Departments of Anatomy, Physiology, and Pharmacology, College of Veterinary Medicine, Auburn University, Auburn, AL 36849, USA 2

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1 Hindwi Publishing Corportion PPAR Reserch Volume 2008, Article ID , 10 pges doi: /2008/ Reserch Article Activtion of Penile Prodipogenic Peroxisome Prolifertor-Activted Receptor γ with n Estrogen: Interction with Estrogen Receptor Alph during Postntl Development Mhmoud M. Mnsour, 1 Hri O. Goyl, 2 Tim D. Brden, 1 John C. Dennis, 1 Den D. Schwrtz, 1 Robert L. Judd, 1 Frnk F. Brtol, 3 Eline S. Colemn, 1 nd Edwrd E. Morrison 1 1 Deprtments of Antomy, Physiology, nd Phrmcology, College of Veterinry Medicine, Auburn University, Auburn, AL 36849, USA 2 Deprtment of Biomedicl Sciences, Tuskegee University, Tuskegee, AL 36088, USA 3 Cellulr nd Moleculr Biosciences Progrm, Deprtment of Animl Sciences, Auburn University, Auburn, AL 36849, USA Correspondence should be ddressed to Mhmoud M. Mnsour, mnsom@uburn.edu Received 23 April 2008; Accepted 14 July 2008 Recommended by Crolyn Komr Exposure to the estrogen receptor lph (ERα) lignd diethylstilbesterol (DES) between neontl dys 2 to 12 induces penile dipogenesis nd dult infertility in rts. The objective of this study ws to investigte the in vivo interction between DESctivted ERα nd the prodipogenic trnscription fctor peroxisome prolifertor-ctivted receptor gmm (PPARγ). Trnscripts for PPARs α, β, nd γ nd γ1 splice vrint were detected in Sprgue-Dwley norml rt penis with PPARγ predominting. In ddition, PPARγ1b nd PPARγ2 were newly induced by DES. The PPARγ trnscripts were significntly upregulted with DES nd reduced by ntiestrogen ICI 182, 780. At the cellulr level, PPARγ protein ws detected in urethrl trnsitionl epithelium nd stroml, endothelil, neuronl, nd smooth musculr cells. Tretment with DES ctivted ERα nd induced dipocyte differentition in corpus cvernosum penis. Those dipocytes exhibited strong nucler PPARγ expression. These results suggest biologicl overlp between PPARγ nd ERα nd highlight mechnism for endocrine disruption. Copyright 2008 Mhmoud M. Mnsour et l. This is n open ccess rticle distributed under the Cretive Commons Attribution License, which permits unrestricted use, distribution, nd reproduction in ny medium, provided the originl work is properly cited. 1. INTRODUCTION Endocrine disruption, originlly limited to steroid receptor signling, now extends to include other members of the 48 reported nucler receptor superfmily [1]. Both peroxisome prolifertor-ctivted receptor gmm (PPARγ) nd estrogen receptor lph (ERα) re trgets for endocrine disrupting chemicls [2 4]. Recently, Goyl et l. showed tht neontl exposure of rts to the estrogenic endocrine disruptor diethylstilbestrol (DES) induced dipogenesis in penile corpus cvernosum by ctivtion of ERα [5 8]. In this model of DES-ERα ctivtion, DES exposure t dose of 0.1 to 0.12 mg/kg bw/dy, on lternte dys, from postntl dys 2 to12, resulted in infertility in 100% of the treted mle rts. Loss of fertility ws ssocited with bnorml ccumultion of ft cells in the corpus cvernosum penis, nd the ssocited loss of cvernous spces pprent s erly s postntl dy 18 (reviewed in [9]). It remins unknown, however, whether this penile ERα-induced dipogenesis is medited by ctivtion of constitutively expressed or DES-induced PPARγ. Both ERα nd PPARγ pthwys re implicted in ft regultion. First, recent findings suggest tht PPARγ nd ERα pthwys involve shred coctivtors tht promote differentition of predipocytes into mture ft cells. For exmple, constitutive coctivtor of PPARγ (PG) is described s bon fide coctivtor tht cross rects with ERα independent of its lignd nd contins four LXXLL motifs tht re chrcteristicofnuclerreceptorcoctivtors[10]. Second, studies hve shown tht forced expression of PPARγ2 or PPARγ1 cn trigger the differentition of fibroblsts to dipocytes resulting in the ctivtion of dipocyte-specific genes nd lipid ccumultion [11].

