 M), E2 (109 M) and P4+E2 (107/109 M) on TNF mRNA transcription (A, B) and TNF protein expression (C, D) by epithelial and stromal cells. All values are presented as n-fold increase relative to the control. Asterisks indicate significant differences (⁄P < 0.05; ⁄⁄P < 0.01) from the respective control, as determined by nonparametric one-way ANOVA Kruskal–Wallis followed by Dunn’s test.
3.1.2. Experiment 1.2. Ovarian steroid effects on TNF production by equine endometrial cells Production of TNF by epithelial cells was increased by P4 and E2 compared to the control group (Fig. 1C; P < 0.05; P < 0.01). Progesterone+E2, E2 and P4 increased TNF production by stromal cells compared to the control group (Fig. 2D; P < 0.01; P < 0.05; P < 0.05). 3.2. Experiment 2. TNF effect on PG production in vitro 3.2.1. Experiment 2.1. TNF effects on PG secretion by equine endometrial cells Basal in vitro PGE2 and PGF2a secretion by epithelial cells was 2.03 ± 0.312 ng/lg DNA and 2.54 ± 0.220 ng/lg DNA, respectively. Basic PGE2 and PGF2a secretion by stromal cells was 1.97 ± 0.361 ng/lg DNA and 2.71 ± 0.236 lg DNA, respectively. Oxytocin increased PGE2 and PGF2a secretion by epithelial and stromal cells compared to the control group (Fig. 2; P < 0.05). Tumor necrosis factor-a increased PGE2 secretion by epithelial cells compared to the control group (Fig. 2A; P < 0.01), while in stromal cells it increased not only PGE2 but also PGF2a (Fig. 2C and D; P < 0.001). 3.2.2. Experiment 2.2. TNF effect on PG secretion and PG synthase mRNA transcription by equine endometrial explants Basal PGE2 and PGF2a production by endometrial tissue incubated in vitro for 24 h was 568.0 ± 82.1 ng/g tissue and 659.3 ± 69.31 ng/g tissue, respectively (Fig. 3A and B). Oxytocin increased PGE2 and PGF2a secretion by endometrial explants compared to the control group (Fig. 3A and B; P < 0.001; P < 0.01, respectively). Oxytocin increased transcription of PTGS-2, PGES and PGFS mRNAs in endometrial explants compared to the control group (Fig. 4A–C; P < 0.01; P < 0.05; P < 0.001, respectively).
The data indicate that the tissue was reactive during the experiment and maintained its physiological properties. Tumor necrosis factor-a stimulated PGE2 as well as PGF2a production in the endometrium after 24-h incubation, compared to the control group (Fig. 3A; P < 0.001; P < 0.05, respectively). Tumor necrosis factor-a also increased transcription of PTGS-2, PGES and but PGFS mRNAs in endometrial explants compared to the control group (Fig. 4A–C; P < 0.05).
4. Discussion The first issue addressed in the study was to determine the ability of epithelial and stromal cells to produce TNF in response to E2 and P4 in the mare endometrium. It was shown that expression of TNF is regulated by ovarian steroids and by itself in human and bovine endometrial cells [9,11]. The fact that endometrial cells can produce TNF helps elucidate the auto-/paracrine modulation processes occurring in the endometrium, especially considering the ability of TNF to induce its own production [9,11]. Besides the effect of P4 on TNF mRNA transcription in epithelial cells, there was a lack of effect of ovarian steroids on TNF mRNA transcription, but they up-regulated TNF protein secretion during 24 h of incubation. 17b-estradiol and P4 increased production of TNF by epithelial cells. In turn P4, E2 and P4+E2 increased TNF production by stromal cells. The reason for differences between mRNA transcription and protein expression is not clear. Vogel and Marcotte [29] demonstrated that production and maintenance of cellular protein requires a vast series of linked processes, from transcription, processing and degradation of mRNAs to translocation, localization, modification and programmed destruction of the proteins themselves. It was shown that about 40% of the
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Fig. 2. Effect of TNF (10 ng/ml) on PGE2 (A, C) and PGF2a (B, D) in vitro secretion by epithelial and stromal cells. Oxytocin (OT; 107 M) was used as a positive control. All values are expressed as n-fold change from control. Asterisks indicate significant differences (⁄P < 0.05; ⁄⁄P < 0.01; ⁄⁄⁄P < 0.001) from the respective control, as determined by a two-tailed Student’s t-test.
