Research Article Interleukins Affect Equine Endometrial Cell Function: Modulatory Action of Ovarian Steroids

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1 Hindwi Pulishing Corportion Meditors of Inflmmtion Volume 214, Artile ID 2813, 11 pges Reserh Artile Interleukins Affet Equine Endometril Cell Funtion: Modultory Ation of Ovrin Steroids Ann Z. Szóstek, 1 Antonio M. Glvão, 1 Tkuo Hojo, 1 Kiyoshi Okud, 2 nd Driusz J. Skrzynski 1 1 Deprtment of Reprodutive Immunology nd Pthology, Institute of Animl Reprodution nd Food Reserh, Olsztyn, Polnd 2 Lortory of Reprodutive Endorinology Grdute Shool of Nturl Siene nd Tehnology, Okym University, Okym, Jpn Correspondene should e ddressed to Ann Z. Szóstek;.szostek@pn.olsztyn.pl Reeived 19 Deemer 213; Aepted 14 Jnury 214; Pulished 27 Ferury 214 Ademi Editor: GrçFerreir-Dis Copyright 214 Ann Z. Szóstek et l. This is n open ess rtile distriuted under the Cretive Commons Attriution Liense, whih permits unrestrited use, distriution, nd reprodution in ny medium, provided the originl work is properly ited. The im of the present study ws to investigte the intertion etween ovrin steroids, interleukins nd prostglndins (PG) in equine epithelil nd stroml ells in vitro. In Experiment 1, ells were exposed to IL-1α (1 ng/ml), IL-1β (1 ng/ml) or IL- 6 (1 ng/ml) for 24 h nd ell prolifertion ws determined using MTT. In Experiment 2, ells were exposed to progesterone (P 4 ;1 7 M); 17-β estrdiol (E 2 ;1 9 M) or P 4 +E 2 for 24 h nd lter medium ws repled with fresh one treted with IL-1α, IL-1β or IL-6 (1 ng/ml, eh) for 24 h. The oxytoin (; 1 7 M) ws used s positive ontrol. In Experiment 3, ells were exposed to P 4 (1 7 M), E 2 (1 9 M) or P 4 +E 2 for 24 h nd the IL reeptor mrnas trnsription ws determined using Rel-time PCR. Prostglndins onentrtion ws determined using the diret enzyme immunossy (EIA) method. Our findings revel funtionl linking etween ovrin steroids nd IL-stimulted PG seretion y equine endometril ells. This intertion ould e one of the mehnisms responsile for endometril lol orhestrting events during the estrous yle nd erly pregnny. 1. Introdution Endometrium is omplex tissue, whih onsists of different ell types. The overriding purpose of endometril yliity is the preprtion for emryo implnttion. Intertions etween prostglndins (PG) nd ovrin steroids ply ruil role in diverse omplex proesses in severl speies. The ovrin steroids ffet the morphologil nd funtionl stte of the endometrium. The 17-β estrdiol (E 2 ) regultes sexul ehvior, enhnes uterine motility, nd promotes seretory tivity of the entire reprodutive trt. In turn, progesterone (P 4 ) ffets endometril seretion, promotes the pregnny mintenne, nd inhiits gondotropin-relesing hormone (GnRH) seretion nd reprodutive ehvior. OvrinsteroidshvelsoeendemonstrtedtoffetPG during the estrous yle in vivo in the mre [1, 2]. Ovrin steroid-stimulted PG seretion y equine endometril ells ws reently evidened in vitro [3, 4]. Prostglndins t lolly, modulting endometril iologil proesses, suh s ell prolifertion, ngiogenesis, emryo implnttion, or peripherlly on orpus luteum (CL) mintenne nd luteolysis [5 1]. Interleukins (ILs) re sereted y numerous immune ells, ting mostly in n uto/prrine mnner. Interleukins suh s IL-1α nd IL-1β or IL-6 re known to prtiipte in the regultion of endometril PG synthesis in mny speies [11 15]. There re two types of IL gonists (IL-1α nd IL-1β) nd oth of them determine iologil responses vi speifi reeptor. Although there re two types of IL-1 reeptors (IL-1RI nd IL-1RII), only IL-1RI trnsdues IL-1 signling response [16]. Interleukin 6 is pleiotropi ytokine, whih is produed y different immune nd nonimmune ell types [17]. Interleukin 6 inds to low-ffinity suunit lled gp8 or IL-6Rα on the surfe of trget ells, promoting til-6/il- 6R lph omplex reruitment of signl-trnsduing suunits lled gp13 or IL-6Rβ [17].