2 2 PPAR Reserch The PPAR fmily consists of three isotypes tht include PPARα (NR1C1), PPARβ (lso known s PPARδ, NR1C2, FAAR, or NUC-1), nd PPARγ (NR1C3) [12 14]. A nucler receptor, PPARγ, is known to ply centrl role in ft metbolism nd dipocyte differentition [15, 16]. The PPARγ is present in two key isoforms, PPARγ1ndPPARγ2. The two isoforms stem from lternte promoters [17]. Compred to PPARγ1, PPARγ2 hs n dditionl 30 mino cids t the N-terminl end nd is distinctively expressed in dipose tissue, where it plys key role in dipogenesis [18]. These nonsteroidl receptors (i.e., do not medite effects of steroids) form prt of clss I nucler hormone receptor superfmily [19] nd function s lignd-ctivted trnscription fctors [20 22]. Ech of the three PPAR isotypes is constitutively expressed in certin reproductive nd nonreproductive rt tissues [23, 24], but their temporl nd cell-specific expression in penile tissue, with the exception of limited demonstrtion of PPARγ in penile corporl smooth muscle cells [25], hs not been shown. Further, no specific link is known between neontl ctivtion of ERα nd penile PPARγ. This is importnt given the expnded definition of the term endocrine disruptors to include ctivtion of metbolic sensors such s PPARs. A number of findings suggest involvement of PPARs in endocrine disruption either through direct receptor ctivtion or indirectly through crosstlk with other nucler receptors. First, in vitro studies demonstrted tht PPARγ nd ERα (the iconic receptor involved in endocrine disruption) re implicted in cross-tlk [26 28]. Second, some endocrine disruptor chemicls, such s monethylhexyl phthlte (MEHP), primry metbolite of diethylhexyl phthlte (DEHP), medite their toxic effect by PPARγ ctivtion [29, 30]. Third, severl nonbiologicl xenobiotics compounds cn ctivte PPARγ. For exmple, ctivtion of PPARγ with synthetic PPARγ ctivtors, such s ntidibetic drugs thizolidinediones (TZDs), improve insulin sensitivity but they undesirbly increse predipocyte differentition nd white dipose tissue mss [31 33]. Consistent with this dipogenic effect, reduced PPARγ level, s in mice with heterozygous (PPARγ +/ ) deficiency, is ssocited with reduced white dipose tissue mss [34]. Findings relted to interction between ERα nd PPARγ in the forementioned DES-penile rt model will illuminte potentil moleculr mechnism by which estrogen exposure t criticl period of development perturbs reproductive tissues. Therefore, we hypothesize tht DES-induced penile dipogenesis is ssocited with ERα-medited ctivtion of PPARγ. Objectives of this study were to (1) determine the bsl expression of PPARs (α, β, ndγ) in rt penis nd (2) evlute the neontl modultory effect of ERαctivtor DES on penile PPARγ s mrker of undesirble dipogenesis. 2. MATERIALS AND METHODS 2.1. Animls nd tretments This DES study ws performed in collbortion with Dr. Hri Goyl t Tuskegee University using mle pups from pregnnt femle Sprgue-Dwley (SD) rts (Hrln Sprgue- Dwley, Indinpolis, Ind, USA). All niml procedures were pproved by Institutionl Animl Cre nd Use Committee t Tuskegee University. In ll experiments, rts were mintined using stndrd housing conditions including constnt temperture of 22 C, d libitum wter nd feeding, nd 12:12 hours light drk cycle. Two experiments were conducted. In experiment 1, three groups of mle pups (n = 5per group, ll were littermtes) received subcutneous injections of 25 μl of olive oil (control), oil contining DES (0.1 mg/kg, Sigm-Aldrich, St. Louis, Miss, USA), or DES plus ICI 182, 780 (16.6 mg/kg, ICI; Tocris Bioscience, Ellisville, Miss, USA) dily on postntl dy 2 to 6. Rts in experiment 1 were scrificed t 28 dy of ge. ICI 182, 780 is high-ffinity estrogen receptor ntgonist (IC 50 = 0.29 nm) nd is lso considered high-ffinity lignd for the membrne estrogen receptor GPR30 (Tocris Bioscience). In experiment 2, two groups of mle pups (n = 4 per group) received DES (1 mg/kg) or olive oil (control) every other dy for 6 dys strting t postntl dy 2. Penile tissues were collected from rts scrificed t 120 dys of ge (dulthood). Smll sections of the penile shft tissue from ech rt in experiment 1 nd 2 were fixed overnight in 4% prformldehyde for IHC or ft stining, nd the reminder of the shft tissue ws frozen in liquid nitrogen nd stored t 80 C