Localization of orexin B and receptor 2 for orexins in testicular cytotypes of the camelid alpaca (Vicugna pacos).

Reprod Dom Anim 2017; 1–7 wileyonlinelibrary.com/journal/rda © 2017 Blackwell Verlag GmbH 

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O R I G I N A L A R T I C L E

Localization of orexin B and receptor 2 for orexins in testicular

cytotypes of the camelid alpaca (Vicugna pacos)

G Liguori

1

_{| C Squillacioti}

1

_{| L Assisi}

2

_{| N Mirabella}

1

_{| E Langella}

3

_{| A Costagliola}

1

_|

A Vittoria

1 1_{DepartmentofVeterinaryMedicineand} AnimalProductions,UniversityofNaples FedericoII,Naples,Italy 2_{DepartmentofBiology,UniversityofNaples} FedericoII,Naples,Italy 3_{SchoolofAgricultural,Forestry,Food} andEnvironmentalSciences,Universityof Basilicata,Potenza,Italy Correspondence LoredanaAssisi,DepartmentofBiology, UniversityofNaplesFedericoII,Naples,Italy. Email:assisi@unina.it

Contents

TheorexinsA(OxA)andB(OxB)aretwohypothalamicpeptidesinvolvedinmany physiologicalfunctionsofthemammalianbody.Theyactthroughthebindingoftwo G-coupledreceptorsnamedreceptor1(OX₁)andreceptor2(OX₂)fororexins.The firstreceptorisspecificforOxA,whilethesecondbindsboththesubstanceswith equalaffinity.Theorexinsandtherelativereceptorshavebeentracedbymeansof differenttechniquesalsoattheperipheryofthebodyandparticularlyintheadrenals, and in gastrointestinal and genital organs. Aim of this work was to investigate the presenceofOxBandOX2bymeansofimmunohistochemistryandWesternblotting analysisinthetestisoftheSouthAmericancamelidalpaca,aspeciesprimarilybreed inChileandEcuadorandrecentlydiffusedinEuropewherethequalityofitswoolis particularlyappreciated.OxBimmunoreactivity(IR)wasfoundinthetubularcompart- mentofthetestiswherespermatogonia(resting),zygoteneandpachytenespermato-cytes,andspermatidsclearlyshoweddifferentlysizedandshapedcytoplasmicpositive structures.OX2-IRwasfoundbothintheinterstitialandtubularcompartmentsofthe testisandparticularlyinLeydigcellsandroundandelongatedspermatids.Western blottinganalysisoftestislysatesshowedthepresenceofaproteinbandwhosemo-lecularweightcorrespondedtothatcurrentlyassignedtoOX₂.Suchfindingseasily translatethehypothesisthatOxBanditsreceptor2playafunctionalrolebothinthe interstitialandtubularcompartmentsofthealpacatestis.

1 | INTRODUCTION

Two decades ago, the peptides orexin A (OxA) and orexin B (OxB) werediscoveredintherathypothalamusbytworesearchgroups(de Lecea etal., 1998; Sakurai etal., 1998) that worked independently. Suchpeptidesderivefromthecleavageofacommonprecursormol-ecule, the prepro-orexin, and bind two G-coupled receptors named

receptor1(OX₁)andreceptor2(OX₂)fororexins.Thefirstreceptor

isspecificforOxA,whilethesecondbindsboththesubstanceswith equalaffinity.

Thehypothalamicorexinsareinvolvedintheregulationofmany functions of the mammalian body such as food intake (Sakurai etal., 1998), sleep–wake cycle (Taheri, Ward, Ghatei, & Bloom,

2000),arterialbloodpressureandheartrate(Shirasaka,Nakazato, Matsukura,Takasaki,&Kannan,1999),sexualbehaviour(Muschamp, Dominguez,Sato,Shen,&Hull,2007),waterassumption(Kuniietal., 1999)andplasmacorticosteronelevels(Kuruetal.,2000).Boththe orexins are also involved in the modulation of the hypothalamic/ hypophyseal–adrenal/gonadal axes acting both indirectly, on the intermediate steps of the chains, and directly, on the final targets (Martyńskaetal.,2006;Voisin,Rouet-Benzineb,Reuter,&Laburthe, 2003).