2 2 Meditors of Inflmmtion Inourstudy,wehypothesizedthtthereisfuntionl link etween IL, PG, nd ovrin steroids, where ovrin steroids modulte PG seretion stimulted y IL. To lrify the intertion of those moleules we investigted: (i) the IL influene on PG prodution nd epithelil nd stroml ell prolifertion, (ii) the modultory effet of ovrin steroids on IL-stimulted prodution of PG, nd (iii) the effet of ovrin steroids on IL reeptors mrnas trnsription. 2. Mterils nd Methods 2.1. Animls nd Endometril Tissue Colletion. Uteri (n = 1) from yli mres t the erly lutel phse of estrous yle were olleted postmortem,fromapriluntiltheendofjulyt lolttoir.themreswerehelthyssttedytheoffiil governmentl veterinry inspetion. The estrous yle phses were identified sed on P 4 nd E 2 nlysis in lood serum nd n ovry mrosopi oservtion [18, 19]. The erly lutel phse is hrterized y the orpor hemorrhgi presene with plsm onentrtion of P 4 > 1 ng/ml. At the mid lutel phse, the developed CL is ssoited with folliles 15 2 mm in dimeter nd P 4 > 6 ng/ml. At the lte lutel phse, regressing CL is present, together with folliles 3 35 mm in dimeter nd onentrtion of P 4 from ng/ml. The folliulr phse is hrterized y n tive CL sene nd follile with vrious sizes presene ut lwys >35 mm dimeter, with onentrtion of P 4 < 1ng/mL [18, 19]. Moreover, the phses were differentited, sine serum E 2 ws present in sl onentrtion in lutel phse (round 2 to 1 pg/ml), ut it rehes vlues ove 2 pg/ml in the folliulr phse [2]. The entire uterus ws olleted within 5 min of n niml s deth, pled in sterile, inomplete (C 2+ nd Mg 2+ free) Hnk s lned slt solution (HBSS) supplemented with gentmiin (2 μg/ml; Sigm-Aldrih, Mdison, USA, #G1272) nd.1% ovine serum lumin (BSA; Sigm-Aldrih, Mdison, USA, #A956), kept on ie, nd trnsported quikly to lortory. Smll piees of endometrium from eh uterus were pled in uffered 4% prformldehyde for histologil nlysis [18], for further lssifition ording to the soring system developed y Kenney [21]. Only ells derived from tegory I endometri of Kenney [21] lssifition were used in the present study. Animl tretment proedures nd tissue olletion were pproved y the Lol Animl Cre nd Use Committee in Olsztyn, Polnd (Agreements No. 51/211) Epithelil nd Stroml Cells Isoltion nd Culture. Atotl of 1 uteri from mres in erly lutel phse of the estrous yle were used. The equine epithelil nd stroml ells were isolted following the methodology reently desried [22]. Cells were ultured t 38 Cinhumidifiedtmosphere of 5% CO 2. The ulture medium ws Duleo s modified Egle s medium/nutrient F-12 Hm (DMEM/Hm s F-12; Sigm-Aldrih, Mdison, USA; D89) supplemented with 1% fetl lf serum (FCS; Sigm-Aldrih, Mdison, USA; #C6278) nd ntiioti nd ntimyoti solution (Sigm- Aldrih, Mdison, USA; #A5955); it ws hnged every 2 to 3 dys. After rehing 9 to 95% onfluene (5 or 7 dys of the inution of stroml or epithelil ells, resp.), ells were trypsinized [22].Further, ells were seeded t density of vile ells/ml for epithelil ells nd vile ells/ml of stroml ells in 24 or 96-well pltes, regrding the experiment. Both ell types viility were over 9%.The ell ulture homogeneity ws evluted using immunofluoresent stining for epithelil nd stroml ell speifi mrkers (ytokertin, vimentin, resp.) s desried efore [22]. The epithelil nd stroml ell homogeneity ws round 97% Experimentl Proedures Experiment 1: The Effet of Interleukin on Epithelil nd Stroml Cell Prolifertion. Stroml (n = 5)nd epithelil (n = 5)ellsderivedfrompssgeIwerepledin96- well plte. After rehing 5% of onfluene, the medium ws repled with fresh DMEM without phenol red supplemented with.1% BSA nd ntiiotis nd ntimyoti solution. Then, ells were inuted with vehile or with IL-1α, IL- 