for RNA extrction nd PCR nlysis. The doses used for end-point evlution t 28 nd 120 dys post-tretment were bsed on previous publictions from our group tht showed DES prentl exposure (between postntl dys 2 to12) t dose rnge of 0.1 to 0.12 mg/kg/dy, or higher (1 mg/kg/dy) result in similr bnorml penile development nd dipogenesis [5, 8] Totl RNA isoltion Totl RNA ws isolted from the body of the penis using TRIZOL regent (Invitrogen-Life Technologies Inc., Crlsbd, Clif, USA), ccording to the mnufcturer s protocol. RNA concentrtions were estimted t 260 nm nd the rtio of 260/280 ws determined using UV spectrophotometry (DU640, Beckmn Coulter Fullerton, Clif, USA). The integrity of ech RNA smple, indicted by the presence of intct 28S nd 18S ribosoml RNA, ws verified by denturing grose gel electrophoresis. RNA smples were treted with DNse (Ambion Inc.) to remove possible genomic DNA contmintion. Smples with 260/280 rtio of 1.8 were used Conventionl end-point nd rel-time PCR Expression of mrna for PPAR (α, β, ndγ) isotypesws initilly determined by conventionl end-point RT-PCR with primers designed using primer quest softwre nd synthesized by Integrted DNA Technology (IDT Inc, Corlville, Iow, USA) from previously published rt sequences (see Tble 1). Subsequently, semiquntittive RT-PCR for complifiction of PPARs nd S-15 (known s Rig; smll subunit ribosoml protein used s house keeping gene) ws performed to determine the reltive expression levels of

3 Mhmoud M. Mnsour et l. 3 Tble 1: PCR primer sets, sequence, product size (bp), nucleotide (nt) loction, nd GenBnk ccession numbers for rt PPARs used in this study. Note tht common ntisense oligoprimer (sequence in bold) ws used for PPARγ1, PPARγ1b, nd PPARγ2. Product/ Product size nt Sense primer Antisense primer ccession# (bp) loction PPARα 5-TTG TGA CTG GTC AAG CTC AGG ACA-3 5-TCG TAC G AGC TTT AGC CGA ATA NM PPARβ U40064 PPARγ NM PPARγ1 AF TAA CGC A CTT CAT CAT A CGA-3 5-TTG ACA GCA AAC TCG AAC TTG GGC TCT A GCA TTT CTG CTC CAC ACT-3 5-ATA CAA ATG CTT TGC CAG GGC TCG CTG ACG AGG TCT CTC TC G GCT G-3 5-AGC AAG GCA CTT CT GAA A GA PPARγ1b AF CAG CGC TAA ATT CAT CTT AAC T-3 5-AGC AAG GCA CTT CTG AA A G A PPARγ AB GAG CAT GGT G TTC GCT GA-3 5-AGC AAG GCA CTT CTG AA A G A-3 AF Y12882 PPARγ/ERα (primers for rel-time PCR were obtined from Superrry Inc) [PPARγ/ERα] 190/179 NM (Sequence re not disclosed by the Compny) respectively [PPARγ] NM [ERα] Gpdh DQ ATG ATT CTA C ACG GCA AG-3 5-CTG GAA GAT GGT GAT G CGT T BC BC Rig/S15 (Ambion) 5-TTC CGC AAG TTC A TAC C-3 5-CGG GGC CGG A TGC T TTA CG BC PPAR isotypes. Verifiction of ccurte PCR products ws confirmed by determintion of the expected size of PCR bnds nd by sequence nlysis of generted mplicons t Auburn University sequencing fcility. The resulting sequences for the three PPAR isotypes were mtched with previously published rt sequences in GenBnk (ccession number NM013196, U40064, nd NM for PPARα, PPARβ, nd PPARγ, resp.) using Chroms 2.31 softwre (Technelysium Pty ltd, Tewntin Qld 4565, Austrli). PPARγ splice vrints or subtypes were identified using specific primers designed for rt PPARγ1 nd PPARγ1b synthesized by IDT Inc. (Tble 1). Liver nd white dipose tissues from dult Sprgue-Dwley rts in experiment 2 were used s positive controls for PPARγ1 [35] ndpparγ2 [18], respectively. The mplifiction protocol ws s follows: initil cycle for 3 minutes t 95 C, nd 30 cycles ech t (95 C for 30 seconds, 55 C for 30 seconds, nd 72 Cfor 30 seconds) followed by finl extension cycle t 72 Cfor 7 minutes. PCR rections were performed on Robocycler (Strtgene Inc, L Joll, Clif, USA) nd products were nlyzed electrophoreticlly on 2% (w/v) grose gels. The intensity of the PCR bnds ws determined using Fluor- S multi-imging nlysis system (Bio-Rd, Hercules, Clif, USA). Level of mrna for PPARs ws normlized to the levels of S-15 housekeeping gene. Quntittive rel-time PCR (Bio-Rd, MyiQ TM ) for determintion of expression levels of PPARγ nd ERα mrna ws performed in 25-μL rection mixture contining 12.5 μlrt 2 rel-time SYBR/Fluorescein Green PCR mster mix, 1 μl first strnd cdna, 1 μlrt 2 vlidted PCR primer set for PPARγ or ERα (Super Arry Bioscience Corportion, Frederic, Md, USA), nd 10.5 μl PCR-grdewter(Ambion Inc). Smples were run in 96-well PCR pltes (Bio-Rd, Hercules, Clif, USA) in duplictes, nd the results were normlized to GAPDH (see primer set in Tble 1) expression. The mplifiction protocol ws set t 95 Cfor15minutes for one cycle, nd 40 cycles ech t (95 C for 30 seconds, 55 C for 30 seconds, nd 72 C for 30 seconds) followed by melting curve determintion between 55 Cnd95 Cto ensure detection of single PCR product. Templte RNA from rt white dipose tissue nd penis were used for determintion of mplifiction efficiencies for (ERα/PPARγ) trgets nd GAPDH by generting stndrd curves. Curves were generted by using seril 10-fold dilutions totl RNA