Morerecently,theorexinsandtheirreceptors,beyondreferred astheorexinergiccomplex,werefoundtobepresentintheadrenals and in other peripheral organs belonging to the gastrointestinal and genital tract. Particularly, the presence of OxA, prepro-orexin

andOX₁hasbeendetectedintheprincipalcellsoftheepididymis andinsometesticularcytotypesoftheratandalpaca(Assisietal., 2012;Liguorietal.,2012,2014;Pavone,Tafuri,Avallone,Staiano, &Vittoria,2009;Tafurietal.,2009,2010),intheurethro-prostatic complexofcattle(Russoetal.,2008)andinthenormal,hyperplas-tic and carcinomatous prostate of humans (Valiante etal., 2013,

2015).TheexpressionofOX₁

mRNAhasbeendescribedinthetes-tis of sheep (Zhang etal., 2005) and chicken (Ohkubo,Tsukada, & Shamoto, 2003) and in the seminalvesicles, penis and epididymis of men (Karteris, Chen, & Randeva, 2004). By contrast, the

pres-enceofOxBandOX₂inthegenitaltractofmammalshasbeenstill

poorly studied.The expression of OxBwas described in rat testis by means of a radioimmunoassay (Mitsuma etal., 2000), and that of prepro-orexin mRNA was found in rat testis (Jöhren, Neidert, Kummer,Dendorfer,&Dominiak,2001)andhumanepididymisand penis (Karteris etal., 2004).The detection of mRNA codifying for

OX₂appearstobespeciesdependentbecauseitwasdemonstrated

in testis, epididymis, penis, seminal vesicles (Karteris etal., 2004) andprostate(Malendowicz,Szyszka,Ziolkowska,Rucinski,&Kwias, 2011)ofhumansanddeniedinthesheep(Zhangetal.,2005)andrat testis(Barreiroetal.,2004;Jöhrenetal.,2001;Zhengetal.,2014). However,itisprobablethatthepresenceofOxBanditsreceptor 2ingenitalorgansismoreabundantthandescribeduptonow.A recent study performed experimentally on the rat testis, in fact, assessedthatboththemoleculesarewidelydiffusedinthegonad alongthemajorityofthestagesofthegermcelldevelopingcycle (Liguorietal.,2017). Thestudyoftheorexinergiccomplexhasneverbeenapplied,at leastinourknowledge,tothegenitaltractofnon-conventionalrumi-nantsofzootechnicalinterest.Forthisreason,webeganaresearch onthepresenceandroleplayedbythecomplexinthetestisofthe camelidalpaca(Vicugna pacos)(Liguorietal.,2012),whosebreeding extendedinthelastyearsfromthelandsoforigintomanycountries intheworld.Thealpacaisacamelidphylogeneticallysimilartollama (Lama glama)andguanaco(Lama guanicoe)nativeoftheAndeanre-gions of South America, from Ecuador to southern Chile. In these countries, the alpaca breeding always played an important role for meat and wool production, and the recent diffusion of the species isparticularlyduetothequalityofitswoolwhichisgreatlyappreci-atedmainlyinUSA,AustraliaandEurope(Schwalm,Gauly,Erhardt,& Bergmann,2007).Atpresent,inItaly,38farmsgainedcreditforman-agementandbreedingofthisspeciesandwereofficiallyrecognizedby theassociationofalpacabreeders(Italpaca).

Obviously, the knowledge of the reproductive physiology of al-pacaisstillpoorlydevelopedbeingfoundedmainlyonpracticeasfar asmatingandselection.Incountrieshavingmoretemperateclimate thanthoseoforigin,thealpacaisconsideredanon-seasonalbreeder becausetheincreasedavailabilityoffoodsurelyenhanceditsfertility (Fowler,1998). Togivefurtherinsighttotheargument,herewedescribethepres-enceofOxBandOX₂ inthealpacatestisbymeansofanimmunohis-tochemicaltechniqueandassesstheexpressionofOX₂usingWestern blottinganalysis.