1β, or IL-6 (1 ng/ml eh). After 24 h, ells prolifertion ws mesured y MTT (3-[4,5-dimethylthizol-2-yl]- 2,5-diphenyltetrzolium romide) method using TOX-1 Kit (Sigm-Aldrih, Mdison, USA, #7H258), ording to the mnufturer s instrutions Experiment 2: The Effet of Ovrin Steroids on Cytokine-Stimulted PG Prodution y Endometril Cells. Epithelil (Figure 1(); n=5)nd stroml(figure 1(); n=5) ells derived from pssge I were pled in 24-well plte in the ulture medium DMEM/Hm s F-12 supplemented with 1% FCS nd ntiioti nd ntimyoti solution. Agin, when ells rehed 9% of onfluene, the medium ws repled with fresh DMEM without phenol red (Sigm- Aldrih, Mdison, USA; D#296) supplemented with.1% BSA nd ntiiotis nd ntimyoti solution. The epithelil ndstromlellswereinutedwithvehileorwithp 4 (1 7 M), E 2 (1 9 M) or P 4 +E 2 (1 7 M/1 9 M) dded to the ulture medium for 24 h. The doses for ovrin steroids were determined sed on our former work [4]. Further on, the medium ws repled with fresh DMEM without phenol red supplemented with.1% BSA nd ntiiotis nd ntimyoti solution. Epithelil nd stroml ells were then inuted with IL-1α/IL-1β/IL-6 (1 ng/ml) for the next 24 h. The oxytoin (; 1 7 M) ws used s positive ontrol. Then, onditioned medi were olleted into tues with 5 μl EDTA, 1% etylsliyli id solution (Sigm-Aldrih, Mdison, USA, #A293), nd frozen t 2 Cuntilfurther PG mesurement. The totl volume of 25 μl TRI Regent (Sigm-Aldrih, Mdison, USA, #T9424) ws dded to eh well ontining ells for single-step DNA isoltion. Cells from four wells were then polled. Deoxyrionulei id ws isolted ording to TRI Regent mnufturer proedure. Deoxyrionulei id ontent ws used to stndrdize the results Experiment 3: The Effet of Ovrin Steroids on IL Reeptor mrnas Trnsription in Epithelil nd Stroml Cells

3 Meditors of Inflmmtion 3 () () () (d) Figure 1: Representtive morphology of ultured equine endometril: () epithelil ells nd () stroml ells. () Epithelil ells identifition y immunofluoresent stining for ytokertin; (d) stroml ells identifition y immunofluoresent stining for vimentin. The sle r = 5 μm (mgnifition: 4). Culture. Stroml (n =5)ndepithelil(n=5) ells derived frompssgeiwerepledin24-wellplte.whentheells rehed 9% of onfluene, the medium ws repled with fresh DMEM without phenol red supplemented with.1% BSA nd ntiiotis nd ntimyoti. Epithelil nd stroml ellswereinutedwithvehileorwithp 4 (1 7 M), E 2 (1 9 M) or P 4 +E 2 (1 7 M/1 9 M) dded to the ulture medium for 24 h. After tht, ulture medium ws removed ndellswerewshedtwiewithpbs.toehwell25μl of Fenozol ws dded nd the ells were removed nd kept frozen until RNA isoltion Methods Totl RNA Isoltion nd DNA Synthesis. Totl RNA ws extrted from epithelil nd stroml ells from Experiment 2, fter ulture using the Totl RNA Prep Plus Kit (A&A Biotehnology, Gdnsk, Polnd) ording to the mnufturer s instrutions. Rionulei id smples were stored t 8 C. Before use, RNA onentrtion nd qulity were determined spetrophotometrilly nd with grose gel eletrophoresis. The sorne rtio t 26 nm nd 28 nm (A 26/28 ) ws pproximtely 2. The mount of 1 μgof RNAwsreversedtrnsriedintoDNAusingQuntiTet Reverse Trnsription Kit (Qigen, #25311) ording to the mnufturer s instrution. The DNA ws stored t 2 C until rel-time PCR ws rried out Rel-Time PCR. Rel-time PCR ws performed with n ABI Prism 73 sequene detetion system using SYBR Green PCR mster mix (Applied Biosystems, Foster City, CA, USA, #439155). The sequenes for equine IL-1RI, IL- 1RII, IL-6Rβ, nd ACTB primers were previously pulished [23]. After preliminry study, ACTB wshosensthe est housekeeping gene. All primers were synthesized y GenoMed(Wrszw,Polnd).Totlretionvolumews 2μL nd ontined 1 μl DNA (1ng/μL), 2 μl forwrd nd reverse primers