4 4 PPAR Reserch nd plotting the log dilution ginst C T (threshold cycle) vlue obtined for ech dilution. The Person s correltion coefficient (r) vlue for ech generted stndrd curve ws 0.98, nd the clculted mplifiction efficiency ws between 98.5 to 99% Immunohistochemistry (IHC) Immunolocliztion of PPARγ in penile tissue ws performed using mouse nti-pparγ IgG1 monoclonl ntibody (sc7273, Snt Cruz Biotechnology Inc, Snt Cruz, Clif, USA) rised ginst C-terminus sequence of humn nd mouse PPARγ (similr to the corresponding rt sequence). The ntibody detects PPARγ1, PPARγ2 nd, to lesser extent, PPARα nd PPARβ of rt, mouse, nd humn by IHC using prplst-embedded tissues. Approximtely 5-mmlong penis sections from the middle of the body of the penis were fixed in 4% prformldehyde for 48 hours, embedded in Prplst (Sigm-Aldrich), nd cut t 5-μm thickness [7]. Mounted penis sections were deprffinized in Hemo- D (Scientific Sfety Solvents, Keller, Tex, USA) nd hydrted to distilled wter (dh 2 O). The slides were trnsferred to rckndplcedin1lof10mmsodiumcitrte(ph6.0). The beker ws plced on hot plte, llowed to come to boil nd tissues were boiled for 20 minutes. When the citrte solution cooled to ner room temperture (RT), the slides were trnsferred to glss stining dish nd equilibrted in phosphte buffered sline (PBS) (Sigm-Aldrich, ST Louis, Miss, USA). After 20 minutes incubtion in blocker (5% norml got serum, Sigm-Aldrich) nd 2.5% BSA (Sigm) in PBS, slides were wshed briefly in PBS. Anti-PPARγ, diluted 1:20 in blocker, ws pplied nd the sections were left to incubte overnight t RT. Next dy, slides were wshed 3x in PBS, 3 minutes ech, nd tissues were incubted with Alex 488-conjugted got ntimouse IgG (Moleculr Probes, Eugene, Ore, USA) for 1 hour t RT. After wshing two times in PBS, 3 minutes ech, slides were mounted with VectShield (Vector Lbortories, Burlingme, Clif, USA), nd the coverslips were seled. The sections were exmined using Nikon TE2000E microscope nd digitl imges were generted using n ttched Retig EX D digitl cmer (Q Imging, Burnby, BC, Cnd). Penile tissue sections from ll 28-dy treted rts were exmined. Representtive microgrphs from different penile histologicl structures were shown for untreted control rts, nd for rts treted with DES or DES + ICI Ft stining Histochemicl demonstrtion of ft ws performed s previously described [7]. Briefly, tissue sections from penile body, pproximtely 5 mm-long, were fixed for 24 hours in 4% formldehyde, followed by en bloc stining of ft for 8 hours with 1% osmium tetroxide dissolved in 2.5% potssium dichromte solution. Specimens were then processed for prplst embedding nd cut t 5-μm thickness. Deprffinized sections were exmined for blck stining indictive of ft cells using light microscopy Sttisticl nlyses Anlysis of rel-time PCR dt for reltive gene expression level (fold chnge of trget reltive to control) ws performed using modifiction of the delt delt Ct method (ΔΔ CT) s described previously [36]. Sttisticl differences between tretment groups were performed using Sigm Stt sttisticl softwre (Jndel Scientific, Chicgo, Ill, USA). Δ CT for rel-time PCR dt [37], nd intensity vlues (for semiquntittive RT-PCR dt) were subjected to nlyses of vrince. Experimentl groups with mens significntly different (P <.05) from controls were identified using Holm-Sidk nd Tukey tests. When dt were not distributed normlly, or heterogeneity of vrince ws identified, nlyses were performed on trnsformed or rnked dt. 3. RESULTS 3.1. Detection nd sequence nlysis of PPAR nd ERα trnscripts in the body of the penis Primer sets used in this study re shown in Tble 1. Trnscripts for three PPAR isoforms (α, β, ndγ) were detected, lbeit