Association study and expression analysis of porcine ESR1 as a candidate gene for boar fertility and sperm quality

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ContentslistsavailableatSciVerseScienceDirect

Animal

Reproduction

Science

journalhomepage:www.elsevier.com/locate/anireprosci

Association

study

and

expression

analysis

of

porcine

ESR1

as

a

candidate

gene

for

boar

fertility

and

sperm

quality

Asep

Gunawan

a

,

Kanokwan

Kaewmala

a

,

Muhammad

Jasim

Uddin

a

,

Mehmet

Ulas

Cinar

a

,

Dawit

Tesfaye

a

,

Chirawath

Phatsara

a,b

,

Ernst

Tholen

a

,

Christian

Looft

a

,

Karl

Schellander

a,∗

a_Institute_of_Animal_Science,_Animal_Breeding_and_Husbandry_Group,_University_of_Bonn,₅₃₁₁₅_Bonn,_Germany b_Department_of_Animal_and_Aquatic_Sciences,_Faculty_of_Agriculture,_Chiang_Mai_University,₅₀₂₀₀_Chiang_Mai,_Thailand

a r t i c l e i n f o

Articlehistory: Received13May2011

Receivedinrevisedform12August2011 Accepted24August2011

Available online 6 September 2011

Keywords: mRNA

Immunofluorescence Spermatogenesis Epididymis Spermatozoa

a b s t r a c t

MalefertilityisimpairedthroughthelackofESR1(EstrogenReceptor1)butlittleisknown abouttheESR1rolesinboarspermatogenesisandfertility.Therefore,thisresearchwas aimedatinvestigatingtheassociationwithspermqualityandboarfertilitytraitsinatotal of300boarsbothfrompurebredPietrainandPietrain×Hampshirecrosses.ASNPincoding regionofESR1g.672C>Tinexon1wasassociatedwithspermmotility(P<0.05)andplasma dropletrate(P<0.01)whilethepolymorphisminnon-codingregionofESR1g.35756T>Cin inton1wasassociatedwithnon-returnrate(P<0.05).Furthermore,toanalysethemRNA andproteinexpressionofESR1inboarreproductivetissues,atotalofsixboarsweredivided intotwogroups[GroupI(G-I)andGroupII(G-II)],whereG-Ihadrelativelybettersperm quality.ESR1expressionwashigherintissuescollectedfromG-Iboarsthanthoseof col-lectedfromG-IIboars,andthedifferenceinmRNAexpressionwassignificant(P<0.01) inheadofepididymis.TheESR1proteinexpressionresultsfromwesternblotcoincided withtheresultsofqRT-PCR.TheESR1proteinlocalizationobservedastrongstaininginthe cytoplasmofSertolicellinthetestis,intheepithelialcellsinheadandtailofepididymis, insmoothmuscleintailofepididymis,andinthepostacrosomalregionandtailofthe spermatozoa.TheseresultswillimprovetheunderstandingofthefunctionsoftheESR1in spermatogenesiswithinthereproductivetractandwillshedlightonESR1asacandidate intheselectionofboarwithgoodspermqualityandfertility.

1. Introduction

Despitebeingthe‘female’hormone,estrogenispresent inlowconcentrationsinmalebloodandinextraordinarily

highconcentrationinsemen(GanjamandAmann,1976).

∗ _{Corresponding}_author._Tel.:₊₄₉₂₂₈_732240;_fax:₊₄₉₂₂₈_732284. E-mailaddresses:agun@itw.uni-bonn.de(A.Gunawan),

kkae@itw.uni-bonn.de(K.Kaewmala),judd@itw.uni-bonn.de

(M.J.Uddin),ucin@itw.uni-bonn.de(M.U.Cinar),dtes@itw.uni-bonn.de

(D.Tesfaye),chirawath.p@cmu.ac.th(C.Phatsara),etho@itw.uni-bonn.de

(E.Tholen),cloo@itw.uni-bonn.de(C.Looft),ksch@itw.uni-bonn.de

(K.Schellander).

The hormone estrogen is mediated through the nuclear

estrogen receptor which functions as ligand-dependant

transcription factor. The increasing interest in the role

ofestrogenin themalereproductivetractis mainlydue

to the demonstration that male fertility is impaired in

mice lacking estrogen receptor 1 (ESR1) (Eddy et al.,

1996). The evidence for ESR1 in different parts of the

male reproductive tract suggests a possible

physiologi-cal rolein spermatogenesisand sperm maturation. The

ESR1knockoutmicehaveprovidedevidencefora

signif-icantandcrucialroleofestrogensinmaintainingnormal spermatogenesis(Eddy et al., 1996). ESR1is involvedin

the reabsorption of luminal fluid during the transit of

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spermatozoa fromthetestistotheheadoftheepididymis whichisimportantfortheirsurvivalandmaturation

dur-ing epididymal storage (Couse and Korach, 1999). The

absenceofESR1leadstoreducedepididymalsperm

con-tent,reducedspermmotilityandfertilizingability(Eddy etal.,1996).Severalstudieshaveshownasignificant asso-ciationofESR1withsemenqualityorfertilityinhumans (Suzukietal.,2002;Guarduccietal.,2006;Lazarosetal., 2010; Safarinejad et al., 2010). Guarducci et al. (2006)

reported a significantassociation ofESR1polymorphism

with lower sperm count in men. Suzuki et al. (2002)

reportedtwosilentpolymorphismsinESR1being

associ-atedwithazoospermiaorsevereoligozoospermiainmen.

To our knowledge, onlyone studywasdevotedto

anal-ysetheassociationbetweenESR1polymorphismwithlitter sizeinsowsbutthisfailedtodetectanystatistically sig-nificantassociation(Munozetal.,2007).TheESR1mRNA

was reported tobe highly expressed in the epididymis,

testis,pituitary,uterus, kidneyand adrenalglandin rats (Kuiper et al., 1996). ESR1 was localized in head, body andtailofepididymisinthebonnetmonkey(Shayuetal., 2004),cat(Nieetal.,2002)andhorse(Hejmejetal.,2005; Parlevlietetal.,2006).Inpigs,ESR1islocatedon SSC1p24-25,whichwasevidencedastheQTLregionfortotalsperm

per ejaculate and close to the QTL for sperm motility

(Xingetal.,2008).Therefore,ESR1couldbeafunctionalas wellasapositionalcandidategeneformalereproduction

in pigs. But no study has yet been devotedto

unravel-ling itsassociationwith boarspermquality andfertility traits.