eh (25 nm) nd 1 μl SYBRGreen PCR mster mix. Rel-time PCR ws rried out s follows: initil denturtion (1 min t 95 C), followed y 4 yles of denturtion (15 s t 95 C) nd nneling (1 min t 6 C). After eh PCR retion, melting urves were otined y stepwise inreses in temperture from 6 to 95 Ctoensure single produt mplifition. The produt speifiity ws lso onfirmed y eletrophoresis on 2% grose gel. The dt were nlyzed using the method desried y Zho nd Fernld [24] PG nd Ovrin Steroids Determintion. The onentrtion of PGE 2 in the onditioned medium ws determined

4 4 Meditors of Inflmmtion using Prostglndin E 2 EIA kit (Cymn) ording to the mnufturer s instrution. The onentrtion of PGF 2α ws determined using the diret enzyme immunossy (EIA) method s desried previously y Uenoym et l. [25] with modifition. The stndrd urve for PGE 2 rnged from 16.5 pg/ml to 1 pg/ml. The intr- nd interssy oeffiients of vrition (CV) were 3.9% nd 8.1%, respetively. The stndrd urve for PGF 2α rnged from.19 ng/ml to 5 ng/ml nd CV were 4.7% nd 9.8%, respetively. The onentrtion of P 4 in lood plsm ws determined using EIA s desried previously [26]. The stndrd urve for P 4 rnged from.925 ng/ml to 25 ng/ml nd intrnd interssy CV were 3.7% nd 8.4%, respetively. The ntiodies (Anti-P4, ode SO/91/4; kindly donted y Dr. S. Okrs,Wrmi-MzuryUniversity,Olsztyn,Polnd)were hrterized previously [27]. The onentrtions of E 2 in lood serum were ssyed y rdioimmunossy (RIA) fter extrtion with diethyl ether (extrtion effiieny: 89%) s desried [28]. The ntiody (Anti-E2, ode BS/88/754; gift from Dr. B. Szfrnsk, Wrmi-Mzury University, Olsztyn, Polnd) ws hrterized previously [29]. The intr- nd interssy Cv verged 4.2%nd8.1%,respetively.TheE 2 stndrd urve rnged from.5 to 8 pg/ml, nd the effetive dose for 5% inhiition (ED5) of the ssy ws 1.98 pg/ml. The intr- nd interssy Cv verged 5.2% nd 9.5%, respetively Sttistil Anlysis. The dt re shown s the men ± SEM of vlues otined in seprte experiments, eh performed in qudruplite. The sttistil nlysis of Experiment 1 nd Experiment 3 ws performed using prmetri one-wy ANOVA followed y Newmnn-Keuls omprison test (GrphPd Softwre version 5, Sn Diego, USA). The sttistil nlysis of Experiment 2 ws performed using nonprmetri one-wy ANOVA Kruskl-Wllis followed y Dunns test. The results were onsidered signifintly different when P < Results 3.1. Experiment 1: The Effet of Ovrin Steroids on Epithelil nd Stroml Cell Prolifertion. Interleukins 1α nd IL- 6 ugmented epithelil ell prolifertion ompred to the ontrol group (Figure 2(); P <.1). In turn, only IL-6 ugmented stroml ell prolifertion ompred to the ontrol group (Figure 2(); P <.1) Experiment 2: The Effet of Ovrin Steroids on Cytokine- Stimulted PG Prodution y Endometril Cells. The sl in vitro PGE 2 nd PGF 2α seretion from epithelil ells ws 1.6 ±.924 ng/μg DNAnd2.63 ±.343 ng/μg DNA, respetively. The sl in vitro PGE 2 nd PGF 2α seretion from stroml ells ws 2.2 ±.332 ng/μg DNAnd1.38 ±.231 ng/μg DNA, respetively. Oxytoin (positive ontrol) ugmented PGE 2 nd PGF 2α seretion from epithelil ells stromlellsompredtotherespetiveontrolgroup(p<.5; Figure 3 to Figure 5). Interleukin 1α ugmented PGE 2 nd PGF 2α y epithelil (P <.1; P <.5; respetively; Figures 3() nd 3())ndstromlells(P <.5;Figures3() nd 3(d)) ompredtotherespetiveontrolgroup.epithelilells pretretment with E 2 or P 4 +E 2 deresed IL-1α-stimulted PGE 2 seretion ompred to the only IL-1α-stimulted group (P <.5; Figure 3()). However, epithelil ells pretretment with E 2 or ugmented IL-1α-stimulted PGF 2α seretion ompred to only IL-1α-stimulted group (P <.1; Figure 3()). Stroml ells pretretment with E 2 or P 4 +E 2 ugmented IL-1α-stimulted PGE 2 (P <.1; Figure 6()) ndpgf 2α (P <.1; Figure 3(d)) seretion ompred to the respetive only IL-1α-stimulted group. Interleukin 1β ugmented PGE 2 nd PGF 2α y epithelil (P <.5; P <.1; respetively, Figures 4() nd 4()) nd stroml ells (P <.5; P <.1; respetively, Figures 4() nd 4(d)) ompred to the respetive ontrol group. Epithelil ell pretretment with ugmented IL-1β-stimulted PGE 2 ompred to only IL-1β-stimulted group (P <.1; Figure 4()). Stroml ells pretretment with E 2 or ugmented IL-1β-stimulted PGE 2 seretionompredtoonlyil-1β-stimulted group (P <.1; Figure 4()). But stroml ells pretretment with P 4 deresed IL-1β-stimulted PGE 2 ompred to only IL-1βstimulted group (P <.5; Figure 4()). In turn, stroml ell pretretment with P 4,E 2,orP 4 +E 2 ugmented IL-1βstimulted PGF 2α ompred to only IL-1β-stimulted group (P <.1; Figure 4(d)). Interleukin 6 ugmented PGE 2 nd PGF 2α seretion y epithelil ells (P <.5; Figures 5() nd 5()) nd PGF 2α seretion y stroml ells (P <.5; P <.1; respetively, Figure 5(d)) ompred to respetive ontrol group. Epithelil ell pretretment with E 2 ugmented IL-6- stimulted PGE 2 seretionompredtoonlyil-6-stimulted group (P <.1; Figure 5()). Additionlly, stroml ell pretretment with E 2 or P 4 +E 2 ugmented IL-6-stimulted PGE 2 seretion ompred to only IL-6-stimulted group (P <.1; Figure 5()). Stroml ell pretretment with P 4,E 2 or P 4 +E 2 ugmented IL-6-stimulted PGF 2α seretion ompred to only IL-6-stimulted group (P <.1; Figure 5(d)) Experiment 3: The Effet of Ovrin Steroids on IL Reeptor mrnas Trnsription In Epithelil nd Stroml Cells Culture. 17-β estrdiol nd/or P 4 upregulted IL-1RI mrna trnsription in epithelil ell ompred to ontrol group (P <.1; Figure 6()). 17-β estrdiol or P 4 upregulted IL-1RII mrna trnsription in epithelil ells ompred to ontrol group (P <.1; Figure 6()). In turn, E 2 downregulted IL-1RI nd P4 + E2 downregulted IL-1RII mrna trnsription in stroml ells ompred to ontrol group (P <.1; Figures6() nd 6(d)). 17-β estrdiol nd P 4 +E 2 upregulted IL-6Rβ mrna trnsription in epithelil ells ompred to ontrol group (P <.1; Figure 6(e)). 17-β estrdiol nd/or P 4 upregulted IL-6Rβ mrna trnsription in stroml ells ompred to ontrol group (P <.5; Figure 6(f)). Additionlly, E 2 nd P 4 +E 2 inresed the IL-1RI/IL-1RII mrna trnsription rtio in epithelil nd stroml ells (P <.5;Figures 7() nd 7()).

5 Meditors of Inflmmtion 5 Cell prolifertion fold of stimultion IL-1α IL-1β IL-6 (1 ng/ml) (1 ng/ml) (1 ng/ml) Cell prolifertion fold of stimultion IL-1α IL-1β IL-6 (1 ng/ml) (1 ng/ml) (1 ng/ml) () () Figure 2: The effet of IL-1α (1 ng/ml), IL-1β (1 ng/ml), or IL-6 (1 ng/ml) on prolifertion of () epithelil ells (men ± SEM; n=5) nd()stromlells(men± SEM; n=5)fter24hinution.allvluesreexpressedsn-fold hnge from ontrol. Asterisks indite signifint differenes ( P <.5; P <.1) from the respetive ontrol, s determined y prmetri one-wy ANOVA followed y Newmnn-Keuls omprison test. 7 7 Prostglndin E 2 (ng/μg DNA) fold of stimultion Prostglndin E 2 (ng/μg DNA) fold of stimultion P 4 E 2 P 4 E 2 IL-1α IL-1α () () Prostglndin F 2α (ng/μg DNA) fold of stimultion Prostglndin F 2α (ng/μg DNA) fold of stimultion P 4 E 2 P 4 E 2 IL-1α IL-1α () (d) Figure 3: The effet of ovrin steroids on IL-1α stimulted PGE 2 nd PGF 2α prodution y epithelil ((), ()) nd stroml ((), (d)) ells. The ells were treted with P 4 (1 7 M), E 2 (1 9 M), or P 4 +E 2 (1 7 /1 9 M) for 24 h. Then, the medium ws repled with fresh medium nd the epithelil ells were stimulted with IL-1α (1ng/mL)for24h.Oxytoin(;1 7 M)wsusedspositiveontrol.Allvluesre expressed s n-fold hnge from ontrol. letters,, nd indite signifint differenes (P <.5) etween groups, s determined y prmetrione-wyanova followedy Newmnn-Keuls omprison test.