t different levels, in penile tissue from norml control dult (120 dys) rts (Figure 1, prts A1 nd A2). Semiquntittive RT-PCR nlysis of PPARs indicted predominnt expression of PPARγ mrna when compred with PPAR (α nd β) isoforms (Figure 1(B)). Sequence nlysis nd lignment with published sequence dt confirmed the identity of ll three PPAR isoforms. Tretment with DES induced over three-fold-increse (3.38) in ERα trnscripts in 28-dy-old rts compred to over two-foldincrese (2.5) in 120-dy-old dult rts when ech ge group ws compred with its respective untreted controls (Figure 2). Similrly, DES induced slightly over seven-foldincrese (7.1) in PPARγ trnscription level in 28-dy-old rts compred with over six-fold-increse (6.8) in 120- dy-old dult rts (Figure 3). The upregultion of PPARγ expression by DES in 28-dy-old rts ws brogted when rts were cotreted with DES nd ICI 182, 780 (Figure 4). The differences in the trnscriptionl level of penile ERα nd PPARγ between the DES-treted rt groups (28 versus 120- dy-old rts) were not significntly different. Becuse of the reltively high expression of penile PPARγ in the 28-dy-old rts subsequent studies for determintion of splice vrints nd PPARγ protein expression were performed in the 28- dy-old rts DetectionofPPARγ splice vrints nd rel-time PCR dt In order to determine which PPARγ splice vrint is expressed in the body of norml nd DES-treted rts, primers (Tble 1) were designed to mplify the two known rt PPARγ1 nd PPARγ1b splice vrints using conventionl end-point RT-PCR. Splice vrint nlyses reveled expression of PPARγ1 in norml 28-dy-old rt penis. However, in ddition to PPARγ1, PPARγ1b nd PPARγ2 were newly induced by DES tretment (Figure 5).

5 Mhmoud M. Mnsour et l. 5 Signl intensity (rbitry units %) bp A B α β γ S n = 3 α β γ PPAR type A α β γ Figure 1: (A1) nd (A2) RT-PCR mplifiction of three PPARs (α, β, ndγ) from the body of the penis of three (1, 2, nd 3) norml dult (120 dys) control rts. (A1) Shows complifiction of PPARs (α, β, nd γ (upper bnds) nd S15 (smll ribosoml subunit protein s housekeeping gene, lower bnds) in two representtive rts (1 nd 2). PCR mrkers were included in lne 1. Expected bnd sizes for S-15, PPARα, PPARβ, ndpparγ were 361, 492, 390, nd 533 bp, respectively. Identities of mplicons were further confirmed by sequence nlysis (see Section 2). Note tht the mpilicons for PPARβ nd S15 in lne 5 nd 6 were overlpped (compre run for PPARα, PPARβ, ndpparγ without S15 shown for rt 3 in (A2). In ll rts note the predominnt expression of PPARγ. 15 μl PCR products were loded per ech lne. (B) Grphic representtion of signl intensity for PPARs showing predominnt expression of PPARγ. Trnscript levels were normlized to the levels of S15 housekeeping gene. To clculte the intensity for PPARβ the men intensity of S-15 in lnes 2, 3, 8, nd 9 in Figure (A1) ws subtrcted from the combined intensity of PPARβ + S15 in lnes 5 nd 6 to obtin the intensity of penile PPARβ for rt 1 nd 2, respectively. P< Immunohistochemistry nd ft stining Immunohistochemistry results reveled PPARγ protein locliztion in trnsitionl epithelium of the urethr, nd the surrounding corpus spongiosum penis. It is lso expressed in stroml, endothelil, neuronl, nd smooth musculr cells of the cvernous sinuses locted in the corpus cvernousm region of norml 28-dy-old rt penis (Figures 6() nd 6(b)). Tretment with DES induced strong stining intensity for PPARγ protein in the peripherlly locted nuclei of newly induced dipocytes (Figure 6(), Pnel (c) with mgnified inset-box view in C2). PPARγ immunostining ws mrkedly reduced by ICI 182,780 tretment (Figure 6(b)). In unstined penile sections from 28-dy-old nd dult DES- ERα fold chnge reltive to GAPDH n = 5 n = 4 CONT-28 d DES-28 d CONT-dult DES-dult DES-post-tretment ge Figure 2: Rel-time PCR showing 3.38 nd 2.5 fold increse in ERα mrna in penile tissue of 28-dy-old (DES-28 d) nd dult rts (DES-Adult) neontlly treted with DES, respectively. Fold chnge ws clculted reltive to respective controls (CONT-28 d nd CONT-Adult). Dt (n = 4-5) re expressed s men ±SE. P<.05. PPARγ fold chnge reltive to GAPDH n = 5 n = 4 CONT-28 d DES-28 d CONT-dult DES-dult DES-post-tretment ge Figure 3: Rel-time PCR showing 7.1 nd 6.8 fold increse in PPARγ mrna in penile tissue of 28-dy-old (DES-28 d) nd dult rts (DES-Adult) neontlly treted with DES, respectively. Fold chnge ws clculted reltive to respective controls (CONT-28 d nd CONT-Adult). Dt (n = 4-5) re expressed s men ±SE. P<.01. treted rts, the new dipocytes were seen s empty spces similr to ft cells nd were specificlly loclized in the corpus cvernosum region of the penis (Figure 7, pnels(b) nd (d)). In ddition, stining with 1% osmium tetroxide confirmed tht the empty spces were cluster of ft cells (stined s blck grnules in Figure 7, pnels (c) nd (e)). No ft cells were seen in penile sections from rts treted with DES+ICI(Figure 7, pnels (f) nd (g)). 