Flow cytometry for the assessment of animal sperm integrity and functionality

14 Pages·2011·1.3 MB·English

by  Md. Sharoare Hossain

AsianJournalofAndrology(2011)13,406–419
(cid:2)2011AJA,SIMM&SJTU.Allrightsreserved1008-682X/11$32.00
www.nature.com/aja
REVIEW
Flow cytometry for the assessment of animal sperm
integrity and functionality: state of the art
Md.SharoareHossain1,2,AndersJohannisson1,MargaretaWallgren1,3,SzabolcsNagy2,4,
AmandaPimentaSiqueira1andHeribertoRodriguez-Martinez5
Flowcytometryisnowarecognizedmethodologywithinanimalspermatology,andhasmovedfrombeingaresearchtooltobecome
routineintheassessmentofanimalsemendestinedtobreeding.Theavailabilityof‘bench-top’flowcytometersandofnewerand
versatilemarkersforcellstructureandfunctionhadallowedtheinstrumentationtomeasuremorespermparameters,fromviabilityto
reactivenesswhenexposedtoexogenousstimuli,andtoincreaseourcapabilitiestosortspermatozoaforpotentialfertilizingcapacity,
orchromosomalsex.Thepresentreviewsummarizesthestateoftheartregardingflowcytometryappliedtoanimalandrology,albeit
keepinganopencomparativeintent.Itcriticallyevaluatesthepresentandfuturecapabilitiesofflowcytometryforthediagnosticsof
potentialfertilityandforthedevelopmentofcurrentreproductivetechnologiessuchasspermfreezing,spermselectionandsperm
sorting.Theflowcytometrymethodswillprobablyfurtherrevolutionizeourunderstandingofthespermphysiologyandtheir
functionality,andwillundoubtedlyextenditsapplicationinisolatingmanyuncharacterizedfeaturesofspermatozoa.However,
continuousfollow-upofthemethodsisanecessityowingtotechnicaldevelopmentsandthecomplexityofmappingspermatozoa.
AsianJournalofAndrology(2011)13,406–419;doi:10.1038/aja.2011.15;publishedonline11April2011
Keywords: animals;fertility;flowcytometry;semenanalysis;spermatozoa;spermfunctionality;spermintactness
INTRODUCTION fact;routinespermiogramevaluationsaresuitabletoidentifyclear-cut
Evaluation of the spermatozoa of animals has either a purpose of casesofinfertilityand,sometimes,toprovidecuesforpotentialsub-
diagnosisofthehealthofthemalereproductiveorgansor,forthose fertility,butarenotabletoprognoselevelsoffertilityofthesemen(or
siresdestinedtobreeding,oftheoddstheirsemenhastofertilize(or, themale)inquestion.7,13,15,16Thereasonisquiteobvious:suchassess-
toleadtofertilizationfailure,dependingontheapproachtotheprob- mentssimplyevaluatebasicvariablesofspermappearanceandviab-
lem).Duringclinicalandrologicalexaminations,evaluationofsper- ility,butcannotdisclosesubjacentstructuraldetails,biochemicaland
matozoaisofparamountimportance,sincesperm‘quality’provides metabolicdetails,orthecapacityofspermatozoatointeractwiththe
anindicationofthenormalityoftesticularfunctionintermsofsperm environment,intermsofmembranereactivity,unlessabatteryoftests
numbers,theirviability(oftensimplydeterminedbysubjectivemot- isused.Neithercantheymeasurewhetherthegenomicmessagesper-
ilityjudgment)andthenormalityofmorphologicalfeatures.Aswell,it matozoadelivertothegametecounterpartduringfertilizationisintact
provides an idea of the normality of the epididymal function, i.e. enoughtowarrantthedevelopmentoftheearlyembryo.Lastbutnot
spermmaturation,withthedisplayofpropermorphologicalfeatures least,mostroutinesemenevaluationsincludesmallsubsamples(often