6 6 Meditors of Inflmmtion Prostglndin E 2 (ng/μg DNA) fold of stimultion P 4 E 2 Prostglndin E 2 (ng/μg DNA) fold of stimultion P 4 E 2 IL-1β IL-1β () () Prostglndin F 2α (ng/μg DNA) fold of stimultion P 4 E 2 Prostglndin F 2α (ng/μg DNA) fold of stimultion P 4 E 2 IL-1β IL-1β () (d) Figure 4: The effet of ovrin steroids on IL-1β stimulted PGE 2 nd PGF 2α prodution y epithelil ((), ()) nd stroml ((), (d)) ells. The ells were treted with P 4 (1 7 M), E 2 (1 9 M), or P 4 +E 2 (1 7 /1 9 M) for 24 h. Then, the medium ws repled with fresh medium nd the epithelil ells were stimulted with IL-1β (1ng/mL)for24h.Oxytoin(;1 7 M)wsusedspositiveontrol.Allvluesre expressed s n-fold hnge from ontrol. letters,, indite signifint differenes (P <.5) etween groups, s determined y prmetrione-wyanova followedy Newmnn-Keuls omprison test. 4. Disussion A short numer of studies hrterized IL in the equine endometrium [23, 3]. However, we hve reently desried IL-1α, IL-1β, nd IL-6 immunololiztion in the equine endometrium [23]. We lso showed tht those ILs stimulted PG seretion through the upregultion of PG synthses mrnas trnsription in endometril explnts in vitro [23]. Additionlly, the equine endometril upregultion of IL-1α nd IL-6 mrnas trnsription together with n upwrd tendeny in IL-1β mrna trnsription ws demonstrted in erly pregnny [3]. However, it hs een shown tht ovrin steroids did not ffet IL-1α nd IL-6 mrnas trnsription in endometril explnts in vitro [3]. Interleukins stimulte PG prodution y endometrium in mny speies esides the mre [12, 31 34]. Tmur et l. [33] nd Hung et l. [34] showedthtil-1β upregulted PGE2 seretion nd PTGS-2 mrna trnsription in humn stroml endometril ells. Furthermore, the ility to produe PG in response to IL-1α in rt stroml ells ws onfirmed y Bny nd Kennedy [31]. In turn, Chen et l. [32]onfirmed tht humn epithelil ells re le to produe PG in response to IL-1α if the ulture medium is supplemented with rhidoni id (AA). Severl previous studies present diverse onlusions [12, 31 35]. Tnikw et l. [12] showed tht IL1α nd IL1β stimulted in dose-dependent mnner oth PGE2 nd PGF2α prodution in ovine stroml ells, ut this stimultory effet of oth IL1s ws not oserved in epithelil ells. Nonetheless, Betts nd Hnsen [35] showed tht IL- 1β hd no effet on PG prodution in ovine stroml ells, ut ugmented PGE2 nd PGF2α prodution y epithelil ells. A omprehensive study onerning IL-6 influene on PG prodution y endometril ells is lking. Our previous dt showed tht IL-6 stimulted PG prodution y equine endometril explnts in vitro [23]. Cyli hnges in the endometrium re omplex proess governed y the interply of severl signling pthwys tht ritilly regulte ell growth nd prolifertion. Ovrin steroids ply key role in these proesses. In the present study, we showed tht ovrin steroids re not only triggering endometril PG prodution, ut they lso modulte endometril ell response to IL. As previously shown, ovrin steroids stimulted PG prodution y equine of epithelil nd stroml ells [4]. The nlysis of our present findings shows tht E 2 n e modultor of endometril ell response to IL. Additionlly, it ws found tht the response to IL-1α nd IL-6 ouldestronglymodulted,whenompredtoil-1β.

7 Meditors of Inflmmtion 7 Prostglndin E 2 (ng/μg DNA) fold of stimultion P 4 E 2 IL-6 Prostglndin E 2 (ng/μg DNA) fold of stimultion P 4 E 2 IL-6 () () Prostglndin F 2α (ng/μg DNA) fold of stimultion P 4 E 2 Prostglndin F 2α (ng/μg DNA) fold of stimultion P 4 E 2 IL-6 IL-6 () (d) Figure 5: The effet of ovrin steroids on IL-6 stimulted PGE 2 nd PGF 2α prodution y epithelil ((), ()) nd stroml ((), (d)) ells. The ells were treted with P 4 (1 7 M), E 2 (1 9 M), or P 4 +E 2 (1 7 /1 9 M) for 24 h. Then, the medium ws repled with fresh medium nd the epithelil ells were stimulted with IL-6 (1 ng/ml) for 24 h. Oxytoin (; 1 7 M)wsusedspositiveontrol.Allvluesre expressed s n-fold hnge from ontrol. letters,, nd indite signifint differenes (P <.5) etween groups, s determined y