4. DISCUSSION This study demonstrted tht three PPAR trnscripts (α, β, nd γ) re constitutively coexpressed in norml rt penis

6 6 PPAR Reserch n=5 Negtive control pnels DES-28 d PPARγ fold chnge reltive to GAPDH 10 A G D 8 CONT-28 d 6 E H B n n 4 2 v n C I F Vl 0 CONT-28 d DES-28 d DES + ICI Tretment-ge Figure 4: Rel-time PCR dt showing ttenution of the effect of DES on PPARγ mrna by ER blocker ICI 182, 780 in 28-dy-old rts treted neontlly with either 25 μl of olive oil (CONT-28 d), oil contining DES (DES-28 d; 0.1 mg/kg bw), or DES plus ICI (DES + ICI; 16.6 mg/kg). ICI tretment significntly inhibited DES-induced PPARγ mrna [DES-28 d versus DES + ICI]. Comprison between control nd DES treted rts showed 7.1 fold increse in expression [CONT-28 d versus DES-28 d]. Letter [] indictes no significnt differences between CONT-28 d nd DES + ICI. Dt (n = 5) re expressed s men ±SE. P <.05. Fc C2 F2 () DES + ICI-28 d γ γ1 γ1b γ2 γ γ1 γ1b γ2 γ γ1 γ1b γ2 S Figure 5: RT-PCR mplifiction of PPARγ (γ, γ1, γ1b, nd γ2) splice vrints in the body of the penis (P) of control (lnes, 2 5) nd DES-treted (lnes, 7 10) 28-dy-old rts. Lnes were mplifiction products from RNA templte obtined from rt liver (L) (used s positive control for PPARγ1b). Lnes were RNA templte from rt white dipose tissue (F) (used s positive control for PPARγ1 nd PPARγ2). Note tht only PPARγ nd PPARγ1 were detected in (P) of norml rts. In contrst, in DES-treted rts enhnced expression of ll PPARγ splice vrints cn be noted. In ddition to PPARγ nd PPARγ1 expression (seen in norml rts), PPARγ1b nd PPARγ2 were induced by DES-tretment. As expected, PPARγ1b nd PPARγ2 were strongly expressed in (L) nd (F), respectively. S-15 (S, lne 20) is housekeeping gene mplified from (P) s control for RT-PCR conditions. The expected mplicon sizes for S, PPARγ1, PPARγ1b, PPARγ2, nd PPARγ re 361, 658, 618, 563, nd 533 bp, respectively. with PPARγ s the predominnt isotype. In ddition, it estblished tht some ERα synthetic lignds, such s DES, cn ctivte PPARγ subtypes when dministered t erly perintl dys. Further, upregultion of ERα by DES ws ssocited with corresponding increse in PPARγ suggesting synergistic interction between the two receptors. CONT-28 d Negtive control pnel L P [P] control [P] DES [L] control [F] control γ γ1 30 μm Vl J bp v M v 30 μm N n v (b) Figure 6: () Representtive immunohistochemicl stining for PPARγ protein in the body of the penis of 28-dy-old DES-treted (A), (B), nd (C) nd control untreted rts (D), (E), nd (F). Note tht PPARγ protein is expressed in DES-treted (DES-28 d) nd norml rts (CONT-28 d) with incresed intensity nd ft cells in DES-treted rts (see pnel (C)). Note expression in trnsitionl epithelium of the penile urethr () nd the surrounding corpus spongiousm () in (A) nd (D) nd in the endothelium of blood vessels nd smooth muscle cells in the dorsl rtery () nd vein (v), nd in nerve fibers of the dorsl nerve (n) of the penis (B) nd (E). Similr stining intensity cn be seen in the endothelium nd smooth muscles of the vsculr lcune (Vl) in the corpus cvernosum penis () in control norml rts (F). Note one contrsting difference is tht the cvernous spces in DES-treted rts in pnel (C) re replced with ft cells (Fc) tht show incresed stining intensity in the cell nucleus locted t cell periphery. Pnels (C2) nd (F2) show closer view of re outlined by insert box. Control sections (minus primry ntibody) were in pnels (G), (H), nd (I). Scle br = 30 μm. (b) IHC stining for PPARγ protein ws significntly reduced by ICI 182, 780 tretment [compre stining in pnels (J) nd (M) with (L) nd (N)]. Pnel (P) is negtive control (minus primry ntibody). Scle br 30 μm.