(displacementofthecytoplasmicdropletfromthenecktotheannu- testedindifferentassays,sothatthesamespermatozoonisnoteval-
lus) and of activated motility after ejaculation. Fertilizing capacity, uatedsimultaneouslybythedifferenttests)ofalarge,heterogeneous
whichisalsoreachedduringepididymalmaturation,isnotmonitored numberofspermatozoaintheejaculate.Thisimpliesthattheresults
byasimplespermiogram,andoftenonlyassessedafterbreedingby obtainedhaveaninherentlargevariability,thuscallingfortheanalyses
naturalmatingorartificialinsemination(AI)ofacertainnumberof of larger sperm numbers.13 It is important to remember that for a
females,thelatterhopefullylargeenoughtokeepinnatevariationat given attribute of a cell only a small amount of an attribute might
minimum levels. Sperm functionality is, in this regard, obviously beneededforthatcelltofunctioncorrectly;foranotherattributehigh
modulated or affected bythe fluids spermatozoa bathe in, either as valuesmightbenecessary.Further,itisillogicaltoassumethatallcells
seminalplasmaorasextendersusedtodecreasespermconcentration, ofacertaintypedotheirtasksthesameway.17
ortoallowcoolingandfreezing–thawingofspermsuspensionsforAI. Asalreadyindicated,anothermajorpointduringsemenevaluation
Thereisaplethoraofexcellentreviewsonthisareaofspermevalu- isthevaryingresilienceofejaculatedspermatozoatosustaincurrent
ation and the reader is advised to consult these.1–14 Most of these andfuturehandlingprocedures,includingextensionorreconcentra-
reviewsandtheliteraturecitedthereinconsistentlyconcurtoabasic tion,cooling,freezingandthawing,high-speedsorting,selection,etc.,
1DepartmentofAnatomy,PhysiologyandBiochemistry,SwedishUniversityofAgriculturalSciences(SLU),SE-75007Uppsala,Sweden;2DivisionofReproduction,SLU,SE-750
07Uppsala,Sweden;3QualityGeneticsHB,SE-24292Ho¨rby,Sweden;4UniversityofPannonia,InstituteofEnvironmentalSciences,H-8200Veszprem,Hungaryand
5DepartmentofClinicalandExperimentalMedicine,Linko¨pingUniversity,SE-58185Linko¨ping,Sweden
Correspondence:ProfessorAJohannisson([email protected])
Received:1September2010;Revised:19November2010;Accepted:26January2011;Publishedonline: 11April2011
Flowcytometryforspermevaluation
MSHossainetal
407
towhichejaculatedspermatozoaarenowadayssubjectedtoforusein Sortertype
assistedreproductivetechnologies,suchasartificialinseminationor Partec(PA-IIandPAS-III),BeckmanCoulter(MoFloXDPandEPICS
invitrofertilizationofchilled,frozen–thawedorsexedsemen.Markers ALTRA),BDBiosciences(BDFACSAria,BDFACSVantageandBD
thatcandeterminehowextensivethedamagesare,andthatcanmon- Influx)sellssortertypeFCs.
itor theiroccurrence canbe usedforaquick, detailedanalyses ofa
largenumberofspermatozoaofagivensample.10,18 Others
Continuous innovation of new fluorescent stains and techniques FCswithnewfunctionsarebecomingavailable;LuminexCorporation
hasfacilitatedthisdevelopment.Moreover,withthesimplificationof (Austin, TX, USA) is dedicated to multiple analysis by suspension
the instrumentation, flow cytometry (FC) is now being introduced arrays (the Flexmap3D and Luminex 100/200 instruments), where
into handling of semen for AI and even into routine andrological verysmallsamplevolumescanbeassayedinasinglemicroplatewell.
