AsianJournalofAndrology(2011)13,139–151 (cid:2)2011AJA,SIMM&SJTU.Allrightsreserved1008-682X/11$32.00 www.nature.com/aja REVIEW Mouse models in male fertility research DuangpornJamsaiandMoiraKO’Bryan Limitedknowledgeofthegeneticcausesofmaleinfertilityhasresultedinfewtreatmentandtargetedtherapeuticoptions.Althoughthe idealapproachtoidentifyinfertilitycausingmutationsistoconductstudiesinthehumanpopulation,thisapproachhasprogressed slowlyduetothelimitationsdescribedherein.Giventhecomplexityofmalefertility,theentireprocesscannotbemodeledinvitro.As such,animalmodels,inparticularmousemodels,provideavaluablealternativeforgeneidentificationandexperimentation.Sincethe introductionofmolecularbiologyandrecentadvancesinanimalmodelproduction,therehasbeenasubstantialaccelerationinthe identificationandcharacterizationofgenesassociatedwithmanydiseases,includinginfertility.Threemajortypesofmousemodels arecommonlyusedinbiomedicalresearch,includingknockout/knockin/gene-trapped,transgenicandchemical-inducedpointmutant mice.Usingthesemousemodels,over400genesessentialformalefertilityhavebeenrevealed.Ithas,however,beenestimatedthat thousandsofgenesareinvolvedintheregulationofthecomplexprocessofmalefertility,asmanysuchgenesremaintobe characterized.Thecurrentreviewisbynomeansacomprehensivelistofthesemousemodels,ratheritcontainsexamplesofhowmouse modelshaveadvancedourknowledgeofpost-natalgermcelldevelopmentandmalefertilityregulation. AsianJournalofAndrology(2011)13,139–151;doi:10.1038/aja.2010.101;publishedonline8November2010 Keywords: infertility;malefertility;mousemodels;spermatogenesis INTRODUCTION follistatin) are involved in the regulation of spermatogenesis.12–15 Infertility is a major medical problem worldwide. In the Western Deregulation of hormonal regulation has been shown to contribute societies,oneineightcouplesofreproductiveageisinfertile(http:// tomale/femaleinfertilityinhuman15andmousemodels.4,16 www.endotext.org/male/male7/maleframe7.htm).Ofthese,malefac- Mammalianspermatogenesiscanbeseparatedintothreecategoriesof torsarethesoleoracontributingcauseoftheinfertilityin.40%of cellular events: the proliferative phase (spermatogonia), the meiotic cases.1Causesofmaleinfertilitymaybebroadlyclassifiedintofour phase(spermatocytes)andthedifferentiationphaseorspermiogenesis categories: (i) defective sperm production; (ii) reproductive tract (spermatids),followedbyaseriesofpost-testicularmaturationprocesses obstruction; (iii) inflammation; and (iv) sexual disorders, for requiredforfullyfunctionalspermatozoa(capableofmotilityandfert- example, erectile dysfunction and ejaculatory failure.2,3 Approxi- ilization).Inadditiontogermcells,thetestiscontainstwootherspecial- matelyhalfofthecausesofmaleinfertilityresultfromdefectivesperm izedcelltypes,SertoliandLeydigcells,whichsupportspermatogenesis.12 production,forexample,completeblockadeofspermatogenesis,low Sertolicellsformtheblood–testisbarrierthroughthepresenceofinter- sperm counts, abnormal sperm motility, morphology or function.3 cellulartightjunctions,whichprovidestheisolatedenvironmentneces- Although the etiology of most cases remains unknown, there is saryfordevelopmentofspermatocytesandspermatids.Sertolicellsalso increasingevidencethatasignificantpercentageofmaleinfertilityis provideparacrinesupporttoalladultgermcelltypes.17Leydigcellsin caused by genetic defects including chromosome aberrations, gene the interstitial tissue of the testis are uniquely positioned to provide mutationsandsingle-nucleotidepolymorphisms.4–11 testosteronetotheseminiferoustubulestodrivespermatogenesis. Insimpleeukaryotes,suchasfliesandworms,itisestimatedthat THECOMPLEXITYOFMAMMALIANMALE over1000genesareinvolvedintheregulationofmalefertility.18,19In FERTILITYREGULATION mice, it has been calculated that the male germ cell transcriptome Followingthecrucialprocessofinuterosexdetermination,theestab- comprises more than 30 000 transcripts.20 The somatic cells of the lishment of male fertility comprises a series of complex and highly testisandthoseinvolvedinendocrineregulationofspermatogenesis structured steps involving cell division, differentiation and cell–cell will contain many others. Theoretically, defects in any one of these interactions.Theproductionoffullyfunctionalspermatozoainmam- genescouldleadtoinfertility. mals takes place within the seminiferous tubules of the testis and is under strict hormonal regulation. Several key hormones secreted by CHALLENGESINDEFININGMALEINFERTILITYGENES thehypothalamus(gonadotrophin-releasinghormone),pituitarygland INHUMANS (luteinizinghormoneandfollicle-stimulatinghormone)andhormones Twocommonapproachesusedtoevaluatethefunctionofageneare secretedbythetestis(forexample,androgens,oestrogens,inhibinsand reversegeneticandforwardgeneticapproaches(Figure1).Inareverse DepartmentofAnatomyandDevelopmentalBiology,TheAustralianResearchCouncil(ARC),CentreofExcellenceinBiotechnologyandDevelopment,MonashUniversity, Melbourne,Victoria3800,Australia Correspondence:DrDJamsai([email protected]) Received:23May2010;Revised:23July2010;Accepted:4August2010;Publishedonline:8November2010 Mousemodelsinmalefertilityresearch DJamsaiandMKO’Bryan 140 Figure1 Reverseandforwardgeneticapproachesforinvivogenefunctionalanalysesinmousemodels.Thereverseapproachbeginsbyselectingacandidategeneof interestfollowedbyevaluatingitsinvivofunctionusinggenemanipulationtechniquesandanimalmodelproduction.Oncegenerated,animalmodelsareusedfor phenotypiccharacterizationtodefinepathologicalabnormalities.Inmanycases,candidategeneshavemultiplerolesindifferenttissues/organs;thus,theanimal modelsoftengiverisetomorethanonephenotypicdefect.Bycontrast,theforwardgeneticapproachisinitiatedbythecreationofanimalmodelsexhibitinga phenotypicdefectofinterestfollowedbydefiningthegeneticalterationresponsibleforthephenotypicdefects. geneticapproach,thefunctionalanalysisofagenebeginsbyaltering The identification of genes required for spermatogenesis, post- gene expression (that is, complete or partial inactivation or ectopic testicularspermmaturation,capacitationandfertilizationisimper- expression), followed by an