2 | MATERIAL AND METHODS

2.1 | Antibodies and chemicals

Mouseanti-OxA(MAB763)andanti-OxB(MAB734)monoclonalanti-bodiesandtheirsyntheticpeptideswereobtainedfromR&DSystems (Abingdon,UK)andfromTocrisBioscience(Bristol,UK),respectively; goatpolyclonalanti-OX₂antibody(sc-8074)anditsblockingpeptide (sc-8074P)fromSantaCruzBiotechnologies(SantaCruz,CA,USA); horseradishperoxidase-conjugatedrabbitanti-goatIgG(A-5420)and bovineserumalbumin(BSA)fromSigmaChemicalCo.(St.Louis,MO, USA); and biotinylated goat anti-mouse (BA-9200), rabbit anti-goat (BA-1000)secondaryantibodiesandavidin–biotincomplex(PK-6105) fromVectorLaboratories(Burlingame,CA,USA).SDS–PAGEandthe semidryapparatuswerepurchasedfromBio-Rad(Hercules,CA,USA); enhanced chemiluminescence kit (RPN 2109) from ECL Amersham (Little Chalfont, Buckinghamshire, UK) and marker proteins from ProSieveQuadColor(London,UK).

2.2 | Animals

The animals employed in this research were kept in optimal sani-taryandnutritionalconditionsinthe“DomusAlpaca”farm(Pratola Peligna,Italy)wheretheywerebredfollowingasemirangecondition. Fivehealthymalesofapproximatelysevenyearsofagewerechosen fororchiectomy.Thesurgicaloperationwasperformedaccordingto Fowler(1998).Thestudyprotocolwasincompliancewithourinstitu-tion’sethicalguidelines.AllprocedureswereapprovedbyItalianlaws regardinganimaluseinresearch(D.Lgs116/92).Allsubjectswere lackingofpreputialadhesionswhichisconsideredasignalofsexual maturity(Tibary&Vaughan,2006).Tissuecollectionwasperformed asdescribedbyLiguorietal.(2013).Afterremoval,thetesteswere finelycutinsmallsampleswhichwereeitherfixedinBouin’sfluidfor immunohistochemistryasdescribedindetailselsewhere(Squillacioti, DeLuca,Paino,Langella,&Mirabella,2009)orfrozeninliquidnitro-genandstoredat−80°CuntilusedforWesternblottinganalysis.

2.3 | Immunohistochemistry

Thefixedmaterialwasdehydratedinaseriesofascendingalcohols, embeddedinParaplastinvacuumandcutinto6-μm-thicksections. Toobtainconsecutivestainedmicroscopicfields,seriesof3-μm-thick sectionswerecutandmountedonnumberedslides.Theavidin–bio-tin–peroxidasecomplex(ABC)methodwasperformedaccordingthe protocolpreviouslydescribedbyDeLucaetal.,2014.Inthespecific step,mousemonoclonalanti-OxBandanti-OxAandgoatpolyclonal anti-OX₂primaryantibodies,1:200diluted,wereappliedonsections overnightat4°C.Thedayafter,theantibodieswereremovedbya triplePBSwashingandthesectionswereincubatedatroomtempera-ture(18–22°C)inbiotinylatedgoatanti-mouseIgGorrabbitanti-goat IgG,both1:200diluted,for30min.Afterwashing,afurtherincuba-tionwasperformedinfreshlypreparedABCreagentfor30min.3-3′ Diaminobenzidine was used as final staining. Some sections were

counterstained with haematoxylin to better localize the positivities. The specificity of the immunoreaction was tested by substituting theprimaryantibodywithPBSorpreabsorbingtheantibodywithan excess(100μg/ml)oftherelativeantigen.Controlsectionsresulted alwaysnegative.Asafurtherspecificitycontrolofthetwoantibod-iesdirectedagainstOxBandOxA,couplesofconsecutive3-μm-thick sectionswerealternatelystainedwiththemandcarefullycheckedfor theeventualcoexistenceofthetwopositivitiesinthesamestructure. Coexistence, infact, could imply cross-reactivity ofanantibody(or both) with the heterologous antigen(s). The preparations were ob- servedbyaNikonEclipseE600lightmicroscope,andmicrophoto-graphsweretakenbyaNikonCoolpix8400digitalcamera.