diagnostics. Itfacilitatesnucleicacidassays,receptor-ligandassays,immunoassays
The present review summarizes the state of the art regarding FC andotherenzymaticassays.CompuCyte(BeckmanCoulter)isalaser
applied to animal andrology, albeit keeping an open comparative scanning solid cytometer which can analyze solid-phase samples
intent.ItcriticallyevaluatesthepresentandfuturecapabilitiesofFC together with tissue sections, cancer tissue imprints and cytology
for the diagnostics of potential fertility and for the development of smears.ThisFCiscapabletomanagehighamountsofcellandtissue
currentreproductivetechnologiessuchasspermfreezing,spermselec- specimens in a solid substrate analysis. CytoBuoy (Woerden, The
tionandspermsorting. Netherlands)is,togetherwithCytoSense,asimplifiedmethod(albeit
averyexpensiveone,thusfar)torecognizespermatozoafromother
PRINCIPLESOFFCANDCOMPARISONTOOTHER cellsanddebrisbyshapeonly.Thiscan,abovetraditionalFC,produce
CONVENTIONALTECHNIQUESTOEXPLORESPERM completesignalcoursesofthemeasuredemissionofeachparticle.Itis
STRUCTUREANDFUNCTION aportableFCsuitableforsubmergedinsitustudiesupto200mdepth
AnFCisaninstrumentthatcanmeasurephysical,aswellasmulticolor togetherwithsilico-imagingandvideoimaging,mainlyapplicablefor
fluorescence properties of particles of cells flowing in a flowing phytoplanktonresearch.ThenewestBDFACSArray(BDBiosciences)
stream.19AnFCisbasicallycomposedoffourmainsystems,fluidics, technology can easily assess cell signaling, cell surface staining and
optics,electronicsandsoftwarehandling,sothatthecellsuspension multiplexed bead assay. It is best suited for protein analysis in cell
biology,immunologyandmorespecificallyforproteomicsstudies.
flowsthroughatubularsystem,itisexposedtolaser(ormercuryarc
lampinsomeolderinstruments)illuminationatparticularspots,and
therecordedemissionsfromthecellsasaresultofthisilluminationis APPLICATIONOFFCFORASSESSMENTOFSPERM
digitizedandcomputer-handledtoprovideunderstandableresults. ATTRIBUTES
Afluidthatismovingunderalaminarflowspecifiesthemovement Enumeratingsperm
andvelocitiesthatcellshavewhenpassingacrossthedetector.During Totalspermperejaculateandtotalspermperstrawarebothimport-
thistransport,cellpropertieslikefluorescence,absorbanceandlight ant.Moreover,withbreedingcompaniesdecreasingspermnumbers
scatteringcanbedetected,makinguseofoneorseveralillumination in the AI doses, accurately accounting how many spermatozoa are
sources,whichmightdifferentlyexciteuploadedmarkers.Inthisway, inseminatedisofutmostimportance.Theissueoftimesincecollec-
FCdetectslabelingbymultiplefluorochromesassociatedwithindi- tionisalsoimportant,sincetotalspermperejaculateperhoursince
vidual spermatozoa, so that more than one sperm attribute can be latestcollectioncangiveinformationaboutproductivityofthetestes,
assessed simultaneously, increasing our capacity to correlate these ifthattimeisappropriatelyshort.20Totalspermperstrawmarksthat
attributesto,forinstance,potentialfertilizingcapacity.Moreover,it the insemination dose is as advertised and it is likely to maximize
makespossibletoacquiredatafromdifferentsubpopulationswithina fertilizingpotentialforthatbulliflittleisknownabouthisresponse
sample,thusevaluatingheterogeneouspopulationsindifferentstates todecreasingnumberofsperm/insemination.However,toprecisely
ofactivation.Thus,theanalysisbecomesobjective,hasahighlevelof determine the number of spermatozoa in a suspension volume is
experimentalrepeatabilityandhastheadvantageofbeingabletowork difficult,unlesstime-consumingmanualchambercountingisdone.