assessment of the phenotypic conse- ativeifwehopetoobtainacompleteunderstandingofmalefertility. quences.Incontrast,aforwardgeneticapproach,orphenotypic-driven Theoutcomesofthesestudieshaveramificationsforthediagnosisand approach, starts by the relativelyundirected generation of and iden- treatmentofhumanmaleinfertilityaswellasthedevelopmentofthe tificationofphenotypicvariantsofinterest.Theunderlyingmutationis much needed male-based contraceptives for humans and animals. identifiedsubsequently.Thelatterapproachisapowerfulmethodfor Male-basedcontraceptives,usingbothhormonalandimmunological revealingcompletelynovelgenesthatfunctioninaparticularprocess approaches, have been under trial with limited success.15,21 Recent asitdoesnotrequireforeknowledgeofagene’sfunction. focushasbeenonidentifyingtargetsfornon-hormonalcontraception Cytogeneticandmolecularstudieshaverevealedthatchromosome bytakingadvantageofthespecificcellularandphysiologicalprocesses abnormalitiesincludingdeletions(forexample,theazoospermiafac- uniquetothereproductiveorgans.Themaingoalistointerfereina torregiononthelongarmoftheYchromosome),translocationsand highlyspecificmannerinkeydevelopmentalorfunctionalprocesses. chromosomeaneuploidies,occurwithanincreasedincidenceininfer- This requires the identification/characterization of many of the tilemencomparedwithfertilemen.4–11Althoughrecentadvancesin unknownmalefertilityregulators.Withtheexceptionofthesperma- molecular biology and the completion of human genome sequence togonialstemcells,thetemporarydisruptionoftesticulargermcells haveenabledthepredictionofcandidategeneswithinthedeletedor is attractive as upon cessation of treatment it should require only translocated regions, the precise functions of the majority of these one wave of spermatogenesis to restore germ cell production and geneshaveyettobedetermined.Inaddition,infertility-causinggenes fertility. and risk factors are identified via mutation screening of candidate genesininfertilecasescomparedwithfertilecontrolmen.However, DIFFERENTTYPESOFMOUSEMODELS theprogressofthisapproachhasbeenslowforanumberofreasons, Given the complexity of male fertility, the entire process cannot be including:(i)inadequatenumbersofpatientandcontrolgroupsfor fullymodeledinvitro.Assuch,animalmodelsprovideaviablealterna- case–control studies; (ii) insufficiently detailed clinical/phenotypic tiveforexperimentation.Micearethemostcommonlyusedanimal information; (iii) the large genetic diversity in human populations models in biomedical research, including reproductive biology, that may contribute to different phenotypic outcomes (effects of because oftheirshortreproductive cycle withalargelittersize and modifiergenes);(iv)thedesignofthemutationscreens,forexample, relativelycheaphousingconditions.Mostimportantly,micearegen- themajorityofmutationscreensfocusonprotein-codingregionsof eticallyverysimilartohumans22andtheirembryosarerelativelyeasy candidategenesthatcaneliminateorbiasagainsttheidentificationof tomanipulateatthegeneticlevel.Moreover,mousespermatogenesis intronic mutations; and (v) follow-up in vivo by gene functional andoogenesisarecomparabletohumans.Overseveraldecades,many analysesinhumansremainsproblematicduetothelimitedavailability types of mouse models have been made available for biomedical ofhumansamplesandtheassociatedethicalrestrictions.Theuseof research,includingknockout/knockinandtransgenicmodels(using mousemodels,however,doesnothavesuchproblemsand,assuch,a thereversegeneticorcandidategeneapproach).Morerecently,chem- substantialamountoffertilityresearchisperformedinthemouse. ical-mutagenized mutant mouse models (using the forward genetic AsianJournalofAndrology Mousemodelsinmalefertilityresearch DJamsaiandMKO’Bryan 141 approach) have been increasingly used to reveal disease-associated performedtodefinepathologicaldefectsduetothecompleteorpartial genes,includingcausesofmaleinfertility.23–28 lossofthetargetedgenefunction. Inadditiontothegenerationofnullalleles,specificdisease-causing Knockout/knockinmiceandgene-trappedmice mutationscanbepreciselyintroducedintothemousegenomebya Themostcommonapproachusedtodefineagenefunctioninvivois ‘knockin’ approach. The production of knockin mice requires two geneablationbyhomologousrecombinationinembryonicstem(ES) roundsofhomologousrecombinationinEScellsinordertoeliminate cells, known as ‘knockout’ or ‘gene targeting’.29,30 This strategy is theremainingsequenceoftargetingvectorsfromthetargetedlocus. designed for the evaluation of a gene function on the basis of the Althoughthegenerationofknockinmiceismorecomplicatedthan completeelimination(nullallele)orpartiallyeliminationofacandid- knockoutmice,thisapproachenablesthecreationofmousemodels ate gene function (that is, deletion of particular domain(s) of the carryingspecificdisease-causingmutationsratherthanagenedeletion encodedprotein). that completely eliminates the protein function. For example, such Generally, targeting constructs are designed to carry a selectable allelescouldcarrymutationsobservedwithinthehumanpopulation. marker to enable the selection of correctly targeted ES clones (for Theproductionofnullallelesbywhichthetargetedgenesareinac- example, kanamycin/neomycin resistance gene), flanked by 59 and tivated in every cell in the animal throughout its life can be very 39homologyregionsofthetargetgene(Figure2).TargetedEScells valuableindefininginvivogenefunction.Thisapproach,however, are injected into blastocysts of a different mouse strain to produce hasalimitationduetoalargepercentageofknockoutmiceexhibiting chimericprogeny,whicharesubsequentlymatedwithwild-typemice unexpectedembryonic/neonatallethality,whichlimitstheanalysisof togenerateheterozygousknockoutmice,thatis,carryingonedeleted thegenefunctioninlaterstagesoflife.Insomeinstances,thegenemay alleleandonewild-typeallele.Homozygousknockoutmice,thatis, contribute to different aspects of health at different stages, for carrying two deleted alleles, are generated by heterozygous inter- example,differentfunctionsinembryostagecomparedwithneonate crosses.31,32 Once generated, the phenotypic