7 Mhmoud M. Mnsour et l. 7 v TA () (b) (c) (d) (f) 30 μm (e) (g) Figure 7: Microgrph sections from penile body of norml rt () nd rts treted neontlly with DES (b) (e) or DES + ICI (f)(g). Pnel () ws from norml dult rt stined with H nd E for demonstrtion of norml histologicl structures of the penis (: dorsl rtery; v: dorsl vein; : corpus cvernosum; : corpus spongiosum; : penile urethr; TA: Tunic lbugine). Pnels ((b), unstined) nd ((c), stined for ft with 1% osmium tetroxide) were from 28-dy-old rt. Pnels ((d), unstined) nd ((e), stined for ft with 1% osmium tetroxide nd presented s mgnified view of nd regions) were from dult rt (120 dys) treted neontlly with DES. Note the empty ppering spces of ft cells in regions in unstined sections (pnels (b) nd (d)). In sections stined with 1% osmium tetroxide (to confirm presence of ft) ft cells pper s blck grnules,. Pnels ((f), unstined) nd ((g), stined with 1% osmium tetroxide) were from 28-dy-old rt treted neontlly with DES + ICI. Note the bsence of empty ppering ft cells nd lck of ft stining in region. Sections from these rts were used for immunolocliztion of PPARγ in Figure 6 prts () & (b). Scle brs = 30 (E) nd in other pnels. Previous studies tht used in situ hybridiztion to determine the distribution of PPARs in rt tissues, including reproductive orgns, showed expression of PPARα nd PPARβ in somtic (Sertoli nd Leydig) nd in germ cells of the testis, but did not ddress expression of these two receptors in penile tissue [23, 24]. The role of PPARα nd PPARβ in the testis, however, remins unknown. Detiled study ddressing expression of PPARγ isotypes in penile tissue is lso lcking, with the exception of study tht showed limited penile PPARγ expression in corporl smooth muscle cells [25]. In this study, PPARγ nd PPARγ1 were detected in norml rt penis. However, DES s ERα ctivtor distinctively induced expression of PPARγ1b nd PPARγ2 splice vrints tht were not present in control untreted penile tissue. The induction of splice vrint PPARγ1b is in greement with previous in vitro studies tht demonstrted ctivtion of PPARγ1 by the endocrine disruptor monoethyl-hexylphthlte in C2C12 mouse skeletl muscle cell line [2], nd with MCF-7 brest cncer cells stimulted with E2, the nturl ERα lignd [38]. Further, the induction of PPARγ2 concurs with incresed dipogenesis observed in the corpus cvernousm penis s PPARγ2 is considered unique mrker for mture dipocytes, nd its forced induction is ssocited with terminl differentition of predipocytes or fibroblst cells to functionl mture dipocytes [11, 22]. The upregultion of PPARγ ws brogted by codministrtion of the type-ii ntiestrogen ICI 182,780, indicting tht DES effects were medited, t lest in prt, vi the estrogen receptor system. It is possible, however, tht ICI my hve directly repressed ctivtion of PPARγ s ICI ws previously shown to inhibit the ction of the selective PPARγ gonist BRL 48, 482 in MDA-MB 231 brest cncer cell culture in the bsence of ER [38]. One importnt difference between this study nd previous in vitro studies tht ddressed signl cross-tlk between PPARγ nd ERα using MCF-7 cells [38 40] is tht the ctivtion of ERα by DES in our study is ssocited with selective induction of PPARγ1b nd PPARγ2. This unique effect resulted in genertion of de novo dipocytes tht provide direct functionl proof for PPARγ2 induction. In contrst to our study, ctivted ERα by E2 lowers both bsl nd lignd-stimulted PPARγ-medited gene reporter ctivity in MCF-7 cncer cell culture [38]. Likewise, ctivtion of PPARγ in MCF-7 cell culture with the nturl PPARγ lignd cyclopentenone 15-deoxy-Δ12,14 prostglndin J2 (15d-PGJ2) inhibited estrogen-responsive elements [40]. Consequently, the MCF-7 cell culture studies suggest tht ERα nd PPARγ negtively regulte ech other. The reson for the difference between our study nd the forementioned in vitro dt could be relted to