witheithersmallorlargesamplesizes. FC of fluorescence-loaded samples appears as a good alternative to
FCscanbebroadlyclassifiedasanalytical(orbench-top),sorterand determinespermtotalspermcountsinceFCcandiscriminatebetween
otherswithmiscellaneousfunctions.AnalyticalFCsareusedsolelyfor cellsanddebris(evenconsideringothercellsthanspermatozoa)yet
theanalysisofcellsorotherparticles,whileFCsortershavetheaddi- countinghighcellnumbers,thusdecreasingvariation.Forinstance,
tionalfeaturethattheyareableto,basedontheresultsoftheanalysis, similarresultsfortotalspermcountbetweenFCandhemocytometry
physicallyisolatethecellsorparticlesofinterest.SorterFCtypescan havebeenrecordedforanumberofspecies.21However,theinherent
either maintain a continuous fluid or split it into droplets after costsofFChavetobeconsidered.Moreover,FCoftenoverestimates
detectoranalysis.Fluidicsortersbetterhandlebiohazardoussamples the proportion of viable unstained spermatozoa while underestim-
than the droplet sorters, while the latter are used, for instance, to atingthatofthedamagedsubpopulation.22Wewilldiscussthisphe-
separatespermatozoabychromosomalsex. nomenon later in this review. Using fluorescent microbeads, total
ThereisanumberofcommercialFCsdevelopedforspecificrequire- spermcountswereassessedbyFC,simultaneouslywithothersperm
ments.Thesemaybebroadlyfunction-groupedasthefollowing. analyses(Figure123–25).However,asthebeadscanintroduceanother
source of variation, alternative FC cell counting methods, like true
volumetriccounting,weredeveloped.26
Analytical
ThemostcommonlyusedFCsaresoldbyPartec(Mu¨nster,Germany),
BeckmanCoulter(Brea,CA,USA),Millipore(Billerica,MA,USA), Spermintactness
Accuri(AnnArbor,MI,USA)andBDBiosciences(FranklinLakes,NJ, Viabilityofspermatozoa. Spermviabilityisaconvenient,yetimprecise
USA). term,becausethereisnocleardemarcationbetweenalivingordead
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sperm.Itisakeydeterminantofspermqualityandaprerequisitefor importanceforcellintegrityandfunction.40Membranestabilityand
successfulfertilization,becomingmoreandmoreimportantwhenlow permeability are two closely interlinked functionalities; thus, evalu-
sperm numbers are used for AI. Although a motile spermatozoon ationofmembranepermeabilityelucidatesitsorganization,41andits
could be defined as viable, the term ‘viable spermatozoon’ is often capability to undergo biophysical changes such water and the
linkedtoanintactplasmamembrane,sincetheplasmalemmaispiv- movement of cryoprotectant agents during cryopreservation.18,42
otal for sperm interactions with other cells and their environment. Cryopreservation causes irreversible damage to the plasma mem-
Therefore,mostviabilityassaykitsjustevaluatewhethertheplasma- brane, leading to cell death in a large number of spermatozoa.43
lemmaisintactornot,byusingimpermeabledyes(thosethatcannot Thus,itisimportanttoknowthepermeabilitycoefficientofthecells
penetrate an intact membrane) alternatively penetrating dyes (to tocryoprotectantagents,aswellastheireffectonthehydrauliccon-
depict those cells with eroded plasmalemma), or a combination, a ductivityofthemembrane.Differentmethods,likeCoultercounter,
matterthatshallbedescribedindetaillater.Whichdyeisusedcan electronmicroscopy,stopped-flowfluorometryanddifferentialscan-
significantlyinfluencetheresults.27Sperm‘viability’canbeassessedby ning calorimetry, have been used for years to evaluate membrane
numerousmethods,somemanualandotherautomated,andnotonly permeability.44Differencesbetweencellpopulationsintheiruptake
directedtotheplasmalemma.Someofthemareslow,assesstoofew of Hoechst 33342 due to altered membrane permeability have also
spermatozoaandarethuspoorlyrepeatable.Thereaderisadvisedto been studied.45 New fluorescent probes (SNARF-1, YO-PRO-1 and
see the reviews by Rodriguez-Martinez and Barth11 or Rodriguez- ethidiumhomodimer)formembranepermeabilityhaveimprovedthe
Martinez13 for a summary of these methods while studying the valueofusingFC42forthispurpose.YO-PRO-1isanimpermeable
spermiogram. membrane probe that can leak in after destabilization of the mem-
brane,asaresultofsilencingofamultidrugtransporterinvolvedin
Plasmamembraneintegrity. Thespermmembraneisdirectlyorindir- transportingitoutofintactcells.Thus,useofYO-PRO-1isauseful
ectlyrelatedwithmanyspermfunctions,warrantingthecapabilityof toolfordetectingearlymembranepermeability.46Thethreeprobesare
thecelltomaintainhomeostasisanddepictmotilityandthecapacity easilydistinguishedbothinFCandinfluorescencemicroscopy.