2.4 | Western blotting analysis

Frozen tissues were homogenized in a buffer (50mm Tris-HCl, pH

7.00; 150 mmNaCl;2%Triton;5mmEDTA;10mg/mlleupeptin;0.1

U/mlaprotinin;1mmPMSF)usinganUltra-Turraxhomogenizerand

centrifugedat16,000gfor20minat4°C.Theresultingsupernatants were characterized for protein concentration by Bio-Rad protein assay. Equal amounts of proteins, solubilized in boiling sodium do-decylsulphate(SDS)samplebuffer(2%SDS;5%L-mercaptoethanol;

66 mmTris,pH7.5;10mmEDTA),wereseparatedona12%SDS–

polyacrylamide gel. After electrophoresis, the gel was transferred to nitrocellulose using a semidry apparatus as described elsewhere (Squillaciotietal.,2012).Themembranewasblockedfor1hrat42°C

with5%BSAinTBSTbuffer(150mmNaCl;20mmTris-HCl,pH7.4;

0.3%Tween20),washedwithTBSTandincubatedfor2hratroom

temperaturewithgoatpolyclonalanti-OX₂antibody1:500dilutedin

2.5%BSA-containingTBST.Themembranewaswashedthreetimes with TBST and re-incubated for 1hr with horseradish peroxidase- conjugatedrabbitanti-goatIgG1:2,000dilutedin1%BSA-containing TBST.Proteinswerevisualizedbyanenhancedchemiluminescence kit.Markerproteinswereusedtoestimatethemolecularweightof each band.

3 | RESULTS

3.1 | Immunohistochemical evaluation of OxB and

OX

₂

in the alpaca testis

Immunohistochemistry allowed us to demonstrate the follow-ing OxB-containfollow-ing cytotypes in the alpaca testis: spermatogo-nia (Figure1a) and preleptotene (resting) (Figure1b), zygotene

F I G U R E   1   OxBIRincytotypes ofthealpacatestis.(a,b):Anisolated spermatogonium(a)andtworesting spermatocytes(b)arecloseincontactwith thetubularmembraneandshowclusters offinepositivegranulesalmostentirely fillingtheircytoplasm.(c,d):Zygotene (c)andpachytene(d)spermatocytes containintheirperinuclearcytoplasm asingle,intenselystained,granular structure,respectively,ovalorroundish inshape(arrows).(e,f):Round(e)and elongated(f)spermatidsshowedtwo differentIRstructuresintherespective cytoplasm:thefirst,arciforminshape, embracedtightlyportionoftheround spermatidnucleuswhilethesecond, punctiforminshape,waslocalizedinthe tailoftheoldercells(arrow).Avidin–biotin immunohistochemicaltechnique.Bars: 20 μm (a) (b) (c) (d) (e) (f)

(Figure1c)andpachytene(Figure1d)spermatocytesaswellasyoung (round)(Figure1e)andmature(elongated)(Figure1f)spermatids.In spermatogoniaandpreleptotenespermatocytes,thepositivemate-rialhadtheshapeofaclusterofmicrogranulesfillingalmostentirely the cytoplasm and driving the nucleus at the periphery of the cell. Couples of positive resting spermatocytes newly formed from the spermatogoniamitosiswereoftenseen.Inzygoteneandpachytene spermatocytes, the OxB immunoreactivity (IR) assumed the aspect of flattened or roundish granules, respectively, which were local-ized in close perinuclear position. Along the spermatid maturation, thepositivematerialchangedinshapeandlocalizationfollowingthe morphologicaltransformationofthecellwhich,asknown,isroundin theyoungelementsandbecameprogressivelyovalandelongatedin theolderones.Youngspermatidsshowedasemilunar-shapedposi-tivestructurecloselyadherenttothenuclearmembrane(Figure1e), whileolderelementscontainedaroundishgranuleattheperipheryof thecytoplasm.Pre-spermatozoaelongatedspermatidsshowedsuch agranuleintheirtailwhichwasconstantlyturnedtowardsthetubular lumen(Figure1f). Couplesof3-μm-thickcontiguoussectionsstainedalternatelyby anti-OxBandanti-OxAantibodiesshowedthatOxBIRnevercoex-istedwithOxA-IRinourpreparations(Figure2a–d). OX₂-IRwasfoundinLeydigcells(Figure3a)andround(Figure3b) andelongated(Figure3c)spermatids. InLeydigcells,thepositivematerialwasfinelygranularinshape anddiffusedinthecytoplasmwithoutanyparticulardistribution.Cells showingdifferentstainingintensitywereclusteredinsmallgroupsor intermingledtonegativeonesinthemajorityoftheinterstitialspaces. TheaspectandlocalizationofthespermatidIRwerecloselysimilarto thatjustdescribedforOxBinthesamecytotype.