In spermatogenesis, beside the reproductive tissues

testis and epididymis, some otheraccessory glands and

tissuesarealsoinvolved.Spermatogenesisisthecomplex

processby whichimmaturegermcellsundergo division,

differentiation and meiosis togive rise to haploid

elon-gated spermatids (O’Donnell et al., 2001). When germ

celldevelopmentiscomplete,thematurespermatidsare

releasedfromtheSertolicellsintothetubulelumenand

proceedthroughtheexcurrentductsystem,knownasthe

rete testis,untiltheyenter the epididymisviathe

effer-ent ducts. During passage through the epididymis, the

spermatids undergo a series of biochemical changes to

becomethemotileandmaturespermatozoacapableof

fer-tilization (O’Donnellet al.,2001).Failurein anyof these

events leads to disturbances of male fertility. The role

ofESR1inboarspermatogenesiswithinthereproductive

tract is poorlyunderstood.For the betterunderstanding

of ESR1rolesin spermatogenesisin pigs,the expression

of ESR1 at different parts of reproductive tract,

includ-ing non-reproductive tissues, are important. Therefore,

the aims of this study were to investigate the

associa-tion ofESR1polymorphism withspermqualityandboar

fertility traits as well as to highlight the roles of ESR1

in boar spermatogenesis within the reproductive tract

by mRNA and protein expression and immunoreactive

ESR1localization.Thisworkmightbehelpful toget

fur-ther insight into the roles of ESR1 in spermatogenesis

in the boar. Theassociationof ESR1polymorphismwith

boar spermquality and fertility will supportthe

candi-dacy of this gene to be used in selection of breeding

boars.

2. Materialsandmethods

2.1. Phenotypes

Samples and phenotypes from 200purebred Pietrain

(PI) and 100 Pietrain×Hampshire crossbred (PI×HA)

boars were used for association analysis in this study.

TheseanimalswereusedforAIincommercialpigherds

inNorth-WesternGermany.Detailsofthepopulationsand

phenotypesweredescribedpreviouslybyWimmersetal.

(2005), Lin et al (2006)and Kaewmala et al. (2011). In

brief, sperm samples from more than 39,000 ejaculates

wererepeatedlycollectedfromtheseboars.Whole

ejacu-lateswereobtainedfrompurebredPietrainandcrossbred

Pietrain×Hampshireboarsagebetweentwoandfiveyears

with an average age of 3.5 years. Sperm quality traits

included sperm concentration (SCON [×108_ml]), _semen

volumeperejaculate(VOL[ml]),spermmotility(MOT[%]),

plasmadroplets rate(PDR[%]) and abnormal

spermato-zoarate(ASR[%])andwereobtainedfromeachejaculate

withlightmicroscopyaccording totheguidelinesofthe

WorldHealthOrganization. Semenwas collected bythe

vinylglovehandmethodtwiceperweek.Foreachboar,

therepeatedmeasurementsofspermqualitytraitswere

available.Fertilitydata(non-returnratedata[NRR]at42 daysafterinsemination[%]andnumberofpigletbornalive [NBA]perlitter)ofeachboarwereavailableasthe devia-tionfromthepopulationmeanswithinsowbreed,parityof sow,farmandseasonclassesasdescribedearlierbyLinetal (2006)andKaewmalaetal.(2011).Thegeneraldescription ofspermqualitytraitsandboarfertilitytraitsareshownin

Table1.

2.2. Polymorphismstudy

Two single nucleotide polymorphisms detected by

Munoz et al. (2007) were used in this study: a cyto-sine(C) transversion toa thymine at g.672C>Tin exon

1 and a thymine (T) to a cytosine (C) transversion at

g.35756T>C in intron 1 of ESR1. For PCR amplification, the forward (5′_{-gttcaaatccctggttgcat-3}′₎ _and _reverse ₍₅′ -ctaggcgtctccccagattag-3′₎_primers_were_designed_from_the exon1 and the forward (5′_{-gacagcttccctgcagattc-3}′₎_and reverse(5′_{-ttcatcatgcccacttcgta-3}′₎_primers_were_designed

from the intron 1 of porcine ESR1 genomic sequence

(GenBankaccessionNo.ENSSSCG00000004083)usingthe

Primer3 tool (Rozen and Skaletsky, 2000). Polymerase

chainreactions(PCR) wereperformedina20␮lvolume

containing100ngofporcinegenomicDNA,1×PCRbuffer

(with1.5134mMMgCl2),0.25mMofeachdNTP,5pmolof

eachprimerand0.1UofTaqDNApolymerase(GeneCraft).

ThePCRreactionwasperformedunderthefollowing

con-ditions:initialdenaturationat95◦_C_for₅_min_followed_by 35cyclesof30s at95◦_C,₃₀_s _at₅₂◦_C, ₃₀_s _at₇₂◦_C_and finalelongationof10minat72◦_C_for_the_polymorphism inexon1.ThePCRconditionswerethesameforthe

poly-morphisminintron1excepttheannealingtemperatureof

30sat56◦_C._After_checking_the_PCR_products_in_1.5%_(w/v)

agarosegels,genotypingwasdonefollowing the

restric-tionfragmentlengthpolymorphisms(RFLPs)analysis.The

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Table1

Means,standarddeviation(S.D.),samplesize,rangesoftraitsinPietrainandPietrain×Hampshire.