prmetrione-wyanova followedy Newmnn-Keuls omprison test. It ws shown tht in the mre IL-1α nd IL-6, expression is upregulted during the folliulr phse of the estrous yle, following E 2 tion [23]. It should e highlighted tht the endometril tissue response to ting ftors results from epithelil nd stroml ell tivity. Our findings showed tht theinflueneofovrinsteroidsonil-1α-stimulted PG prodution is dependent on the ell or PG types. Additionlly, we showed tht IL-6-stimulted PG prodution is strongly inresed y E 2. Interestingly, lthough we hd seen no effet of IL-6 on PGE 2 seretion y stroml ells, the pretretment with E 2 nd P 4 +E 2 used n inrese of PGE 2 seretion. In turn, the modultion of IL-1β influene on PG prodution y ovrin steroids is less evident, when ompred to IL-1α oril-6.however,itshouldenotedthtil-1β lone hs the strongest influene on PG prodution from endometril ells ompred to IL-1α or IL-6. Regrding the present findings, the intertion etween PG, IL, nd ovrin steroids my e ruil for the lol regultion of equine endometrium. Bring in mind tht endometril IL-1α nd IL-6 expression re upregulted in folliulr phse of the estrous yle, their promotion of endometril ell prolifertion nd lso tht E 2 enhned their support of PG prodution; it my e ssumed tht these ytokines my ply role in lol hnges, suh s ngiogenesis, ell prolifertion, nd other proesses tking ple in endometrium. Additionlly, the ross-tlk etween PG, IL, nd ovrin steroids is highly likely to e determinnt for implnttion. 17-β estrdiol positively ffeted IL-1α- nd IL-6- stimulted PGE 2 prodution my e possile mehnism responsile for the ngiogenesis regultion nd ell prolifertion in endometrium. Prostglndin E 2 ts in n uto-/prrine mnner on prongiogeni ftors suh s vsulr endothelil growth ftor (VEGF) seretion nd ngiopoietin-1 nd ngiopoietin-2 expression [36 38]. Additionlly, PGE 2 nd PGF 2α influened epithelil ells prolifertion in humn endometril ells [7, 8]. Tsujii nd DuBois [9] showedthtpge 2 enhned prolifertion. It seems tht IL-1α nd IL-6 t on ell prolifertion diretly nd indiretly y PG stimultion. One Hned et l. [3] ould not detet the link etween ovrin steroids tion nd IL-1α nd IL-6 expression round implnttion period, it is possile tht this intertion on tht stge of the pregnny is relted to PG prodution. Synhronized development of the emryo to the lstoyst stge, endometrium differentition to the reeptive stte, nd ross-tlk etween the lstoyst nd uterine luminl

8 8 Meditors of Inflmmtion 25 IL-1RI 15 IL-1RI 2 Fold indution 15 1 Fold indution P 4 E 2 P 4 E 2 () () 4 3 IL-1RII IL-1RII Fold indution Fold indution 2 1 P 4 E 2 P 4 E 2 () (d) 25 2 IL-6Rβ 8 6 IL-6Rβ Fold indution Fold indution 4 2 P 4 E 2 P 4 E 2 (e) (f) Figure6:TheeffetofP 4 (1 7 M), E 2 (1 9 M), nd P 4 +E 2 (1 7 /1 9 M) on IL-1RI ((), ()), IL-1RII ((), (d)), nd IL-6Rβ ((e), (f)) mrna trnsription in epithelil ells(n = 5) nd stroml ells(n = 5) fter 24 h inution. Results re normlized ginst ACTB. Dt re presented s fold indution reltive to ontrol. Asterisks indite signifint differenes ( P <.5; P <.1; P <.1) fromthe respetive ontrol, s determined y nonprmetri one-wy ANOVA Kruskl-Wllis followed y Dunns test. epithelium re fundmentl to the implnttion proess [39]. In mie nd rts, E 2 is essentil for preprtion of the P 4 -primed uterus to the reeptive stte [39]. Vrious ftors inluding ytokines, growth ftors, homeoox trnsription ftors, nd PG prtiipte in emryo implnttion through uto-/prrine nd/or juxtrine mehnisms [39]. In ontrst to other speies, in the horse, the role of PG nd IL on the emryo implnttion is not desried. It ws demonstrted tht the trgeted disruption of PTGS-2 is the useofmultiplefiluresinmurinefemlereprodutive proesses tht inlude ovultion, fertiliztion, implnttion, nd deiduliztion [4, 41]. The onentrtions of PGs reelevtedintheresofinresedendometrilvsulr permeility ssoited with the initition of implnttion [42, 43] nd exogenous PGs n reverse, t lest prtilly, theeffetsofindomethinonimplnttioninrt[44].