3.2 | Expression of OX

₂

in tissue extracts

TheexpressionoftheOX₂proteininthealpacatestiswasdetected byWesternblottinganalysisusingagoatpolyclonalantibodyraised againstapeptidemappingneartheC-terminusofOX₂ofratorigin. Asacontroltissueofourexperimentwaschosentheratbrainnotori-ouslycontaininghugeamountofthereceptor(Cluderay,Harrison,& Hervieu,2002).Tissueextractsofalpacatestisandratbrain(Figure4) reactedwiththeanti-OX₂ antibodywhichrecognizedinthemapro-teinbandweighting40kDa,whichisthemolecularweightcurrently assignedtotheorexinreceptor2(Figure4,lanes1and2,respectively).

4 | DISCUSSION

Apreviousstudyfromourresearchgroupdescribedthepresence ofOxAanditsreceptor1inthealpacatestis(Liguorietal.,2012). Thepresentworkreportstheexpressionofthetwocounterpartsub-stances,OxBandOX₂,inthesameorgan,andthus,theexistenceof theorexinergiccomplexinthemalegonadoftheSouthAmerican camelid is definitely demonstrated. In particular, the presence of OxB in the alpaca testis tissue is supported by the detection of prepro-orexinpreviouslydemonstratedbyus(Liguorietal.,2012), andinthepresentstudy,wedemonstratethepresenceofthespe-cificreceptor,OX₂. F I G U R E   2   Specificitycontrol experimentoftheanti-OxBandanti- OxAantibodies.(a–d):Thestainingof twocouples(a,bandc,d)of3-μm-thick consecutivesectionswiththeanti-OxB(a, c)andanti-OxA(b,d)antibodiesshowed thatthematerialpositivetothefirst antibodywasnotboundbythesecond. Avidin–biotinimmunohistochemical method.Bars:20μm (a) (b) (c) (d)

The presence of OX₁ in mammalian Leydig cells suggested that OxAcouldplayaroleintesticularsteroidogenesis(Assisietal.,2012; Barreiroetal.,2004;Liguorietal.,2012).Particularly,itwashypoth-esizedthatOxA,mainlyproducedbythetubularSertolicells,could act on its same source by an autocrine modality of action decreas-ingtheproductionofMüllerianinhibitingsubstance(MIS),whichisa steroidolitic compound basically synthesized in these cells (Teixeira, Fynn-Thompson,Payne,&Donahoe,1999).Theconsequentstimula-tionoftestosteroneproductionwasexperimentallyassessedthrough the sequential in vitro addition of OxA and MIS, and vice versa, to slicesofratandalpacatestis(Assisietal.,2012;Liguorietal.,2012). TotestapotentialsteroidogeniceffectofOxBinthemalegonad,an experimentalstudywasperformedonin vitroculturedrattestisslices (Liguorietal.,2017)inwhichthesteroidogenicactivityofOxAwas confirmed,but,conversely,OxBwasfoundtobeineffectiveontes-tosteroneproduction. Alsotheadrenalsteroidogenesishasbeenprovedtobeinfluenced bytheorexinergiccomplex.Boththeorexins,infact,stimulatedcor-tisol and corticosterone production from dispersed rat and human

adrenocorticalcellsthroughactivationoftheadenylatecyclase(AC)- dependentsignallingcascade(Malendowicz,Tortorella,&Nussdorfer, 1999).Thesamemetabolicpathwaywasinvolvedincortisolandaldo-steronereleasestimulationinducedbyOxAonin vitroculturedpor-cineadrenocorticalcells(Nanmokuetal.,2002).

At present, also the role played by the orexins in the activation of intermediate metabolic pathways is poorly known. Karteris etal. (2004)incubatedcellmembranefragmentsfromhumantesticularly-sateswiththetwoorexinsinanin vitroexperimentalassay.Boththe peptideswerefoundtostimulatethephospholipaseC(PLC)/inositol phosphatepathway,andOxBprovedtobemoreefficientthanOxA indisclosingsuchaway.Morerecently,Zhengetal.(2014)demon-stratedthatOxAactivationofOX₁

upregulatestestosteroneproduc-tion via the ERK1/2 and p38 MAPK signalling pathway in primary culturesofratLeydigcells.Theroleplayedbytheorexinergiccomplex in the metabolism of other types of steroidogenic cells is, similarly,

poorlyknown.ActivationofOX₁inChinesehamsterovary(CHO)cells

ledtoarapid,strongandlong-lastingincreaseinERKphosphoryla-tion,whichwasmediatedbyCa++_{intracellularinflux(Ammounetal.,}