Population Traits Samplesize Mean S.D. Minimum Maximum

Pietrain(PI) SCON(108_/ml) _29,161 _3.03 _0.94 ₁ ₆

VOL(ml) 30,772 237.03 57.32 25 522

MOT(%) 30,118 84.72 4.37 65 95

PDR(%) 30,239 5.41 3.33 0 15

ASR(%) 30,528 6.53 4.18 0 20

NRR42(%)a ₂₀₀ _0.28 _7.06 ₋_24.07 _17.68

NBA(perlitter)a ₂₀₀ _0.02 _0.57 ₋_1.69 _1.37

Pietrain×Hampshire(PIHA) SCON(108_/ml) ₉₁₂₃ _2.95 _0.97 ₁ ₆

VOL(ml) 9673 297.50 81.62 56 546

MOT(%) 9395 85.46 4.03 70 95

PDR(%) 9409 5.76 3.14 0 15

ASR(%) 9543 4.95 4.00 0 20

NRR42(%)a ₁₀₀ _0.97 _4.18 _−12.23 _13.79

NBA(perlitter)a ₁₀₀ _0.05 _0.52 ₋_2.97 _0.83

PIandPIHA SCON(108_/ml) _38,284 _3.00 _0.95 ₁ ₆

VOL(ml) 40,445 256.87 75.54 25 546

MOT(%) 39,513 85.01 4.37 65 95

PDR(%) 39,648 5.51 3.28 0 15

ASR(%) 40,071 5.99 4.26 0 20

NRR42(%)a ₃₀₀ _0.41 _6.23 _−24.07 _17.67

NBA(perlitter)a ₃₀₀ _0.01 _0.55 _−2.97 _1.37

a _Fertility_(NRR42,_NBA)_corrected_with_factors:_parity,_farm,_season_and_breed.

AluIforg.672C>Tandg.35756T>C,respectively(BioLabs).

The digestion was carried out in 10␮l of reaction

mix-tureofeachsampleandincubatedovernightat37◦_C_for g.35756T>Cand65◦_C_for_g.672C_>_T._Detection_of_RFLPs_of

300boarswascarriedoutbyelectrophoresisin3%(w/v)

agarosegels.

2.3. Statisticalanalysisforspermqualitytraits

TheassociationofESR1withboarspermqualitytraits

wasanalysedbyvarianceanalysis(PROCMIXED)usingthe

SASsoftwarepackage(ver.9.2;SASInstituteInc.,Cary,USA) asdescribedearlierbyKaewmalaetal.(2011).

yijklm=+breedi+seasonj+genotypek+agel

+ejaculation_m+ε_ijklm [Model 1]

wherey_ijklmarethespermqualitytraits(SCON,VOL,MOT, PDR,ASR);istheoverallpopulationmean;breed_iisthe fixed effect of the i-th breed (i=PI; PIHA, PIand PIHA); seasonjisthefixedeffectofthej-thseason(j=1through8; fourseasonsperyear,intotaleightseasonswithin2years

from January2000toDecember2001);genotype_k isthe

fixedeffectofthek-thgenotype(k=1,2and3);age_listhe effectofboarage(covariable);ejaculationmisthe

perma-nentenvironmentaleffectofthem-thboar(random)and

εijklmistheresidualerror.

2.4. Statisticalanalysisforfertilitytraits

TheassociationanalysisbetweenESR1andthefertility traitswascarriedoutusingthefollowinggeneralizedlinear model(PROCGLM)inSAS(Kaewmalaetal.,2011).

y_ijkl=+breed_i+genotype_j+year_k+ε_ijkl [Model 2]

whereyijk istheboarfertilitytraits(NRRandNBA);is the overallpopulationmean;breediisthefixed effectof

thei-thbreed(i=PI,PIHA,PIandPIHA);genotypej isthe fixedeffectofthej-thgenotype(j=1,2and3);year_kisthe fixedeffectofthek-thboaryearofbirth(k=1through3: boarbornbefore1996,in1996–97andin1998–99),ε_ijkis theresidualerror.

Achi-square(2)testwasconductedtotestthe

popula-tionsforHardy–Weinbergequilibrium.Leastsquaremean

valuesfortheESR1genotypeswerecomparedbyt-testand P-valueswereadjustedbytheTukey–Kramercorrection.

2.5. SelectionofanimalsformRNAandprotein expression

The reproductive (testis, head, body and tail of

epi-didymis, vas deferens, bulbourethral gland, vesicular

glandsandprostategland),non-reproductive(brain, mus-cleandliver)tissuesfrom6breedingboarswithdivergent

phenotypes were collected from the AI station (SuisAG,

Sempach,Switzerland)for mRNAand proteinstudy. For

differential expression study between reproductive and

non-reproductivetissuesbysemi-quantitativePCRstudy,

mRNAfromall6boarswaspooledtogetheraccordingto

thetissues.Ontheotherhand,thedifferentialmRNAand proteinexpressionstudyindifferentreproductivetissues

fromtwodivergentgroupsofanimalswasperformedby

Table2

Means,standarddeviation(S.D.),numberofboarsandrangesoftraits selectedformRNAandproteinexpressionstudy.

Traits Selected animals (n=6)

G-I (n=3)

G-II (n=3)

Mean S.D. Mean S.D. Mean S.D.

SCON(106_/ml) _262.32 _87.97 _335.94 _50.78 _188.70 _22.54

[image:4.595.41.554.94.322.2] [image:4.595.307.551.686.768.2]

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semi-quantitativePCR,qRT-PCRandwesternblot, respec-tively. Forthesepurposes,the6boarsweredividedinto two groups:groupI(G-I) andgroupII(G-II),eachgroup containing3boarsonthebasisofSCON,SVOLandSMOT (Table 2). TheSCON (average spermconcentration) was highly negatively (r=−0.8) correlated with SVOL

(aver-agesemenvolume),whereasSCONwashighlypositively

(r=0.7)correlated with SMOT (average spermmotility).

Moreover,SVOLwashighlynegatively(r=−0.8)correlated

with SMOT.Forthe animalsof G-Ithe SCONwashigher

thanthe mean(262.32×106_ml),_SMOT_was_higher_than

the mean (76.59%) and SVOL waslower thanthe mean

(215.24ml/ejaculation)of respectiveparameters.Forthe animalsofG-IItheseparameterswereviceversa.The sig-nificantdifferencebetweenthetwogroupswascalculated usingproct-testinSAS.ThereweredifferencesforSCON (P<0.05) and for SVOL (P<0.01) between G-I and G-II,

whereasforthe SMOTthedifferencewasnotsignificant

(P=0.12).

2.6. Semi-quantitativePCR

TotalRNAwasisolatedusingTRIreagent(Sigma)from

different reproductive and non-reproductive tissues of

breedingboars mentioned above(Section2.5). RNA was

purified using RNeasy Mini Kit (Qiagen) according to

the manufacturer’s instructions. Total RNA was treated

using on-column RNase-Free DNase set (Promega) and

quantifiedsphectrophotometrically(NanoDrop,ND8000).