characterization is andadultstages.Inthecaseofinfertilityandcontraceptiveresearch, Figure2 Genetargetingstrategyforthegenerationknockoutmice.Atargetingcassetteisdesignedtocontainhomologyarmsflankingadrugselectablemarker(for example,kanamycin/neomycin)tofacilitatetheidentificationoftargetedESclones.Dependinguponthedesignofthetargetingcassette,anullallelecanbegeneratedby: (i)eliminationofsequencescriticalfortranslationalinitiation;(ii)creationofaframeshiftinthecodingregionandprematurestopcodon(indicatedby*);and(iii)creation ofunstablemRNA.Untranslatedregionsaredepictedbyunfilledboxes;protein-codingregionsaredepictedbyfilledboxes;ATG:initiationcodon.ES,embryonicstem. AsianJournalofAndrology Mousemodelsinmalefertilityresearch DJamsaiandMKO’Bryan 142 thisisaseriouslimitation.Toovercomethislimitation,theablationof shouldbeawareoftheexistenceoftheInternationalKnockoutMouse atargetgeneataprecisetimeand/orspecifictissuecanbeachievedby Consortium(http://www.knockoutmouse.org),whichaimstomutate usingtheconditionalknockoutapproach,whichallowsthemouseto allprotein-codinggenesinthemousegenomeusingacombinationof developuptoacertainstageofdevelopmentpriortoinactivationof genetrappingandgenetargetinginC57BL/6mouseEScells.38The thegene.Thisapproachutilizesthesite-specificrecombinationsys- International Knockout Mouse Consortium includes the following tems,Cre/LoxPorFlp/FRT.33 programs:theKnockoutMouseProject(http://www.knockoutmou- Inadditiontoknockoutandknockinapproaches,ahighthrough- se.org/about/komp) (USA), the European Conditional Mouse putmethodforrandomgenedisruption,knownas‘genetrapping’, Mutagenesis Program (http://www.eucomm.org),39 the North hasbeendeveloped.34Thisapproachisbasedontheuseofagene- American Conditional Mouse Mutagenesis Project (http://www. trappingvectorcontainingaconserved39acceptorsplicesite,aeukar- norcomm.org/index.htm) (Canada) and the Texas A&M Institute yotic drug-selectable marker (and/or markers for gene expression forGenomicMedicine(http://www.tigm.org)(USA). analysis)andtranscriptionaltermination(polyA)signaltorandomly insert and interrupt gene transcription in the ES cell genome Transgenicmice (Figure 3). If the insertion of a gene trap cassette occurs within an Atransgenicorganismreferstoanorganismthathashaditsgenome intronofagene,itwillresultinalternativesplicingviatheuseofthe39 modifiedtocarryacopy(ormorethanonecopy)ofapieceofDNAof acceptor splice site within the cassette. The transcription of the foreign origin. These models enable researchers to evaluate in vivo trapped gene will terminate at the poly A signal and result in the phenotypic defects as a result of ectopic expression of a gene, for productionoftruncatedproteinornoproteindependinguponwhere example,overexpressionofawild-typegeneandexpressionofadom- thecassetteisinserted.InsertioninanintronpriortotheATGstartsite inant-negativeformtodiminishthenormalfunctionofagene.The islikelytogeneratenullalleles.34 production of transgenic mice is achieved by direct injection of a Throughtheuseofhigh-throughputtechnologies,ESclonescon- purified fragment of a transgene of interest into fertilized oocytes tainingthousandsofdifferenttrappedgeneshavebeenproduced.The (Figure4).Followinginjection,thetransgeneisinsertedintothegen- collectionofESgenetrapcelllineshasbeenmadepublicallyavailable ome of fertilized oocytes, which are subsequently used to generate and centralized through the International Gene Trap Consortium, transgenic mice. The fact that transgene integration usually occurs whichaimstogeneratealibraryofmousemutantEScellscovering randomly, and the expression level of transgenes can be influenced mostofthegenesinthemousegenome.35–37Furthermore,researchers bypositionaleffectandtransgenecopynumber. Figure3 Ahigh-throughputgenetrappingapproach.AgenetrapcassetteisdesignedtocontainthefeaturesnecessaryforitsintegrationintotheEScellgenomeand terminationoftranscriptionofthetrappedallele.ThesefeaturesincludeSAsiteandpolyAsignal.Ifinsertedwithinanintronofagene,thenativesplicingpatternis affectedbytheSA,resultinginfusionoftheupstreamexonswiththetrappedcassette.TranscriptionofthetrappedmRNAissubsequentlyterminatedatthepolyAsite. Ifinsertedwithinthe59UTR,thetrappedmRNAwillproducenofunctionalprotein.Ifinsertionoccurswithinanintrondownstreamofthetranslationalstartsiteofa gene,atruncatedprotein(fusedwiththetrappedcassette)maybeproduceddependinguponthestabilityofthetrappedmRNA.ES,embryonicstem;polyA, polyadenylation;SA,slicingacceptor;UTR,untranslatedregion. AsianJournalofAndrology Mousemodelsinmalefertilityresearch DJamsaiandMKO’Bryan 143 Figure4 Transgenesisstrategy.Atransgenecassetteisdesignedtocontainfeaturesnecessaryforgeneexpression,forexample,promoterandpolyadenylation signal.Additionalmarkerssuchasfluorescentproteintags(forexample,GFPandYFP)canbeincludedtofacilitatethedetectionoftransgeneexpression.The transgeneisintroducedintothegenomeoffertilizedmouseoocytes.Integrationofthetransgeneoccursrandomlyandmultiplecopiesofthetransgenemaybe integratedinthesamechromosomallocationorondifferentchromosomes.GFP,greenfluorescentprotein;YFP,yellowfluorescentprotein. Chemical-inducedpointmutantmice detailsonanENUmutagenesisapproachusedtodiscovermalefer- Tofacilitateunbiasedgenediscoveryinthemouse,aforwardgenetic- tilityregulators,readersarereferredtoRefs23–28. based approach using the potent mutagen, N-ethyl-N-nitrosourea Incontrasttoacandidategeneapproach(knockout/knockinand (ENU)hasbeendeveloped.40–42ENUprimarilyactsasanalkylating transgenesis),ENUmutagenesisprovidesapowerfultoolforthedis- agent,transferringitsethylgrouptoanyofanumberofnucleophilic coveryofpreviouslyunsuspectedgeneswithinbiologicalprocesses.In nitrogenoroxygensitesoneachofthefourdeoxyribonucleotides.40–42 addition to defining gene function, critical domains/regions of the ThetransferredethylgroupconstitutesaDNAadductthat,duringcell mutatedproteincanbeidentified.Thefirstlarge-scaleENUmutagen- proliferation and DNA replication, results in heritable mutations. esisbeganin1997.43,44Currently,thereareatleast20ENUconsor- ENU inducesthehighestmutation rateofany