tointeractwiththeenvironment,includingtheliningepitheliumof Annexin V is a calcium-dependent probe that has recently being
the female genital tract or the oocyte–cumulus cell complex.7 used for tracking externalization of phosphatidylserine (PS) in the
Althoughthespermplasmamembranecoverstheentirecell,itcon- spermmembrane.SincePSisnormallylocatedexclusivelyattheinner
sists of several distinct membrane compartments, like the one that faceofthelipidbilayer,suchexternalizationmonitorsearlychangesin
coverstheouteracrosomemembrane,onethatcoversthepostacro- membranestabilityandintactness,detectableearlierthanwhenPIis
some portion of the sperm head down to the annulus, and lastly used,forinstance(Figure3).CombiningSNARF-1,YO-PRO-1and
anotheronecoveringtheprincipalpieceandtherestofthetail.The ethidiumhomodimerofferssomeadvantagesovertheAnnexinV/PI
firstfluorescentprobesappliedtoevaluateplasmamembraneintegrity assay.WhereasintheAnnexinV/PIassay,thereisalwaysanunstained
werefluoresceindiacetate28andcarboxyfluoresceindiacetate.29These subpopulation,thetriplestainlabelsallthespermatozoainthesam-
dyesarenon-fluorescent,butconvertedtofluorescentderivativesby ple.Thesubpopulationoflivecellsusingthenewtriplestainingcon-
intracellularesteraseenzymes,andtrappedbytheintactplasmamem- curs with the subpopulation of live cells using the well-validated
brane,resultinginafluorescingcell. AnnexinV/PIassay.Also,thepercentageofearlydamagedspermato-
Themostcommonlyusedcombinationofdyesusedformembrane zoa was higher with the Annexin V/PI assay. This might reflect an
integritychecksisSYBR-14/propidiumiodide(PI)(Figure2).Inthis increase in membrane permeability, preceding the transposition of
combined stain, the nuclei of viable spermatozoa fluoresce green, PSduringanevolvingcryodamage.However,itcanalsobe,inayet
whilethosewitherodedplasmalemmaarecounterstainedred,includ- tobedeterminedphysiologicalchange,averyearlystep,whilechanges
ingamoribundcellsubpopulation(green–red).Propidiumiodideis in cell volume regulation and movement of ions occur, during the
normallyusedastheviabilityprobeofchoiceinFCasthissupravital initiationofapoptosis47orcryoinjury.48Inaddition,anearlierinac-
stain rapidly penetrates non-viable spermatozoa when their plasma tivation of enzymes involved in maintaining membrane asymmetry
membraneisdisrupted.30 thanthoseinvolvedintransportingamphipathicsmallmoleculeslike
This FC procedures (available as a live/dead kit from Invitrogen YO-PRO-1mightexplainthisfact.