Furthermore,RNAintegritywascheckedby2%agarosegel

electrophoresis.First-strandcDNAweresynthesizedfrom

individualRNAusingSuperscriptIIenzyme(Invitrogen).

cDNA amplification was performed by using specific

forward and reverse primers (forward: 5′

-agggagagg-agtttgtgtg-3′ _and _reverse: ₅′_{-tctccagcagcaggtcatag-3}′₎

derivedfromporcineESR1sequence(GenBankaccession

AF035775). Amplificationwas performedwith an initial

heatingat95◦_C_for₅_min_followed_by₃₅_cycles_of₉₅◦_C_for 45s,annealingtemperatureat60◦_C_for₁_min_and₇₂◦_C_for

1min,onthePCRThermalCycler(BioRad).PCRproducts

wereelectrophoresedona1.5%agarosegelandvisualized

upon staining with ethidium bromide. Amplification of

Glyceraldehyde 3-phosphate dehydrogenase (GAPDH)

servedashousekeepinggene.

2.7. QuantitativeReal-TimePCR(qRT-PCR)

ForqRT-PCR,totalRNAwasisolatedusingTRIreagent

(Sigma) from different reproductive tissues of the two

divergent groupsof animals (G-Iand G-II)as previously

described (Section 2.5). Total RNA isolation and cDNA

synthesis wasdescribedinthe previoussection (Section

2.6).Thesameprimerpairusedinsemi-quantitativePCR

was also used in qRT-PCR. Nine-fold serial dilution of

plasmid DNA was prepared and used as a template for

the generation of the standard curve. In each run, the

96-wellmicrotiterplatecontainedeachcDNAsample,

plas-midstandardsforthestandardcurvesandano-template

control.To ensurerepeatabilityofthe experiments,each

sample was run in three replications. qRT-PCR was set

upusing2␮lfirst-strandcDNAtemplate,7.6␮ldeionized

H2O, 0.2␮M of upstream and downstream primers and

10␮l 1× Power SYBR Green I master mix with ROX as

reference dye (BioRad). The thermal cycling conditions

were3minat94◦_C_followed_by₄₀_cycles_of₂₀_s_at₉₄◦_C

and 1min at 60◦_C. _Experiments _were _performed _using

theABIprism®₇₀₀₀_(Applied_Biosystems)_qRT-PCR_system.

An amplification-based threshold and adaptive baseline

were selected as algorithms. The housekeeping gene

GAPDH(forward:5′_{-acccagaagactgtggatgg-3}′_and_reverse: 5′_{-acgcctgcttcaccaccttc-3}′₎_derived_from_porcine_sequence

(GenBankaccessionNo.AF017079)wasusedforthedata

normalization.Finalresultswerereportedastherelative

abundancelevelafternormalizingwithmRNAexpression

levelofthehousekeepinggene.DifferencesinESR1mRNA

expressionwereanalysedwiththesimplet-testinSAS soft-ware(SASInstituteInc.,ver.9.2).Valuesof P<0.05were consideredtoindicatestatisticallysignificantdifferences.

2.8. Westernblotting

Theproteinwasextractedfromdifferentreproductive

tissues(testis,head,bodyandtailofepididymis)fromthe twodivergentgroupsofbreedingboarsasusedinqRT-PCR. However,forwesternblotstudy,proteinsfromthethree

G-Iboarswerepooledtogetherandproteinsfromthethree

G-IIboarswerepooledtogetheraccordingtothetissues.

Theproteinextractedfromtissueswasseparatedby

SDS-PAGE(gradient 4–18%). Subsequentlythe proteins were

transferred onto a nitrocellulosemembrane (Amersham

Biosciences).Afterblockinginblockingbuffer(20mMTris

pH7.5,150mMNaCl,0.05%Tween-20and1%

Polyvinylpy-rolidone)atroomtemperaturefor1h,themembranewas

incubatedwiththeanti-ESR1antibodypurifiedfromrabbit polyclonalantibody(Cat.nr.543;SantaCruz)inthe block-ingmedium(diluted1:500)overnightat4◦_C._Non-specific

bindingof antibodywas washedoffwith sixchangesof

0.1% PBST (10min totime). The horseradish peroxidase

conjugatedgoatanti-rabbitIgGsecondaryantibody(Cat.nr.

Sc2004;SantaCruz)wasusedasthe secondaryantibody

(diluted 1:5000). The membrane was incubated for 1h

atroomtemperaturewith secondaryantibody,followed

by washing with six changes of 0.1% PBST (10min to

time).ThechemiluminescewasdetectedbyusingtheECL

plus westernblottingdetection system (Amersham

Bio-sciences)andwasvisualizedbyusingKodakBioMaxXAR

film(Kodak).GAPDHwasusedasaloadingcontrolandfor

normalization.Themembranewasstrippedbyincubation

in2%SDS,100mMTris–HCl,0.1%␤-mercaptoethanolfor

30minat60◦_C_and_re-probed_with_GAPDH_antibody_(Cat.nr. Sc20357;SantaCruz).

2.9. Proteinlocalizationbyimmunofluorescence

Due to the limitations of fresh samples from G-I

and G-II boars, we collected different fresh

reproduc-tivetissueswerecollectedfrom ahealthybreedingboar

afterslaughteringforproteinlocalizationby

immunofluo-rosence.Immunofluorescencestainingwasperformedon

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Table3

Genotype,allelefrequenciesandthechi-squaretestoftheporcineESR1gene.