agenttestedinmice tiumsaroundtheworld.45Suchprogramshaverevealedmanycritical (on average 0.0015 point mutations per locus per gamete in the genesforhumandiseases,includingmaleinfertility.46–49 C57BL/6 strain). An ENU mutagenesis program involves injecting adult male mice with a series of ENU intraperitoneal injections. MOUSEMODELSFORMALEINFERTILITY Followingatransientperiodofsterility(10–12weeks,dependingon In recent years, the identification of infertility genes spanning all mouse strain and doses of ENU used), owing to the depletion of aspectsofspermatogenesis,spermmaturation,capacitationandfert- differentiatedspermatogonia,micearesubjectedtocontrolledbreed- ilizationhasbeenacceleratedbymousemodelstudies.4,50–52Atleast ingschemestopropagatetheinducedmutations.Ultimatelydescen- 400fertilityessentialgeneshavebeenmodeledinmice.Themajorityof dentsofthesemicearescreenedforaphenotypeofinterestandlinkage these genes were identified using knockout models and to a lesser analysisusedtoidentifytheunderlyingmutation.Mutationsareoften extent,knockin,transgenicandENU-mutagenizedmodels.Thecur- introducedontoonemousestrainandoffspringareoutcrossedtoa rentreviewisbynomeansacomprehensivelistofthesemousemod- different strain to enable mapping of the mutated chromosomal els,ratheritcontainsexamplesofhowmousemodelshaveadvanced region.Outbreedingcanoccureitherduringthegenerationofmice ourknowledgeofmalefertilityregulation.Herein,wefocusonsingle- forphenotypicscreening(Figure5)oronceaphenotypeofinteresthas gene defects that affect the process of spermatogenesis and post- beenidentified.Linkageanalysis(thelocalizationofthecausalmuta- testicular maturation and fertilization. Defects in genes involved in tion to a relatively small region in the whole genome) can be done hormonal regulation of spermatogenesis are not included in the usinganumberoftechniques.Themajority,however,exploitsmall review.Readersarereferredtoacomprehensivelistofthesegenesin differencesingenomicDNAsequencebetweenindividualstrainsof Refs4and16. mice.Once thelinkageregionis defined,themutation is identified throughthesequencingofcandidategenes.Candidategeneswithin Premeioticdefects the linkage interval are generally selected based on gene expression Inmammals,althoughspermatogenesisbeginsinfetallife,germcells profile,predictedproteinfunctionandavailablemouse/humandis- undergomitoticarrestpriortoregainingtheirproliferativeanddiffer- easephenotypes.Mostrecently,mutationidentificationwithoutthe entiativecapacityatpuberty.Thestemcellsinvolvedintheprocessof needforcandidategeneselectionhasbeenachievedbywholegenome spermatogenesisarecalledspermatogonia.Spermatogoniadifferenti- sequencing technologies (O’Bryan MK, pers. commun.). For more ate and undergo numerous cycles of mitosis at the basement of AsianJournalofAndrology Mousemodelsinmalefertilityresearch DJamsaiandMKO’Bryan 144 seminiferoustubules.Inmice,spermatogoniabegintoproliferate(as wellasself-renew)anddifferentiateat,4daysafterbirthanddivide continuously through mitosis to give rise to spermatocytes. Premeioticdefectscanleadtothecompletedisruptionofspermato- genesisandresultinaphenotypeequivalenttothatofthe‘Sertolicell- only’syndromeseenin,15.7%ofmenwithnospermintheirejacu- lates.53 Several genes involving mouse spermatogonia self-renewal, apoptosisandcellcycleregulationhavebeenshowntocontributeto maleinfertility.ExamplesofsuchgenesarelistedinTable1. Meioticdefects Meiosisisaspecialcelldivisionwherebydiploidparentalcellsproduce genetically diverse haploid sperm (or eggs). The reduction in chro- mosomenumberisachievedbyoneroundofDNAreplicationfol- lowedbytwosuccessiveroundsofchromosomesegregation(meiosisI andII).MeiosisIinvolvesthesegregation/separationofhomologous chromosomes from each other, whereas meiosis II involves the segregation of sister chromatids and therefore resembles mitosis.61 Malegermcellsinthemousetestisentermeiosisinthesecondweek of life. During meiosis, germ cells are termed spermatocytes. SpermatocytespassthroughG andSphaseandsubsequentlyenter 1 themeioticprophase,duringwhichtimechromosomecondensation andtheformation ofDNAdouble-strand breaks(DSBs)(leptotene spermatocytes), followed by the initiation of pairing (synapsis) betweenhomologouschromosomes(zygotenespermatocytes)occur. The completion of synapsis of homologous chromosomes and the repairofDSBs,usinghomologouschromosomesastemplates,occur fromthemidzygotenethroughthepachytenespermatocyteperiods. Theculminationofthisprocessresultsinthereciprocalexchangeof geneticinformationbetweenhomologues,andiscompletedthrough Figure5 Exampleofathree-generationbreedingschemeusedtoidentify themidpachytenetodiploteneperiods.62Geneticexchangetakesplace recessivemutations.Toscreenforrecessivemutations,athree-generation throughtheformationofDSBsfollowedbyacrossover(synapsis)of breedingschemeisrequired.Foundermalemouse(referredtoasgeneration geneticmaterialbetweenhomologouschromosomepairs.Thisleads 0(G ))ofaninbredstrain(forexample,C57BL/6)isinjectedwithENU.TheENU- 0 tothereassortmentofmaternalandpaternalallelesandtheproduc- treatedmaleissubsequentlymatedwithwild-typefemalesofadifferentinbred strain(forexample,CBA)toproduceG offspring.G progenycanbeproduced tionofgeneticallydivergedhaploidsperm.Theformationandrepair 1 2 fromG littermateintercrossesorfromG 3wild-typeCBAcrosses(asshown ofmeioticDSBsisapivotalprocessthatdrivesgeneticdiversity. 