9 Meditors of Inflmmtion IL-1RI/IL-1RII rtio 3 2 IL-1RI/IL-1RII rtio P 4 E 2 P 4 E 2 () () Figure7:TheeffetofP 4 (1 7 M), E 2 (1 9 M), nd P 4 +E 2 (1 7 /1 9 M) on IL-1RI:IL-1RII mrna trnsription rtio in epithelil ells ((); n=5) nd stroml ells ((); n=5) fter 24 h inution. Results re normlized ginst ACTB. Asterisks indite signifint differenes ( P <.5; P <.1) from the respetive ontrol, s determined y nonprmetri one-wy ANOVA Kruskl-Wllis followed y Dunns test. In turn, IL-1α nd IL-1β re involved in prodution of leukemi inhiitory ftor (LIF) [45], grnuloytemrophge olony-stimulting ftor (GM-CSF), nd olony stimulting ftor-1 (CSF-1) prodution [46]. Interleukin 1 stimultes prodution of metlloproteinses (MMP) nd omponents of the plsminogen tivtor (PA)/PA-inhiitor sde [47, 48] nd lso dereses onnexin 43 in humn endometril stroml ells [49]. It hs lso een suggested tht IL-6 my ontriute to tropholst growth nd plentl development in humns [5]. In our present work we pointed out the potentil intertion etween E 2, IL, nd PG. We suggest tht E 2 modultion of IL-1α- nd IL-6-stimulted PG prodution during the preimplnttion period my prtiipte in event orhestrtion, inluding differentition of endometril ells nd the vsulr endothelil ell hnges ompnying implnttion nd susequent plentl development. However, further studies re required to onfirm these ssumptions. The pretretment with omintion of P 4 nd E 2 ugmented PG prodution in response to IL in endometril ells. However, this enhnement, with one exeption, ws lwys followed y E 2 inrese of IL-stimulted PG prodution. Thus, one my onlude tht E 2 minly inreses IL-stimulted PG prodution. These results suggest tht the mehnism responsile for enhnement of ILstimulted PG prodution y steroids is different in epithelil nd stroml ells. In epithelil ells, this mehnism is ssoited to IL-1RI mrna trnsription fter ovrin steroids tretment, suggesting tht ovrin steroids inrese IL-1 effet on PG prodution vi upregultion of IL-1RI expression. In stroml ells, single tretment with E 2 nd P 4 did not ffet IL-1RI mrna trnsription. Possile meditors of E 2 tion in stroml ells re protein kinse A(PKA), nulerftor-κb (NF-κB), nd/or extrellulrsignl-regulted kinses 1/2 (ERK1/2), whih hve een reported to e involved in the ontrol of PGE2 seretion nd PTGS-2 expression in response to IL-1β in humn stroml ells [51] nd the upregultion of PTGS-2, through PKA tivtion in different types of tissues [52, 53]. As n exmple, E 2 hs een shown to intert with NFκB nd to modulte its tivity [54], stimulting PKA tivity in hippompl neurons [55, 56]. However, new studies re required to lrify the enhnement of IL-stimulted PG prodution y steroids. Additionlly, our findings onfirmed tht E 2 regultes IL-6-stimulted PG prodution through upregultion of IL-6Rβ mrna.however,theinflueneof E 2 on IL-6Rα expression should e investigted in the future. In summry, it hs een shown for the first time tht E 2 enhne IL-1α nd IL-6-stimulted PG prodution. It my e one of the mehnisms responsile for lol orhestrting events in endometril tissue during estrous yle nd implnttion. Additionlly, we suggest tht E 2 influene on IL-1- nd IL-6-stimulted PG prodution my result from tivtion of IL-1RI nd IL-6Rβ mrnas trnsription in endometril ells. Conflit of Interests The uthors delre tht there is no onflit of interests regrding the pulition of this pper. Aknowledgments This work ws supported y Grnt MAESTRO of the Ntionl Reserh Center nd dedited to DJS (no. 211/2/A/NZ5/338) nd AZS nd KO were supported y jointnpolish-jpnese projet under greement of PAS nd JSPS.

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12 MEDIATORS of INFLAMMATION The Sientifi World Journl Hindwi Pulishing Corportion Volume 214 Gstroenterology Reserh nd Prtie Hindwi Pulishing Corportion Volume 214 Journl of Hindwi Pulishing Corportion Dietes Reserh Volume 214 Hindwi Pulishing Corportion Volume 214 Hindwi Pulishing Corportion Volume 214 Interntionl Journl of Journl of Endorinology Immunology Reserh Hindwi Pulishing Corportion Disese Mrkers Hindwi Pulishing Corportion Volume 214 Volume 214 Sumit your mnusripts t BioMed Reserh Interntionl PPAR Reserh Hindwi Pulishing Corportion Hindwi Pulishing Corportion Volume 214 Volume 214 Journl of Oesity Journl of Ophthlmology Hindwi Pulishing Corportion Volume 214 Evidene-Bsed Complementry nd Alterntive Mediine Stem Cells Interntionl Hindwi Pulishing Corportion Volume 214 Hindwi Pulishing Corportion Volume 214 Journl of Onology Hindwi Pulishing Corportion Volume 214 Hindwi Pulishing Corportion Volume 214 Prkinson s Disese Computtionl nd Mthemtil Methods in Mediine Hindwi Pulishing Corportion Volume 214 AIDS Behviourl Neurology Hindwi Pulishing Corportion Reserh nd Tretment Volume 214 Hindwi Pulishing Corportion Volume 214 Hindwi Pulishing Corportion Volume 214 Oxidtive Mediine nd Cellulr Longevity Hindwi Pulishing Corportion Volume 214