Polymorphism Breed Numberofboars(n) Genotypefrequency Allelefrequency Chi-squaretest

CC(n) CT(n) TT(n) C T 2

g.672C>T Pietrain 200 0.73(146) 0.09(19) 0.18(35) 0.78 0.22 0.55 Pietrain× Hampshire 100 0.61(61) 0.18(18) 0.21(21) 0.70 0.30 0.33 PIandPIHA 300 0.66(200) 0.15(44) 0.19(56) 0.74 0.26 0.38

Polymorphism Breed Numberofboars(n) Genotypefrequency Allelefrequency Chi-squaretest

TT(n) TC(n) CC(n) T C 2

g.35756T>C Pietrain 185 0.83(154) 0.14(26) 0.03(5) 0.90 0.10 0.05 Pietrain× Hampshire 94 0.80(75) 0.17(16) 0.03(3) 0.88 0.12 0.03 PIandPIHA 279 0.82(229) 0.15(42) 0.03(8) 0.89 0.11 0.04

temperaturewith5%bovineserumalbumininPBS(50nM sodiumphosphate,pH7.4;0.9%NaCl).Sectionswere incu-bated overnight at 4◦_C _with _the _ESR1 _rabbit_polyclonal primaryantibody(Cat.nr.543;SantaCruz)dilutedat1:50 inPBSTfollowedbysixtimes(10minpertime)washing withPBS. Then,thesectionswereincubated 1hatroom temperaturewiththebiotinylateddonkeyanti-rabbit IgG-B as a secondary antibody (Cat.nr. Sc2090; Santa Cruz) (dilution 1:200) which was conjugatedwith fluorescein isothiocynate (FITC). Later on sections werewashed six times (10min per time) with PBS. Finally, the samples werecounterstainedwithvectashieldmountingmedium (VectorLaboratories)containing40,6-diamidino-2-phenyl indole(DAPI)andcoveredwithacoverglassslip.The stain-ingwasobservedby confocallaserscanningmicroscope (Carl Zeiss). In case of negative controls, PBS was used insteadoftheprimaryantibody.

3. Results

3.1. Polymorphismstudy

Twosinglenucleotidepolymorphisms(SNPs)were con-firmedinexon1andintron1ofESR1inthePIandPI×HA populations.Animalsofthesepopulationsweregenotyped atg.672C>Tinexon1andatg.35756T>Cwhichwerethe SNP segregatingwithin the populations.The SNPs were confirmed by PCR-RFLP. The DNA restriction fragments obtainedforg.672C>TofESR1-BstNIpolymorphismwere: 190,117,50,32and17bpfortheCCgenotype;190,117,50, 32,17and12bpfortheCTgenotype,and190,117,50and 12bpfortheTTgenotype.TheDNArestrictionfragments obtainedforg.35756T>CofESR1-AluIpolymorphismwere: 93,48,40and5bpfortheTTgenotype;133,93,48,40and 5bpfortheTCgenotype,and133,93,and5bpfortheCC genotype.

The calculated genotype and allele frequencies of porcine ESR1 gene are shown in Table 3. In this study,

threegenotypeCC,CTandTTwerefoundforbothSNPsat

g.672C>Tand g.35756T>C inour populations.

Homozy-goteCCwasmorefrequentandhomozygoteTTwasrare

atSNPg.672C>TwhereasincaseofSNPg.35756T>C,the

homozygoteTTwasthemorefrequent,andhomozygoteCC

wasrareinourpopulations.Thechi-squaretestrevealed

thatthelocusofESR1wasinHardy–Weinbergequilibrium

inbothPIandPI×HApopulations(Table3).

3.2. Associationanalysis

Association analysis of g.672C>T withsperm quality andfertilitytraitsrevealedsignificant(P<0.01)association

withMOTandPDR.SemenfromanimalswithgenotypeCT

hadsignificantlylowerspermmotilityandhigherplasma

dropletratethananimalswith CCand TT(Table4). The

results also indicated that there were dominant effects

(P<0.05)onMOTandPDRinourpopulation.Inthecase ofg.35756T>C,theassociationanalysisshowedsignificant (P<0.05)associationwithNRR.AnimalswiththeTC geno-typehada significantly(P<0.05)higherNRRthanthose withgenotypeTTandCC(Table4).

3.3. mRNAexpressionbysemi-quantitativePCR

ESR1gene expressionwas higherin headof the

epi-didymis than in body and tail, moderate in testis, vas

deferens,bulbourethralglandsandlowerexpressionwas

foundinnon-reproductivetissue(brainandliver)except skeletalmuscle.Thesemi-quantitativereverse transcrip-tionPCRresultofGAPDHshowednoremarkabledifferences amongtissues(Fig.1).

3.4. mRNAandproteinexpressionstudyintissuesfrom G-IandG-IIboars

TheESR1mRNAwasexpressedintestis,bodyandtail

of epididymis from both the G-I and G-IIboars but the

higherexpressionwasshowninheadofepididymisofG-I

thanthatofG-IIboarsinsemi-quantitativePCR(Fig.2a).

ThesemRNAexpressionresultsofsemi-quantitativePCR

appeared to be consistent with the results of the

qRT-PCR. The ESR1 mRNA expression was higherin headof

epididymisofG-Iboars(P<0.01),whereastheexpression

[image:6.595.39.558.94.228.2] [image:6.595.311.546.659.744.2]

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Table4

AssociationbetweenESR1genotypeswithspermqualityandfertilitytraits.

Polymorphism Trait ESR1genotype(± S.E.) Effect(± S.E.)

CC CT TT Additive Dominance

Spermquality Numberofobservations#

26,078 5971 7507

g.672C>T SCON(108_/ml) _3.00_±_0.04 _2.98_±_0.09 _2.95_±_0.08 _0.02_±_0.04 ₋_0.01_±_0.10

VOL(ml) 258.90 ± 3.76 258.59± 7.30 258.80± 6.74 0.05± 3.76 −0.26± 8.13 MOT(%) 85.02± 0.27a _83.47_±_0.53b _84.73_±_0.49ab _0.14_±_0.27 _1.40_±_0.59*

PDR(%) 5.34± 0.20c _6.88_±_0.40d _5.97_±_0.37cd ₋_0.31_±_0.20 ₋_1.22_±_0.45*

ASR(%) 6.18± 0.23 6.98± 0.55 6.66± 0.41 −0.24± 0.23 −0.56± 0.50

Fertility Numberofboars Additive Dominance

200 44 56

NRR42(%) 0.64± 0.44 −0.64± 1.13 −2.24 ± 2.60 1.42± 1.30 −1.41± 1.65 NBA(perlitter) 0.20± 0.11 0.10 ± 0.09 0.02± 0.08 −0.09± 0.14 −0.09± 0.11

Polymorphism Trait ESR1genotype(± S.E.) Effect(± S.E.)