1 1 here).Finally,G3progenyaregeneratedfromG2littermateintercrossesand/orG2 Due to the complexity of the meiotic process, a large number of females3G1fathers.Duringeachstepofcrossing,eachprogenywillhavedif- genes are proposed to be involved in its regulation. Defects in this ferentcombinationsofchromosomesfromtheENU-treatedmousestrainandthe process can lead to meiosis failure, the production of aneuploid strainusedforsubsequentoutcrossing.Thesedifferencesenableresearchersto gametes and infertility. Furthermore, gamete aneuploidy can result mapthe region containingtheENU-induced mutation causing phenotypic defectsofinterest(indicatedby*).Inthiscase,mutationsareintroducedinto inembryonicdeathordevelopmentaldefectsintheoffspring.62Alist theC57BL/6genome.CBA,cytometricbeadarray;ENU,N-ethyl-N-nitrosourea. ofsomecrucialmeioticgenesidentifiedbytheuseofmousemodelsis showninTable2.Manyofthesegenesareinvolvedintheinitiationof programmedDSBformation,meioticrecombination,DSBrepairand Table1 Examplesofgenesessentialforpremeioticgermcelldevelopmentimplicatedbymousemodelstudies Gene Proposedfunction Knockoutphenotype Fertilitystatus Reference Etv5(Erm)(Etsvariantgene5) Transcriptionfactor Azoospermia;failedtomaintainspermatogonia Maleinfertility 54 Bax(BCL2-associatedXprotein) Regulationofapoptosis Premeioticgermcellarrest Maleinfertility 55 Pi3k(phosphoinositide-3-kinase) Phosphatidylinositol39-kinasesignaling Impairedspermatogoniaproliferationand Maleinfertility 56 pathway increasedapoptosisofspermatogonia Nanos2(Nanoshomolog2 Germcelldifferentiation Germcellapoptosisandcompletelossof Maleinfertility 57a,58 (Drosophila)) spermatogonia Ddx4(Vasa)(DEAD(Asp–Glu–Ala– Germcellproliferationanddifferentiation Impairedpremeioticgermcellsdifferentiationand Maleinfertility 59 Asp)boxpolypeptide4) increasedapoptosisofspermatogonia Dazl(Deletedinazoospermia-like) Germcellproliferationanddifferentiation Azoospermia;fewspermatogoniaentermeiosis, Male/femaleinfertility 60 andthosethatdofailtoproceedbeyond pachytene aTransgenicmousemodel. AsianJournalofAndrology Mousemodelsinmalefertilityresearch DJamsaiandMKO’Bryan 145 e c n ere 80 ef 3 a4 5 6 7 89 0 1 23 4 56 7 8 9, 1 2 3 4 5 6 7 8 9 a9 R 6 6 6 6 6 66 7 7 77 7 77 7 7 7 8 8 8 8 8 8 8 8 8 4 y n e g ertility uspro dieby ertility ertility ertility subf zygo ales subf subf subf Fertilitystatus Male/femaleinfertility Male/femaleinfertility Male/femaleinfertility Male/femaleinfertility Maleinfertility/female Male/femaleinfertilityMale/femaleinfertility Male/femaleinfertility Maleinfertility Male/femaleinfertilityMale/femaleinfertility Male/femaleinfertility Male/femaleinfertilityMaleinfertility Maleinfertility Male/femaleinfertility Maleinfertility;hetero ofhomozygousfem midgestationMaleinfertility/female Male/femaleinfertility Male/femaleinfertility Male/femaleinfertility Maleinfertility/female Maleinfertility/female Male/femaleinfertility Male/femaleinfertility Male/femaleinfertility Maleinfertility Knockoutphenotype ImpairedDSBformationandrecombinationinitiation ImpairedDSBformationandrecombinationinitiation Impairedchromosomalsynapsisandchromosomefragmentation Impairedchromosomalsynapsis Meiosisarrestedatpachytenestage ImpairedDSBrepairImpairedmeioticrecombination Impairedchromosomalsynapsis Impairedchromosomalsynapsis ImpairedchromosomalsynapsisImpairedchromosomalsynapsisandsynaptonemalcomplexes formation Dynamiclossofchiasmataandapoptosis MeiosisarrestatprophaseIandatrophyofthetestesandovariesMeiosisprophaseIarrestandincreasedgermcellapoptosis Meiosisarrestatpachytenestage Meiosisarrest Meiosisarrestandepigeneticdefects MeiosisprophaseIarrest Impairedchromosomalsynapsisandsexbodyformation Abnormalsynaptonemalcomplexes Impairedchromosomalsynapsis Impairedaxialelementformation Impairedaxial/lateralelementsandsynaptonemalcomplexes formation ImpairedDSBrepair Impairedchromosomalsynapsisandsexbodyformation Impairedsisterchromatidcohesionformationandchromosomal synapsis Meioticarrest;spermatocytesfailedtoexitprophaseG2/MIviatransition siae. s c vi mplicatedbymousemodelstudie Proposedfunction ProgrammedDSBformation ProgrammedDSBformation DSBsensingandrepairing Meioticrecombination AssemblyofspecificDSBrepair complexes MeioticrecombinationandDSBrepairMismatchrepair Mismatchrepair Mismatchrepair MismatchrepairMismatchrepair Mismatchrepair CellcycleregulationCellcycleregulation Homologouschromosomessynapsis Homologouschromosomessynapsis DNAmethylation Proteinubiquitinationanddegradation HistoneH3methyltransferasethat controlsepigeneticeventsrequiredfor meioticprophaseSynaptonemalcomplexformation Synaptonemalcomplexassembly,meioti recombination,andXYbodyformation SynaptonemalcomplexassemblyandchromosomalsynapsisAxial/lateralelementsandsynaptonemal complexformationandchromosomalsynapsis DSBrepair Synaptonemalcomplexformation, homologousrecombinationandDSBrepair Segregationofchromosomes Translationalregulation coli;S.cerevisiae,Saccharomycescere a Table2Examplesofgenesessentialformeiosisi Gene (Sporulationprotein,meiosis-specific,SPO11homologSpo11())S.cerevisiae(Meiosisdefective1)Mei1(Ataxiatelangiectasiamutatedhomolog(human))Atm(Dosagesuppressorofmck1homolog,meiosis-specificDmc1homologousrecombination(yeast))(H2Ahistonefamily,memberX)H2afx (Thyroidhormonereceptorinteractor13)Trip13(MutLhomolog1())Mlh1E.coli(MutLhomolog3())Mlh3E.coli(Postmeioticsegregationincreased2())Pms2S.cerevisiae(MutShomolog4())Msh4E.coli(MutShomolog5())Msh5E.coli (Exonuclease1)Exo1(Cyclin-dependentkinase2)Cdk2(CyclinA1)Ccna1(FK506-bindingprotein6)Fkbp6()(Proteasome(prosome,macropain)Psmc3ipHop2,Tbpip26Ssubunit,ATPase3,interactingprotein)(DNA(cytosine-5-)-methyltransferase3-like)Dnmt3l (Seveninabsentia1A)Siah1()(PRdomaincontaining9)Prdm9Meisetz (Rec8homolog(yeast))Rec8(Synaptonemalcomplexprotein1)Sycp1 (Synaptonemalcomplexprotein2)Sycp2 (Synaptonemalcomplexprotein3)Sycp3 (Synaptonemalcomplexcentralelementprotein1)Syce1(Synaptonemalcomplexcentralelementprotein2)Syce2 (Structuralmaintenanceofchromosomes1B)Smc1b (Eukaryotictranslationinitiationfactor4,3)Eif4g3c aChemical-mutagenizedpointmutantmousemodels. Abbreviations:DSB,double-strandbreak;E.coli,Escherichi AsianJournalofAndrology Mousemodelsinmalefertilityresearch DJamsaiandMKO’Bryan 146 cellcycleregulation,andplayapivotalroleinbothmaleandfemale body function and in initiating the growth of the axoneme; the meiosis. Catsper genes, which are involved in Ca21 regulation and onset of hyperactivatedmotility;111–114Slo3,whichisinvolvedinK1flowand Postmeioticorspermiogenesisdefects in regulating capacitation and hyperpolarization;115 and Gapds and Uponthecompletionofmeiosis, haploidgerm cellstermed‘round Pgk2, which are involved in the establishment of glycolytic pathways spermatids’areproducedandsubsequentlyundergoaseriesofdiffer- on the spermtail accessory