differences between in vitro nd in vivo milieu or to the deletionl mutnts used in the in vitro studies compred with the in vivo wild type receptors in our study. Another reson for the disgreement could be due to differences in coctivtors nd corepressors present in MCF-7 nd penile tissue cells or more importntly to differences in the lignds used. One plusible hypothesis, however, for the incresed trnscriptionl ctivtion of PPARγ1b nd PPARγ2 by DES-ctivted ERα is tht exposure of rts to DES t criticl neontl period of dys 1 to 12 is uniquely ssocited with reprogrmming of penile stroml or predipocytes to mture dipocytes [5 8]. In support of this concept, it is known tht postntl dys 1 to 5 in rodents coincide with period for reproductive trct nd dipocyte differentition [41]. Further, dt from other lbortories indicted tht neontl exposure of rodents to DES is ssocited with incresed whole body ft t dulthood

8 8 PPAR Reserch [42]. This novel dipogenic effect of DES ws proposed s model for the study of wht is clled developmentl obesity medited by erly exposure to endocrine disruptors [43]. The moleculr mechnism involved in DES-ERα-PPARγ trnsctivtion could be relted to two fctors. First, ctivted-erα could directly bind to PPAR response elements (PPREs) becuse the two receptors shre the cpcity to bind to the AGGTCA hlf-sites consensus sequences contined s plindrome or direct repet in estrogen response elements (EREs) nd PPRE sequences, respectively [44]. This mechnism could result in bidirectionl ctivtion of shred trget sequences between ERα nd PPARγ depending on ctivted receptor involved. Second, it is known tht estrogen could induce enzymtic conversion of prostglndin D2 (PGD2) nd the endogenous metbolites of the ltter cn directly ctivte PPARγ [45]. The ltter effect, however, ws not ssocited with induced PPARγ mrna [46] suggesting tht the first mechnism could be in ply in our study. The strong PPARγ protein expression in norml trnsitionl epithelium of the urethr nd the dorsl rtery nd vein of the penis indictes possible physiologicl role for PPARγ in the penis vsculture nd the urothelium of the urinry trct. Although this study did not ddress functionlity of PPARγ in the penis, current evidence suggests tht its constitutive expression in some tissues is linked to eicosnoids nd prostglndins (PGs) ctions [47, 48]. In this regrd, the terminl metbolite of the J series of PG, 15d-PGJ2, is considered the nturl ctivtor of PPARγ [48]. Sources of penile PGs could include synthesis by locl penile cells nd/or cells of the renl medull where PGs cn be trnsported vi the ureter nd pelvic urethr to the penis [49]. Among other functions, PGs re importnt meditors of inflmmtion, vsculr homoeostsis, nd pin ll of which my be relevnt to the pthophysiology of the penis. Stining with osmium confirmed the presence of new lipid-lden dipocytes in penile tissues of DES-treted rts. Previously, our group showed tht Sprgue-Dwley rts treted neontlly with DES ccumulted ft in the corpus cvernous penis [5 8] just s observed for the rts in the present study. The histologicl demonstrtion of DESinduced lipid buildup in the corpus cvernosum penis concurs with the newly induced dipocyte mrker PPARγ2 detected with RT-PCR. In penile tissue direct phrmcologicl ctivtion of PPARγ by the ntidibetic TZD pioglitzone reportedly blocked corporl veno-occlusive dysfunction in rt model of type 2 dibetes mellitus [25]. However, this effect ws ssocited with ft buildup suggesting tht direct ctivtion of penile PPARγ by TZDs or indirectly by ERα lignds, s in this study, could be potentil pthwy for development of undesirble dipogenesis. In conclusion, PPARs re currently considered potentil drug trgets for diverse conditions including, vsculr homoeostsis, dibetes mellitus, hyperlipidemi, inflmmtion, cncer, nd infertility [50 54]. 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