(Carlsbad,CA,USA))havebeensuccessfullyappliedformanyspecies, TheFCevaluationofplasmamembraneintegrityusedmanyfluor-
as human,31 bovine,32 porcine,13,31,32 ovine,31 Lagomorpha,31 mur- escentprobesandnewprobesareemergingrapidly.TheAnnexinV/PI
ine,31,33avian,34,35honeybees36,37andfish.38Furthermore,itisable assayworksfortrackingPSexposureincryopreservedsamples,but
to simultaneously evaluate sperm cell viability together with some due to the presence of cell debris and egg yolk particles, this probe
otherattributes,e.g.,incombinationwithfluorescentlylabeledplant combinationmightoverestimatetheunstainedsubpopulationwhen
lectinsforsimultaneousassessmentofplasmamembraneandacro- usingFCfordetection.Consideringthislimitation,useofYO-PRO-1
someintegrity.39Themainadvantageofthisdyecombinationisthat in combination with SNARF-1 and ethidium homodimer has been
bothfluorochromeshavethesameintracellulartarget(DNA);how- testedincryopreservedboarspermatozoawithgoodcorrelationwith
ever,itmeansitsmainweaknessaswell:sincemostDNAisfoundin Annexin V/PI as a result.49 Another alternative for assessing early
thespermhead(andlessinthemid-pieceinthemitochondria),the changes was to use another triple fluorochrome combination of
assay indicates the integrity of the plasma membrane of the head Merocyanine540,YO-PRO-1andHoechst33342,50provingeffective
domainonly.Moreover,theprobementioned abovewillnotassess forseveralspecies.51
theintegrityoftheplasmalemmacoveringneithertheacrosome,the
mid-piecenortherestofthetail. Acrosomeintegrity. Theacrosomeisamembrane-enclosedstructure
coveringtheanteriorpartofthespermnucleus.Powerfulhydrolyzing
Assessing sperm membrane permeability and stability. As mentioned enzymesbelongtothatstructure,whichisabasicfeatureofthesperm
above, the intactness of the sperm plasma membrane is of utmost head of all mammals.52 Acrosomal integrity is a prerequisite for
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Figure3 AnnexinV/PIlabeling.Densityplotshowsnon-alteredspermatozoa
Figure1 Colordotplotrepresentingthesimultaneousevaluationofviability
(lowerleftquadrant),PI-negativespermatozoawithexposedPS(lowerright
(SYBR141/PIstaining)andcellconcentration.Viablespermatozoaareindicated
quadrant),PI-positivespermatozoawithoutexposedPS(upperleftquadrant)
bygreencolor,deadonesareindicatedbyredcoloranddyingspermatozoaare
andPI-positivespermatozoawithexposedPS(upperrightquadrant).PI,propi-
indicatedbybluecolor.Fluorescentmicrobeadsareindicatedbymagentacolor.
diumiodide;PS,phosphatidylserine.
PI,propidiumiodide.
isothiocyanate-labeled Pisum sativum agglutinin (FITC-PSA)57 and
fertilization,12 essential for sperm penetration of the zona pellucida.
FITC-concanavalinA(FITC-ConA).58,59Electronmicroscopicstudies
Biologically,theplasmamembraneandtheouteracrosomemembrane
havedemonstratedtheultrastructuralmorphologyofacrosomeswith
fuse and vesicate during the acrosome reaction when activated by
thestainingpatternsofspermatozoaafterlabelingwiththeantibodies
spermbindingtothezonapellucida.Acrosomeintactnessistradition-
byPietrobonetal.60Duetohighcostsandneedfortrainedpersonnel,
allyexaminedinvitrousingphase-contrastmicroscopyordifferential
such approach is rarelyusedand suitable only for research purpose.
interference-contrastmicroscopyonunstainedorstainedsamplesfor
Although epifluorescence microscopy is being used, Pen˜a et al.61
lightmicroscopy.2Fluorescentmicroscopy-basedstainingarechlorte-
observedthatitwaslessprecisefordetectingthepercentageofsper-
tracyclinestaining,53,54paramagneticbeads,55quinacrine,56fluorescein
matozoawithdamagedacrosomescomparedtoFC.
The FC can advantageously be used instead of epifluorescent
microscopy,10 allowing determination of acrosome reaction and its
temporaloccurrence,andtheexaminationoflargespermnumbers.