structuresand the generation of ATP for entiationprocessescollectivelyknownas‘spermiogenesis’togiverise axonemefunction.116,117Foracomprehensivereviewofmousemodels to highly polarized spermatozoa. Spermiogenesis involves dramatic withabonormalspermtailfunction,readersarereferredtoEscalier.52 morphological alterations, which include chromatin reorganization Spermiationisahormonallyregulatedprocessbywhichspermato- andcondensation,acrosomeformation,cytoplasmicremoval,sperm zoa are released into the lumen of the seminiferous tubules. This tailassemblyandspermiation. involvesthedetachmentofelongatedspermatidsfromtheSertolicells Duringspermheadformation,thecharacteristicshapeofthesperm and the removal of most of the cytoplasm within the nascent sper- ineachspeciesisformedthroughthecoordinatedrepackagingofthe matozoaasresidualbodies.118,119Severalknockoutmodelsofgenes chromatin and the sculpting ofthe sperm head throughprocessing involving in chromatin packing, nuclear condensation, cytoplasmic involvingtheacroplaxome–manchettecomplex.Specifically,histones exclusion, and signal transduction and protein transport have been that package DNA in the majority of cell types are removed and showntocontributemaleinfertility(Table3). replaced first with transition proteins (TPs) and then protamines (Prms) to form a highly condensed and transcriptionally inactive Post-testicularmaturationandfertilizationdefects nucleus.AsevidenceinbothTP(Tp1andTp2)andPrm(Prm1and Spermappearmorphologicallymaturefollowingspermiation;how- Prm2)knockoutmodels,theattainmentofthecorrectratioofthese ever, they do not gain the capacity for fertilization until they have proteinsisessentialfortheformationof‘normal’lookingspermand undergone transit through the epididymis in a poorly understood for fertility.90–93 In agreement with mouse model studies, clinical process known as ‘epididymal maturation’. Epididymal maturation associationdatasuggestthatattainmentofthecorrectratioofPrms isdependentuponfactorsproducedbytheepididymisandischar- isessentialforhumanmalefertility.94,95 acterized by extensive post-translational protein modifications, Inparallelwiththechangesinnuclearpackaging,mountingdata changes in membrane composition and the progressive attainment suggestthattheelongatingspermatidnucleusisshapedthroughpro- offunctionalcompetence.120–123Foramoreextensivereviewofthese cessesinvolvingtheacroplaxome–manchette.96Theacroplaxomeisan importantprocesses,readersarereferredtoRefs124–126. F-actin-containingseriesofloop-likestructuresthatformaroundthe Subsequenttoepididymalmaturation,spermmustundergothepro- elongating spermatid and appear to be involved in the progressive cessofcapacitationinthefemalereproductivetract(orinmedia)tobe extrusion of the head, as well as in the anchoring of the growing fully capable of fertilization. Capacitation refers to a series of signal acrosome.97,98Inaddition,theacroplaxomeiscoupledtotheman- transduction and protein modification processes that occur during a chette, which in turn is composed of a marginal ring around the period of time following the ejaculated sperm into the female repro- nucleusandafringe-likemicrotubulestructurethatextendsintothe ductivetract.Capacitationconfersuponspermtheabilitytobindthe distal cytoplasm. Several mouse models, and microscopic studies, zonapellucidaoftheoocyteandundergotheacrosomereaction.Sperm stronglysuggestthatthesestructuresareintegraltotheformationof capacitation correlates with a massive increase in the tyrosine phos- normalspermheadshape.99–101Dataalsosuggestthatthemanchette phorylation of many sperm tail and head proteins,127–129 an increase isinvolvedinthetransportofproteinsrequiredforspermtailforma- inmembranefluidity,cholesteroleffluxandanincreaseinintracellular tioninaprocessknownasintramanchettetransport.100–102 Ca21andcyclicAMPconcentration.130–132Capacitationisalsocorre- Mousemodelshavealsobeencriticallyimportantindefiningthe latedwiththeabilityofspermtomanifesthyperactivatedmotilityand mechanismsofspermtailformationandhaverevealedsomesurpris- forthemtobindtotheoutervestmentsoftheoocyteduringacrosome ingphenomenaandtheetiologyofseveralsignificanthumanpathol- reaction.133–135Hyperactivatedmotility,whichischaracterizedbyhigh- ogies.52Duringthelastfewyearsinparticular,researchhasbegunto amplitude asymmetrical bending of the sperm tail, is proposedtobe illustrate the importance of axoneme function, not only for sperm importantforthespermtodetachitselffromthemucosalmembraneof motilitybutalsoforciliafunctioninarangeofsomatictissuesand theoviduct,wheretheyaretemporarilystored,andalsotopenetratethe manyaspectsofhumanhealth.Theaxonemeisa912microtubule zona pellucida of the oocyte during the acrosome reaction.135 Ion structure that runs as a core through the sperm tail (modified fla- channels including the CatSper channels play a central role insperm gella).103Thesamestructureisconservedinallmotilecilia(andfla- hyperactivationviatheregulationofCa21flow.111–114 gella) from all kingdoms ranging from trypanosomes and TheacrosomereactionisalsocharacterizedbyaCa21influxthat Chlamydomonas,tomiceandhumans.WhileAfzeliusetal.104recog- triggersfusionofanumberofsitesoftheinitialsperm–zonapellucida nizedseveraldecadesagothatabnormallyformedaxonemesleadto interaction.136,137Thisinteractioninvolvescomplementary,butasyet bothsterilityanddisordersinotherciliatedtissues,includingthelung, incompletelydefined,sitesonthespermouteracrosomalmembrane brainandkidneyinasyndromeknownasprimaryciliarydyskinesia,it and ZP3 on the egg plasma membrane.138–140 The fusion allows isonlyinthelastdecadethatsomeoftheunderlyinggeneshavebeen development of vesicles, containing hydrolytic enzymes, which are revealed.105–107 In addition, and largely through the use of mouse releasedinaprocesscalledacrosomalexocytosistobreakdownthe models, it is now recognized the abnormal cilia (sperm tail) func- egg cumulus mass. This exposes the inner acrosomal membrane so tion/formation at least in mice can be induced by a range of other thatitcanbindthezonapellucidaandanappropriatelyreactedsperm genes involved