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Fig. 3. Influence of TNFa (10 ng/ml) on (A) PGE2 and (B) PGF2a secretion from mid-luteal phase endometrial explants. All values are expressed as n-fold change from the control. Asterisks indicate significant differences (⁄P < 0.05; ⁄⁄P < 0.01; ⁄⁄⁄P < 0.001) from the respective control, as determined by a two-tailed Student’s t-test.
variation in protein concentration can be explained by differences in mRNA transcription [30,31]. This may result from the combination of different post-transcriptional regulation processes [29] and might be the explanation for differences in TNF mRNA transcription and protein production in response to ovarian steroids. In the mare, TNF expression and its influence on PG expression have most recently become subjects of interest [14,15]. Nevertheless, the action of endometrial TNF has been studied in domestic animals for at least a decade [4–6,32,33]. In the mare, knowledge concerning this issue is sparse. In the cow, endometrial TNF is known as a luteolytic agent, as well as being luteotropic depending on its concentration, which was established by several in vivo studies [4,25]. Our present and previous [15] findings suggest that TNF, produced by and acting on the equine endometrium, acts as an
autocrine-/paracrine factor, activating potential luteotropic mechanisms that induce PGE2 production in mid-luteal phase of the estrous cycle. Firstly, it was shown that endometrial expression of TNF, TNFRI and TNFRII was up-regulated at the mid-luteal phase of the estrous cycle [15]. Secondly, the present findings show that TNF stimulated PGE2 secretion in endometrial cells and tissues. The in vivo antiluteolytic action of PGE2 was confirmed by Vanderwall et al. [34]. Nonpregnant mares were continuously infused with PGE2, on Days 10–16 postestrus. This intrauterine administration of PGE2 prolonged CL function [34]. Additionally, Lukasik et al. [35] showed that PGE2 stimulated P4 secretion from equine luteal steroidogenic cells in vitro. In the present study, the results from Experiment 1 indicated that stromal cell production of TNF increased in response to P4, the level of which is highest at the mid-luteal phase of the estrous cycle. Even though our present
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porcine luminal epithelial cells during early pregnancy, PTGS-2, PGES-1 and PGES-2 mRNA transcription was up-regulated in response to TNF [5]. In summary, our study provides better understanding of TNF production and action, as well as interactions among TNF, PG and ovarian steroids, in the equine endometrium. Our results indicated that TNF is produced by epithelial and stromal cells of the equine endometrium and that its secretion is up-regulated by ovarian steroids. Furthermore, it was confirmed that epithelial as well as stromal cells are able to produce PG in response to TNF exposure. Additionally, it was shown that TNF stimulated PG secretion via up-regulation of PG synthases mRNA transcription. Taking into consideration all these findings, it is suggested that local production of TNF is closely related to ovarian steroid action and the interaction between TNF and PG regulates physiologic processes in the equine endometrium. Thus, TNF plays auto-/paracrine roles in the equine endometrium during the estrous cycle.
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Fig. 4. Influence of TNFa (10 ng/ml) on (A) PTGS-2, (B) PGES and (C) PGFS mRNAs transcription in mid-luteal phase endometrial explants incubated in vitro. All values are presented as n-fold increase relative to the control. Asterisks indicate significant differences (⁄P < 0.05; ⁄⁄P < 0.01; ⁄⁄⁄P < 0.001) from the respective control as determined by nonparametric Mann–Whitney U test.
findings support the hypothesis that TNF acts as a luteotrophic agent, nevertheless, it requires confirmation by in vivo studies. Considering that TNF stimulates PG in other species, it should be pointed out that one of the purposes of this study was to determine which cell type responds to TNF exposure [32,33]. Our results indicated that equine epithelial cells are able to produce PGE2 and PGF2a in response to TNF. Nevertheless, PGE2 is only produced by epithelial cells in response to this cytokine. It is very interesting and worth notice that this response seems to be different depending on the species [32,33]. In the cow, Murakami et al. [32] showed that TNF strongly stimulated PGF2a secretion by stromal cells but did not affect epithelial cells. Our findings indicate that TNF-stimulated PG secretion by equine endometrium occurs via PG synthases mRNA transcription modulation. The up-regulation of PG synthases mRNA transcription by TNF in porcine endometrial cells was confirmed by others [5]. Also in the sow, on Days 12,13 and 15,16 of the estrous cycle, TNF up-regulated endometrial PGFS mRNA transcription [36]. In
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