TT TC CC Additive Dominance

Spermquality Numberofobservations#

26,015 5837 637

g.35756T>C SCON(108_/ml) _3.02_±_0.04 _3.21_±_0.10 _2.96_±_0.23 _0.02_±_0.11 ₋_0.21_±_0.15

VOL(ml) 257.27 ± 3.49 253.35± 7.67 257.45± 7.24 −0.09± 0.70 4.01± 1.59 MOT(%) 84.67± 0.24 84.93± 0.54 85.87 ± 1.20 −0.62± 0.60 0.31± 0.80 PDR(%) 5.72± 0.18 5.93± 0.41 4.47± 0.91 0.63± 0.46 −0.80± 0.61 ASR(%) 6.45± 0.21 6.26± 0.47 5.76± 0.21 0.35± 0.53 −0.14± 0.71

Fertility Numberofboars Additive Dominance

230 41 8

NRR42(%) 0.11± 0.48e _2.50_±_1.02f _0.20_±_0.28a ₋_0.14_±_1.73 ₋_3.24_±_2.18

NBA(perlitter) 0.01± 0.04 0.04 ± 0.09 0.25± 0.06 −0.01± 0.10 −0.06± 0.11

#_Repeated_{measurements.}

a,b_P_<_0.01;c,d_P_<_0.001;e,f_P_<_0.05._*_P_<_0.05.

differences werenon-significant in the testis, body and tailofepididymisinbetweenG-IandG-IIboars(Fig.2b).

ESR1proteinwith66kDamolecularweightwasdetected

intestis,head,bodyandtailoftheepididymisinbothof G-IandG-IIboars(Fig.2c).Thewesternblotresultshowed thattheESR1proteinexpressionwashigherinheadofthe epididymisinG-Iboars.Thisproteinexpressionseemedto beconsistentwiththeresultsoftranscriptionlevels.

3.5. LocalizationofESR1proteininboarreproductive tissuesbyimmunofluorescence

Sections of testis, head, body and tail of epididymis

werestainedthroughthesameopticalpanelfortheESR1

proteinexpression(Fig.3).ImmunoreactiveESR1protein

was observed as strong staining in cytoplasmof Sertoli

cells but poorly in germ cells and Leydig cells in testis (Fig.3a).Spermatozoawithinthelumenoftheepididymis

headwerefoundtobepositivelyimmunostainedwithESR1

(Fig.3b).Similarly,spermatozoawithinthelumenofthe

epididymisbodyandtailwerepositivetoimmunoreactive

ESR1protein(Fig.3candd).Theimmunofluorescenceof ESR1 proteinwas specificallylocalizedin the post

acro-somalregion(arrow)and tailofthesperm(arrowhead)

whenspermatozoawithintheepididymiswereexamined

(Fig.3e).Moreprecisely,theepithelialcellsintheheadand

tail ofthe epididymis expressedhigherimmunoreactive

ESR1protein(Fig.3bandd),whereasthe epithelialcells

inthe epididymisbodypoorlyexpressedthetarget

pro-tein(Fig.3c).Inthecaseoftheepididymisheadandbody, thesterociliacellswereremarkablyhigherimmunostained whileintheepididymistailtheproteinwasstrongly local-izedinthesmoothmuscle(Fig.3b–d).

4. Discussion

4.1. AssociationofSNPwithboarspermqualityand fertilitytraits

ThisstudyrevealedassociationofESR1withsperm qual-ityandfertility traitsinboars (Table4).Theexonic SNP g.672C>Twas found tobe significantly associated with spermqualitytrait:spermmotilityandplasmadropletrate. AssociationstudyofESR1israreinboars,butLazarosetal. (2010)reported twoSNPsofESR1at397(T>C)and351 (A>G)hadapositiveeffectonspermqualityinhumans.

Moreover,Suzukietal.(2002)reportedapolymorphism

ofESR1inexon4associatedwithidiopathicazoospermia

in humans.Theexistence ofESR1at the postacrosomal

[image:7.595.44.561.94.439.2]

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Fig.2.mRNAandproteinexpressionofESR1inreproductivetissues(testis,head,bodyandtailoftheepididymis).(a)ESR1mRNAexpressionindifferent reproductivetissuesfromG-IandG-IIboarsbysemi-quantitativePCR.(b)ESR1mRNAexpressionindifferentreproductivetissuesfromG-IandG-IIboars byqRT-PCR.(c)ProteinexpressionindifferentreproductivetissuesfromG-IandG-IIboarsbywesternblotting.(T:Testis,HE:Headofepididymis,BE: Bodyofepididymis,TE:Tailofepididymis.)

estrogensinmalegametematurationandmotility.Ithas

beenreportedthatestrogenproducedlocallyisinvolvedin theregulationofspermmotility(Lazarosetal.,2010).ESR1

polymorphism may influence theselocally acting

estro-gen levelswith effect onsperm motility (Carreauet al.,

2002). Abnormal sperm production and reduced

fertil-ity have been reported in transgenic male mice lacking

ESR1 (Eddy et al., 1996). Infertility from lacking ESR1is reportedmainlyduetothedisruptionoffluidreabsorption

in efferentductuleswhichincreasedthebackpressureof

theaccumulatingluminalfluidsthatleadstoaprogressive degenerationofthetesticulartissueandseverelyimpaired

spermatogenesis (Eddy et al., 1996; Hess et al., 1997;

CouseandKorach,1999).TheidentifiedSNPisconfirmed

in exon whichmay playimportant rolein transcription

process.Polymorphisminthecodingregioncouldhavea

directeffectthroughchangingthenucleotidesequenceand structureofgene,possiblyleadingtochangesinmRNA syn-thesis,splicing,maturation,transportation,translationor degradation(IidaandAkashi,2000).

TheintronicSNPg.35756T>Cwassignificantly associ-atedwiththefertilitytraitnon-returnrate(NRR)(Table4).