in sperm tail/cilia development or in transducing enterstheperivitellespace.Thespermandtheplasmamembraneof environmentalsignalstotheaxoneme.Suchgenesandmodelsinclude theeggbindandfuse.141–143 Tektin2(Tekt2),whichisinvolvedindyneinarmformation inthe Severalgeneshavebeenimplicatedtoplayaroleinpost-testicular axoneme;108 Alms1,109,110 and several other genes involved in basal maturation,capacitationandfertilizationusingmousemodelstudies AsianJournalofAndrology Mousemodelsinmalefertilityresearch DJamsaiandMKO’Bryan 147 e eferenc 44 45 0 1 2,9346 47,148 49 50 51a00 01 a08 5217 16 5354 5511–114 b6 56 57 5859 15 60 61 62 R 1 1 9 9 91 1 1 1 1 1 1 1 11 1 11 11 4 1 1 11 1 1 1 1 y ar m Fertilitystatus Maleinfertility Maleinfertility Chimeramale/female infertility Malesubfertility MalesubfertilityMaleinfertility Maleinfertility Maleinfertility Maleinfertility Maleinfertility Maleinfertility Maleinfertility Maleinfertility MaleinfertilityMaleinfertility Malesubfertility MaleinfertilityMaleinfertility MaleinfertilityMaleinfertility Maleinfertility Maleinfertilityandpri ciliarydyskinesia Maleinfertility MaleinfertilityMalesubfertility Maleinfertility Maleinfertility Maleinfertility Malesubfertility forspermiogenesis,epididymalmaturation,capacitationandfertilizationimplicatedbymousemodelstudies ProposedfunctionKnockoutphenotype SpermatidcytoplasmicremovalDeformedspermheadandtailstructureandimpairedspermiogenesis AcrosomebiogenesisFragmentedacrosomes;globozoospermia(round-headedspermatozoa) SpermchromatincondensationHaploinsufficiencyeffect;spermderivedfromspermatogeniccellswithonecopyofthe mutatedalleleareimmotileandunabletofertilizeeggs SpermchromatincondensationSubtlespermmorphologyabnormalitiesandreducedmotility SpermchromatincondensationImpairedchromatincondensation,spermheadabnormalitiesandreducedspermmotilityChromatinremodelingAbnormalspermheadmorphology,reducedDNAcompactioninspermatozoaand impairedspermmotility Celladhesion,differentiationandapoptosisSpermatidmaturationarrest,degeneratedandsloughedoffintothelumen CelladhesionAbnormalspermheadandspermtailmidpiecestructure,impairedzonabinding,andlackofoocytepenetrationSpermheadmorphogenesisOligospermia(lowspermcounts)andglobozoospermia Ligand-inducedtranscriptionfactorSloughingofimmaturegermcellsinthelumenofseminiferoustubules AcrosomeandtailbiogenesisImpairedacrosomeandtailformation;thebasaltailcuffatthepointofinsertionbutfailto addaxonemestoelongatethetailstructureSpermtailbiogenesis,assemblyoftheAbsentspermflagella,abnormalspermhead,disruptedmicrotubularorganelleessentialaxonemes/flagellaforspermheadandflagellaformationSpermtailbiogenesis,dyneinarmformationinBendingofthespermflagellaandmarkeddefectsinmotilityandprimaryciliarydyskinesia theaxoneme SpermmotilityNormalspermcountsandmorphologywithseverelyimpairedmotilityGenerationofenergysupplyforspermmotilityNormalspermcountsandmorphologywithimpairedmotility(lossofforwardprogression motility) GenerationofenergysupplyforspermmotilityImpairedprogressivemotility AcrosomebiogenesisGlobozoospermiaVesicletraffickingfromtheGolgiapparatustoFragmentedacrosomesGlobozoospermia theacrosome IontransportforspermtailformationNormalspermcounts,abnormalspermtailstructureandmarkedlyreducedmotilitySpermmotilityandfertilization(ionchannels)Normalspermcounts,normalmorphology,markedlyreducedmotilityandunableto fertilizeintacteggs SpermatidcytoplasmicextrusionExcesscytoplasmandimpairedmotility Ciliaandflagellafunction;axonememovementNormalspermcounts,normalmorphology,markedlyreducedforwardmotility MembranefusionDefectinsperm–eggfusion Spermtailassembly(scaffoldprotein)Normalspermcounts,normalmorphology,markedlyreducedmotilityAcrosomereactionandsperm–egginteractionImpairedfertilization Capacitationandacrosomereaction(ionImpairedmotility,abnormalspermmorphology(hairpinshape),impairedspermtransportation)capacitationandacrosomereactionCelladhesions(plasmamembraneprotein)Defectinspermtobindtothezonapellucida FoldingandtransportofintegralmembraneImpairedsperm–zonapellucidaadhesion proteins(testis-specificchaperone)Celladhesions(plasmamembraneprotein)Impairedsperm–eggmembraneadhesion,sperm–eggfusion,migrationfromtheuterus intotheoviduct,andbindingtotheeggzonapellucida Table3Examplesofgenesessential Gene (Spermmaturation1)Spem1(Golgi-associatedPDZandcoiled-coilGopcmotifcontaining)and(Protamine1and2)Prm1Prm2 (Transitionprotein1)Tnp1(Transitionprotein2)Tnp2()(Testis-specificserinekinase6)Tssk6Sstk ()(Celladhesionmolecule1)Cadm1Tslc1()(Poliovirusreceptor-related2)Pvrl2Nectin2 (Caseinkinase2,alphaprimeCsnk2a2polypeptide)(Retinoicacidreceptor,alpha)Rara()(Platelet-activatingfactorPafah1b1Lis1acetylhydrolase,isoform1b)(Meiosis-expressedgene1)Meig1 ()(Tektin2)Tekt2Tektin-t ()(Adenylatecyclase10)Adcy10sAC(Glyceraldehyde-3-phosphateGapdsdehydrogenase,spermatogenic)(Phosphoglyceratekinase2)Pgk2()(ArfGAPwithFGrepeats1Agfg1Hrb)(ProteininteractingwithCkinase1)Pick1 (Voltage-dependentanionchannel3)Vdac3,,,Catsper1Catsper2Catsper3Catsper4(Cationchannel,spermassociated1–4) (Cappingprotein(actinfilament)Capza3muscleZ-line,alpha3)(Dynein,axonemal,heavychain1Dnahc1) (Izumosperm–eggfusion1)Izumo1(Akinase(PRKA)anchorprotein4)Akap4(Proproteinconvertasesubtilisin/kexinPcsk4type4) ()(Potassiumchannel,subfamilyKcnu1Slo3U,member1)(Cyritestin)Adam3(Calmegin)Clgn (Fertilin-)Adam2b aGene-trappedmousemodels.bENUmutagenizedmousemodel. AsianJournalofAndrology Mousemodelsinmalefertilityresearch DJamsaiandMKO’Bryan 148 (Table3).Incontrasttopremeioticandmeioticdefects,themajority www.europhenome.org),theEuropeanMousePhenotypingResource of genes critical for postmeiotic and post-testicular defects exhibit of Standardised Screens (EMPReSS: http:empress.har.mrc.ac.uk) male-specificinfertilitydefect. and the European Mouse Disease Clinic (EUMODIC: http://www. eumodic.org).167,168 Within our lab, we broadly categorize the type FACTORSTOBECONSIDEREDWHENEVALUATING ofmaleinfertilityusingthestrategyoutlinedinBorgetal.169 MOUSEMODELS EMPReSSprovidescomprehensivestandardoperatingprocedures (SOPs)coveringthemainphysiologicalsystemsformousephenotypic