The most commonly used probes are plant lectins labeled with a
fluorescent agent (usually FITC), most commonly PSA or peanut
agglutinin (PNA). PSA is a lectin from the pea plant that binds to
mannose and galactose moieties of the acrosomal matrix. As PSA
cannot penetrate an intact acrosomal membrane, only acrosome-
reactedordamagedspermatozoawillstain.62Arachishypogaeaagglu-
tinin (PNA) is a lectin from the peanut plant which is believed to
displaylessnon-specificbindingtootherareasofthespermatozoon,
leadingsomeworkerstofavorthisoverPSA63anditisthelectinof
choicewhenevaluatingspermextendedineggyolk-containingmedia,
as PSA has a nonspecific binding affinity to egg yolk64 and several
nonspecific binding sites on the sperm cell surface.63 Carver-Ward
et al.65 proposed that PNA is the most reliable lectin compared to
PSA and ConA. Only PNA gives a specific comparison between
unreacted and reacted sperm, and the differences between the two
markers are merely a matter of magnitude. Petrunkina et al.66
observedthatFITC-PNAbindstotheouteracrosomalmembrane,67
sothatitcanbettermonitoracrosomalintegrity.Oneofthefrequently
Figure2 ViabilityevaluationbySYBR14/PIlabeling.Three-dimensionalcontour
usedfluorochromecombinationsforthesimultaneousassessmentof
plotshowsdistinctpeaksforviableanddeadspermatozoa;moreover,dying
spermatozoaandcelldebriscanbeeasilyrecognizedaswell.PI,propidium plasma membrane integrity and acrosomal integrity are FITC-PSA
iodide. andPI.61Thisdoublestainingformembraneintegrityandacrosomal
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integrityisrelativelyreliableforfreshandinvitrocapacitatedsperm.68 fluorescent detectors are needed to evaluate one sperm attribute;
Problems can arise when frozen–thawed spermatozoa are analyzed. therefore, it is less applicable in multicolor FC experiments.
Eggyolkparticlesfromtheextenderhavelowfluorescenceand,there- Moreover, Garner and Thomas84 found the presence of aggregates
fore,canbeassessedasliveacrosome-intactspermusingthePI/FITC- onlymarginallydetectableaftercryopreservation.Theyhypothesized
PSAdouble-labelingmethod.However,itisyetarguedthatlectinsdo that cryopreserved sperm cells have damaged metabolic capacity,
notspecificallybindtotheacrosomeregion69andthatotherbinding probablyduetoglycerol.Wedevelopedanalternativelabelingtech-
sites can be easily distinguished by epifluorescence microscopy, nique,usingMitoTrackerDeepRed,78whichneedsonlyonedetector
whereasFConlyidentifiesonesignalfromtheentirespermatozoon. toidentifyspermatozoawithhighandlowmitochondrialmembrane
A newly developed triple staining (SYBR-14, phycoerythrin-conju- potential (Figure 6). Unfortunately, the spectral properties of this
gated PNA and PI; Figure 4) procedure was developed and in our particulardyedonotfittothesmallerbenchtopFCsequippedwith
handwasfoundasanefficientmethodforevaluatingacrosomalinteg- a488-nmlasersourceonly.