2006). In the same cellular type, also the OxB-induced OX₂

activa-tion promoted ERK phosphorylaactiva-tion and the intermediate pathway

seemedtoinvolvetheG_q

/PLC/proteinkinaseC(PKC)pathwaystimu-lation(Guo&Feng,2012).InhumanadrenocorticalH295Rcells,OxA andOxBenhancedsteroidogenesisbyregulatingtheactivationofthe

Ca++_{/PKCandERK1/2MAPKpathways(Ramanjaneyaetal.,2009).}

Finally,inCHOandadrenocorticalcellsofratandhumanorigin,the

ligand activation of OX₁ was followed by AC/PLC/PKC (Kukkonen,

Holmqvist,Ammoun,&Akerman,2002)andAC/PKA(Holmqvistetal., 2005)signallingpathwaystimulation,respectively. F I G U R E   3   OX₂-IRincytotypesofthe alpacatestis.(a):SmallclusterofLeydig cellscontainingdifferentquantityof smallpositivegranulesscatteredintheir cytoplasm.(b,c):Round(b)andelongated (c)spermatidsshowedintheircytoplasm arciformorroundishpositivestructures which,respectively,embracedportion ofthenucleusoftheyoungercellsor werelocalizedinthetailoftheolder ones.Avidin–biotinimmunohistochemical technique.Bars:20μm (a) (b) (c) F I G U R E   4   OX₂expressiondetectedbymeansofWestern blottinganalysisoftestishomogenate.Lane1:alpacatestis.Lane 2:ratbrain.MolecularweightmarkersareexpressedinkDaand reportedontheright

Inthealpacatestis,OxB-IRwasdescribedinmanytubularcyto-types:spermatogonia,preleptotene(resting),zygoteneandpachytene spermatocytesaswellasindevelopingspermatids.OX₂-IRwasfound bothintheinterstitialandthetubularcompartments.Theseresults alsodemonstratethewidedistributionofthetwopeptidesinthemale gonadinlinewithapreviousstudyshowingthelocalizationofOxB andOX₂inrattestis(Liguorietal.,2017).Ourresearchalsohighlights asimilaritybetweentheorexinergicsystemofthealpacatestisand thatdescribedintherattestisandsuggeststhatthissystemcouldplay aroleintheregulationofspermatogenesis.

5 | CONCLUSIONS

Inconclusion,theresultsobtainedinthisworkdefinitelydemonstrate that,inadditiontoOxAandOX₁(Liguorietal.,2012),alsoOxBand OX₂arewidelydiffusedinthealpacatestis.Althoughfurtherstudies areneededtofullyunderstandtheroleplayedinthemammaliantestis bytheorexinergiccomplex,weretainthatthepresentknowledgeof theargumentissufficienttoprospectforthecomplexapivotalrolein determinismofmanyphysiopathologicalaspectsofthemalegonad. Abettercomprehensionofsuchrolecouldbeimportantalsoforde-velopingnewtherapeuticapproachestotesticulardysfunctionsboth inanimalsandmen. ACKNOWLEDGEMENTS WethankMrAntonioCalamoandMrsSabrinaAlìforthetechnical assistance. CONFLICT OF INTEREST Weconfirmthattherearenoknownconflictofintereststodeclare andtherehasbeennosignificantfinancialsupportforthisresearch. AUTHOR CONTRIBUTIONS AVcontributedtotheexperimentaldesign,analysisandinterpreta-tionofresults,andcriticalreviewandfinaleditingofthearticle;GL contributed to the experimental design, performed the immunohis-tochemistry, contributed to the acquisition and the interpretation ofresults,andwrotethemanuscript;CSandNMcontributedtothe collection of the surgical specimens, Western blotting method, and acquisitionandtheinterpretationoftheresults;LAandELcontrib-utedtoreagentsandanalysistools;ACcontributedtotheacquisition andtheinterpretationofresults.Alltheauthorsdiscussedtheresults andcontributedtowritethefinalversionofthemanuscript.Allthe authorsapprovedthefinalversionofthemanuscript. REFERENCES

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How to cite this article:LiguoriG,SquillaciotiC,AssisiL,etal.

LocalizationoforexinBandreceptor2fororexinsintesticular cytotypesofthecamelidalpaca(Vicugna pacos).Reprod Dom