Reproductive efficiency of boars is usuallymeasured by

non-returnrate, whichisdefined aspercentage of sows

notappointedforasecondinseminationwithinaperiod

ofdaysafterthe firstinsemination.Male fertilitycanbe regardedasaresultofbothfertilizingabilityofthesperm cellsaswellasthe numberoffertilizedoocytesandthe

viability of the embryo. But, under the field conditions

onlytheoutcomeoftheinseminationscanberecorded,i.e. whetherornotthefemaleshavereturnedforrepeat insem-ination(Stalhammaretal.,1994).However,littleisknown

aboutthepolymorphismandassociationofporcineESR1

withfertilitytraitlikeNRRin pigs.AnESR1genevariant atc.1227C>Tinexon5showedapositiveeffectonlitter sizeinsows(Munozetal.,2007).It isimportanttonote that,thedetectedSNPatESR1g.35756T>Cwasinintron 1whichissubjectedtolessfunctionalconstraintandmay changetheprimarystructureoftheESR1.Though‘silent’, itcouldaffectESR1functionbyalteringthemRNAstability (Caponetal.,2004).Moreover,thereisincreasingnumber ofreports describingthe roleofintronin regulatingthe expressionlevelofageneortissuespecificexpression pat-tern(JiangandLe,2000;Paganietal.,2004).Traitsrelated tofertilityofboarsareoflowheritability(h2₌_0.01–0.06)

and are stronglyaffected by environmental and genetic

effectsofthe boaritself,the sow andthe offspring(See, 2000). Therefore, effects of single loci are expected to

be low and require a higher number of animals to be

[image:8.595.129.467.68.417.2]

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Fig.3. LocalizationofESR1proteinindifferentpartsofboarreproductivetissues.(a)ImmunofluorescencedetectionofESR1incytoplasmofSertolicells andgermcells.SertolicellswerestainedwithESR1(arrows)andthenucleiwerecounterstainedwithDAPI(arrow).(bandc)ESR1proteinlocalization inepithelialcellsintheheadandbodyoftheepididymisandinthespermatozoawithinthelumenoftheepididymis.(d)LocalizationofESR1proteinin epithelialcells,inspermatozoawithinthelumenoftheepididymisandremarkableexpressionwasinsmoothmuscleinthetailoftheepididymis.Smooth muscleinthetailoftheepididymiswasstainedwithESR1(arrows).(e)TheESR1localizedinacrosomalregion(arrow)andtailofspermatozoa(arrowhead) withinepididymis.(f–j)ThecellnucleiwerecounterstainedwithDAPI.(k–o)Mergedimages.(p–t)Negativecontrol.(T:Testis,HE:Headofepididymis, BE:Bodyofepididymis,TE:Tailofepididymis.)

moderate to medium heritability (h2₌_0.19–0.37) ₍_See,

2000).However,twoSNPsof ESR1areidentifiedin both

exonandintronwhichmighthaveanimportanteffectson

spermqualityandfertilitytraitsinpig.

4.2. ESR1mRNAandproteinexpressioninboar reproductivetissues

Thephysiologicalroleofestrogens inmale

reproduc-tion is now extensively revisited (Carreau et al., 2002) butexpressionstudyisrareinboars.Inthisstudy,mRNA

expressionanalysisbysemi-quantitativePCRandqRT-PCR

demonstratedthattheporcineESR1geneexpressionwas

higher in reproductive tissuesthan in non-reproductive

tissues(Fig.1).Importantly,ESR1expressedhigherinthe

headofepididymiscollectedfromG-Iboarsthanthatof

G-II boars. The highest expression of ESR1 in the head

of the epididymis was in agreement with the previous

reportsdescribingthatthehighestconcentrationof estro-genreceptorwasfoundintheepidydimisheadinmiceand macaques(Schleicheretal.,1984).Itiswelldocumented thatmaleestrogenisinhighpresenceintheefferentducts

in the headof epididymis (Hess and Carnes, 2003). The

presenceofESR1intheheadoftheepididymishasimmense importancesinceestrogenplaysacrucialfunctioninfluid

reabsorptioninthisregioninthemale(Hessetal.,1997). Thisfluidreabsorptionisimportantforspermmaturation andformaintainingthenumberofspermintransit(Janulis etal.,1996).Anydisruptionoftheestrogenreceptorgenein malemiceresultedwithdecreasedfertilityandtesticular

weightandverylowspermcounts(LubahnandGolding,

1993). Moreover, ESR1 abundance is detected in

differ-entpartsof malereproductivetract andspermatozoain

mice(Zhouetal.,2002),humans(Aquilaetal.,2002)and rodents(Pelletier et al., 2000)which coincides withour findings.Theevidenceofestrogenindifferentpartsofthe malereproductivetractsuggestsapossiblephysiological

roleof estrogen in spermatogenesisand sperm

matura-tion.Furthermore,thelowerESR1transcriptabundancein theheadoftheepididymisofG-IIboarsinthisstudymay

indicate alack ofestrogen actionwhich maycontribute

tothe poor spermqualityand fertility. It isknown that

spermanditsmembraneproteinsundergoseveral

modifi-cationsduringmaturationwithintheepididymis.Estrogen

antagonist treatment in mice showed that lack of ESR1

functionintheepididymisheadcausesfailureoffluid

reab-sorption, impairedsperm production and loss of sperm

motility(Shayuetal.,2004).InfertilityinlackingESR1is

reportedmainlydue tothedisruption offluid

[image:9.595.54.552.70.383.2]

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degenerationofthetesticulartissueandseverelyimpaired spermatogenesis(Eddyetal.,1996;Hessetal.,1997;Couse andKorach,1999).Moreover,swellinganddamageof sem-iniferoustubulesandthedilutionoftheessentialproteins secretedfromepididymisalsocontributedtothe infertil-ity(Eddyetal.,1996;Hessetal.,1997;CouseandKorach, 1999).