Althoughtheuseofanimalmodelsisidealforinvivogenefunction screens.Todate,,150SOPshavebeendeveloped.Additionally,SOPs analysis,phenotypicdefectsobservedingeneticallymodifiedanimal for histology, pathology and gene expression analyses are available. models can influenced by different genetic backgrounds and envir- EuroPhenomeprovidesphenotypingdatafromSOPscontainedinthe onmentalfactors.Itisimportantthatthesefactorsareappropriately EMPReSS database together with the development of novel mouse addressedtopreventmisinterpretationofresults. phenotypingapproaches.EUMODICaimstoperformprimaryphe- Inthecaseofknockout/knockinmice,theEScellsandtherecipient notyping of 650 mouse mutant lines derived using the European blastocystareoftenobtainedfromanimalscarryinggenesofdifferent ConditionalMouseMutagenesisProgram.39Mouselineswithpheno- coatcolorstofacilitatetheinitialselectionofchimericprogeny.The typic defects of interest will undergo specialized phenotypic assess- mostcommonlyusedEScellsarethosederivedfromthemousestrain ment.DatafromtheEUMODICprojectarebeingintegratedwithin 129,whichhasanagouticoatcolor.Oncetargeted,theseEScellsare theEuroPhenomewebinterface.Thesepublicallyavailableresources microinjected into blastocysts obtained from a mouse strain with will enable researchers to precisely assess the phenotypic defects of differentcoatcolors(forexample,C57BL/6mice,whichhaveablack mousemodels. coatcolor).Offspringwithahighdegreeofagouticoatcolor,asan indicationofthetransmissionof129EScell-derivedgene,aremated CONCLUSION toproduceheterozygousknockoutmice.Thesemiceareintercrossed Theavailabilityofcompletegenomesequencesofhuman,mouseand togeneratehomozygousnullprogeny,whichgiverisetoacohortof anumberoforganismshasopenedupaneweraofgenefunctional miceonamixedgeneticbackground(forexample,129andC57BL/6). analysis.Usingmousemodels,reproductivebiologistshavesuccess- Similarly, mice generated through genome-wide ENU mutagenesis fullyrevealedhundredsofgenesessentialforfertilityandmanyarein may be on a mixed genetic background as a consequence of the theprocessofbeingidentifiedandcharacterized.Thenextchallengeis founder mouse strain used for ENU treatment and a mouse strain thetranslationoftheknowledgeobtainedfromstudiesonthemouse usedforoutcrossingforlinkageanalysisandmutationidentification into the human infertility clinic to serve the ultimate goals of: (i) beingdifferent.Inmanycases,theinitialphenotypiccharacterization improvingtheexistinginfertilitytreatments;(ii)providingalternative oftheknockout/knockinandpointmutantmutagenizedmiceisper- choices of treatment; (iii) developing precise diagnostics; and (iv) formedonamixedgeneticbackground,whichcanresultinarangeof formulatingcontraceptivesformaleswithminimalsideeffects. phenotypicvariations.Geneticmodifiers(forexample,single-nucleo- tide polymorphisms and flanking gene effect) are proposed to be a criticalfactorthatcontributestothephenotypicdiversity.163 COMPETINGFINANCIALINTERESTS Tominimizetheeffectofgeneticbackgroundvariations,knockout Theauthorsdeclarenoconflictofinterest. mice generated by 129 ES, for example, cells can be backcrossed to produceacongenicline,thatis,astrainthatdiffersfromanotherinthe ACKNOWLEDGMENTS regionofthegeneofinterestandashort-linkedchromosomalsegment TheauthorsthankMsAnneO’Connor(DepartmentofAnatomyand DevelopmentalBiology,MonashUniversity)forassistanceinthereviewofthe aroundthegene.Theproductionofacongeniclineinvolvesbreeding manuscript.Grantfunding:DJisaNationalHealthandMedicalResearch of the knockout mice with the strain of mice used for the initial Council(NHMRC)ofAustraliaPeterDohertyPostdoctoralFellowand chimerabreeding(oranewinbredstrainofchoice)foratleast10gen- MKO’BisanNHMRCSeniorResearchFellow. erations.Thisconventionalbackcrossingapproachcantakeupto3 yearstogenerate99%oftherecipientgenomeatthetenthbackcross- ing(N10).Recently,anewhigh-speedcongenicapproachusinground spermatidsretrievedfromimmaturemales(22–25daysofage)and 1 LeifkeE,NieschlagE.Maleinfertilitytreatmentinthelightofevidence-based polymorphicmarkeranalysiswasreported.164Thisapproachhasbeen medicine.Andrologia1996;28Suppl1:23–30. successfullyusedtoproducecongeniclinewithina6-monthperiod. 2 deKretserDM.Maleinfertility.Lancet1997;349:787–90. 3 de Kretser DM, Baker HW. Infertility in men: recent advances and continuing Analternativeapproachtoavoidcongeniclineproductionistouse controversies.JClinEndocrinolMetab1999;84:3443–50. C57BL/6EScellsfortheinitialgenetargeting.Althoughhomologous 4 MatzukMM,LambDJ.Thebiologyofinfertility:researchadvancesandclinical recombination in the C57BL/6 ES cells is lower than that of the challenges.NatMed2008;14:1197–213. 5 O’FlynnO’BrienKL,VargheseAC,AgarwalA.Thegeneticcausesofmalefactor 129 strain, it has been shown that a significant number of targeted infertility:areview.FertilSteril2010;93:1–12. ESclonesareproducedusingC57BL/6EScells.165 6 ReijoR,LeeTY,SaloP,AlagappanR,BrownLGetal.Diversespermatogenicdefects inhumanscausedbyYchromosomedeletionsencompassinganovelRNA-binding In addition to genetic background, several environmental factors proteingene.NatGenet1995;10:383–93. canhaveaprofoundinfluenceontheobservedphenotypeininbred 7 MorrisRS,GleicherN.Geneticabnormalities,maleinfertility,andICSI.Lancet1996; andcongenicstrains.Thesefactorsincludeviralandbacterialinfec- 347:1277. tions,exposuretoloudsounds,anddietaryandmaternaleffects.166 8 ThielemansBF,SpiessensC,D’HoogheT,VanderschuerenD,LegiusE.Genetic abnormalitiesandmaleinfertility.Acomprehensivereview.EurJObstetGynecol ReprodBiol1998;81:217–25. 9 VicdanA,VicdanK,GunalpS,KenceA,AkarsuCetal.Geneticaspectsofhumanmale INTERNATIONALMOUSEPHENOTYPINGCONSORTIUMS infertility: the frequency of chromosomal abnormalities and Y chromosome Researchersshouldbeawareoftheexistenceofresourcesthatprovide microdeletionsinseveremalefactorinfertility.EurJObstetGynecolReprodBiol 2004;117:49–54. accesstodataandproceduresformousephenotyping.Theseinclude 10 MaduroMR,LambDJ.Understandingnewgeneticsofmaleinfertility.JUrol2002; theEuropeanMouse PhenotypingResource (EuroPhenome: http:// 168:2197–205. AsianJournalofAndrology