Inthisstudy,theproteinexpressionanalysisby

west-ernblottingshowedthatESR1antibodyrecognizedaband

at66kDainalltissues.Thebandissimilartothe

molecu-larweightofhuman(66kDa)andmouse(66kDa)ESR1and

veryclosetothatofthestallion(65kDa).Although,the lev-elsofproteinindifferenttissuesfromG-IandG-IIwerenot

asdistinguishableasmRNA,theESR1proteinexpression

wasremarkablyhigherintheheadoftheepididymisofG-I boarsthanthatofG-IIboars(Fig.2b).Thevasdeferensinthe headoftheepididymisarethemajorsitesforESR1 expres-sionwhichregulatesthetesticularfluidreabsorptionand

increasesthe spermcellconcentrationasthey enterthe

epididymis(Hessetal.,1997).Moreover,theepididymisis involvedindifferentproteinabsorptionaswellasin pro-teinsecretion(Syntinetal.,1999).Theheadandbodyofthe epididymisarereportedtosecrethigheramountsof differ-entproteinsinboarsincomparisontootherfarmanimals (Syntinetal.,1999).Theseproteinsecretionsareregulated

by estrogen and the proteinsare involved in the sperm

membraneremodelling,intheinitiationofspermmotility, andespeciallyinsperm-egginteraction(Pearletal.,2007). Ithasbeencalculatedthat50–90%ofthetotalprotein leav-ingthetestisisabsorbedbytheefferentductsinthehead oftheepididymis(Clulowetal.,1994).

4.3. ProteinlocalizationofESR1

ESR1wasfoundtobeexpressedstronglyin the

cyto-plasmofSertolicells,expressedongermcellsandLeydig cells in testis (Fig. 3a). These results are in agreement with previous results localizingESR1in Sertoliand Ley-digcells(HessandCarnes,2003)andinLeydigandgerm cellsinrat(Pelletieretal.,2000).Pelletieretal.(2000) sug-gestedthatintheLeydigcellstheESR1mightbeinvolved

in the secretion and maturation of germ cells. Estrogen

arereportedtobeinvolvedinmaintainingtheSertolicell function (O’Donnellet al., 2001)as well asin

establish-ingSertoli–germcelladhesion(MacCalmanandBlaschuk,

1994).However,thecellulardistributionpatternsofESR1 proteinbetweenspeciescouldbedifferent.

We localizedESR1 in the head, body and tail of the

epididymis. Whenspermare releasedfrom their ‘nurse’

(Sertoli) cells in the testis, theyare transported in fluid secretedbythe Sertolicellstoacollectingarea,therete

testis. Fromthere, the dilute suspensionof spermenter

thethin-walledefferentductsintheepididymisheadand theepitheliaofefferentductsexpressenormousestrogen receptors(Fisheretal.,1997).Thespermpassthroughthe

epididymis wheretheymatureand progresstoward the

vas deferens. Moreover, the ESR1 in the epididymis are

reportedtomodulatesecretionofproteinssuchasoscilin thatpromotesthematurityandviabilityofthe spermato-zoa(MowaandIwanaga,2001).Themostintensesignals ofESR1arereportedinepithelialcellsintheheadandtail

oftheepididymisinratsandmonkeysandsuggestedtobe responsibleforsemenconcentration(Fisher etal.,1997; Hessetal., 1997).Our studyidentified astrongsignalof ESR1inthesmoothmusclelayerinthetailoftheepididymis

whichisinaccordancewithaprevious

immunohistochem-icalstudyinhumanandrabbitwhichstatedthatestrogen isessentialfortransportingthespermatozoaby influenc-ingepididymalsmoothmusclecontractilityaswellasfor ejaculationwiththehelpofoxytocin(Filippietal.,2002). ItisimportanttonotethattheexpressionofESR1in

dif-ferentpartsof theepididymiscouldvaryamong species

sinceithasbeenlocalizedinallpartsofepididymisinthe rat(Pelletieretal.,2000),mouse(Zhouetal.,2002),bonnet monkey(Shayuetal.,2004),horse(Parlevlietetal.,2006), cat(Nieetal.,2002)anddog(Nieetal.,2002).

We found that the ESR1 is remarkably localized in

spermwithinthelumenoftheepididymis.ESR1was

espe-ciallyimmunolocalizedinthe postacrosomalregionand

tail of the sperm in this study (Fig. 3e). This result is

ingoodagreementwithpreviousstudieswhichlocalized

immunoreactiveESR1inthepostacrosomalregionofthe

spermhead(Solakidietal.,2005)andinthetailpieceofthe sperm(Durkee etal.,1998)inhumans.Thepost

acroso-malregionofspermisinvolvedinthesperm–eggplasma

membranefusion(Liuetal.,2008).ThelocalizationofESR1

in the post acrosomal region implies its involvementin

thefertilizationprocess(Ramalho-Santosetal.,2002).Itis importanttonotethatthereareotherproteinsidentified

inthepostacrosomalregionofspermsuchasequatorin

andoscilinwhichareimportantforsuccessfulfertilization (Montagetal.,1998;Solakidietal.,2005).Ourlocalization ofESR1inthetailofporcinespermcoincidedwithprevious reportsinhumans(Durkeeetal.,1998;Aquilaetal.,2002). Moreover,Aquilaetal.(2002)reportedthattheESR1might beinvolvedinspermsurvivalandmotility.Impaired motil-ityofspermhasbeenreportedinmicelackingfunctional ESR1insperm(Eddyetal.,1996)andinESR1knockoutmice (HessandCarnes,2003).Lowfertilizationrateshavebeen reportedincaseofimmotilespermatozoa(Nijsetal.,1996).

5. Conclusion

Associations of ESR1 gene polymorphisms with boar

spermqualityand fertilitytraitshavebeen describedfor

thefirst time,providingevidencethatESR1mightbean

importantcandidategeneforboarspermqualityand fertil-ity.However,thisstudyhastobevalidatedinotheranimal populationsinordertoevaluateitspotentialinselective breeding.Finally,theESR1mightplayarolein spermatoge-nesisvalidatedthroughassociationstudyandbyprofiling

ofmRNAandproteinexpressioninnon-reproductiveand

reproductivetissuesincludingspermatozoa.

Acknowledgments

This research was financially supported by FBF

(Förderverein Biotechnologieforschung e. V., Bonn,

Germany). We thank Dr.A. Niggemeyer, Dr.S. Brüning,

Dr. M. Friedrichs and Dr. A. Riesenbeck (GFS Artificial

InseminationStation:Die GenossenschaftzurFörderung

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Hofer, Dr.H. Lutherand Dr.K. Caspari(SuisAG Artificial

Insemination Station, Sempach, Switzerland) for their

support during the sample collection and the

arrange-ments ofthereproduction records.We arealsothankful

to Prof. Dr. C. Knorr (Institute of Veterinary Medicine,

Georg-August-University, Goettingen, Germany) for his

co-operation.

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