ritytogetherwithviability.39,70ThiscombinationlabelseveryDNA- Therearesomeothermitochondrialstainslike3,3’-dihexyloxacarbo-
containingeventandthecolorsofthefluoroprobesfittothestandard cyanine iodide and 2-(4-(dimethylamino)styryl)-N-ethylpyridinium
filtersetofmostcommercialFCs.Thephycoerythrin-conjugatedPNA iodidewhichstainmitochondriaoflivingcellsthatoughttobetested
canalsobepreparedinthelaboratory.71 in andrology. A recent study showed accumulation of cationic lipo-
philic probe 3,3’-dihexyloxacarbocyanine iodide in combination with
Mitochondrialstatus PI could assess mitochondrial membrane potential effectively in pig
Mitochondria, located in the sperm mid-piece generate a major spermatozoa.88Otherstudiesindicate,however,that3,3’-dihexyloxacar-
part of the ATP required for sperm metabolism, membrane func- bocyanineiodideisnotspecifictomitochondrialmembranepotential.89
tion and motility, alongside with anaerobic glycolysis in the
cytoplasm.19,72–74 Besides, mitochondria are the coordinators of Chromatinintactness
apoptosis mechanisms in a number of cell systems,75,76 and they EvaluationofspermDNAintegrityisofutmostimportanceowingto
areinvolvedinspermmaturation72andprotectionagainstdamage earlyembryodevelopmentdependingonitsnormality.12,13Although
induced by cryopreservation.74 Recently mitochondrial proteins spermDNAispackedinahighlycompactedandstableform,90chro-
were found to be involved in the capacitation-dependent tyrosine matinabnormalitiesandDNAdamageexist,derivedfromeitherpre-
phosphorylation in spermatozoa.77 meiotictesticularinsults,duringspermiogenesis,whenDNAispacked
Thereareapproximately100mitochondriainthemid-pieceofthe orduringfurtherchromatinbuilding(protaminedominance)during
spermatozoon,dependingonthespecies.78Mostofthesedyesapplied epididymalmaturation.91–93Alternatively,itcouldbetheresultoffree
intheevaluationofmitochondrialstatusworkbydiffusingintoliving radical-induced damage94 or a consequence of apoptosis, in some
cells and accumulating in mitochondria. The most widely used species.95 Sperm DNA fragmentation can be assessed with terminal
mitochondrial-specific probe, rhodamine 123 (R123), is a cationic deoxynucleotidyl transferase-mediated dUTP nick end labeling
compound that accumulates in the mitochondria as a function of (TUNEL)assay,whichcanidentifyDNAstrandbreakswithmodified
transmembranepotential.79Itwashistoricallyappliedtospermatozoa nucleotides.Theseincorporatedlabelednucleotidescanbedetectedin
incombinationwithethidiumbromide.80TheR123accumulatesin spermatozoabyFC,fluorescencemicroscopyorlightmicroscopy.96
the mitochondria and fluoresces green, the fluorescence intensity TUNELcansimultaneouslydetectsingle-anddouble-strandbreaks.
depends on total amount of functioning mitochondria. Sper- ByTUNELthedegreeofDNAdamagewithinacellcannotbequan-
matozoa with damaged membranes are usually identified by the tified,whichonlyrevealsthenumberofcellswithinapopulationwith
uptake of ethidium bromide or PI, to discriminate between those DNAdamageAnadvantageoftheTUNELassayisitsapplicationin
living and dead.80 Unfortunately, this stain does not differentiate FC,97 although Domı´nguez-Fandos et al.98 found 2.6 times greater
betweenmitochondriathatexhibithighrespiratoryrates.Whenmito- spermdamagebyFCthanthatoffluorescentmicroscopy,thussug-
chondrialmembranepotentialislost,R123iswashedoutofthecells. gestingthatitmayoverestimatedamage.ChromomycinA3(CMA3)
The main weakness of R123 is that it has low sensitivity and mito- isafluorochromethatdetectsprotaminedeficiencyinlooselypacked
chondriahaveseveralenergy-independentbindingsites.81 chromatin, which is correlated to the extent of nicked DNA.10 The
ThemitochondrialprobeMitoTrackerGreen82isnon-fluorescent CMA3isausefultooltoassessthepackagingqualityofspermchro-
inaqueoussolution,butfluorescesgreenuponaccumulationinthe matin and allow indirect visualization of protamine deficiency.
mitochondriaregardlessofmitochondrialmembranepotential.83The Traditionally fluorescence microscopy is being used to evaluate
mitochondrialstain5,59,6,69-tetrachloro-1,19,3,39-tetraethylbenzimi- DNA integrity by CMA3, but recently it was reported that this
dazolylcarbocyanineiodide(JC-1;Figure5)thatexistsasamonomer CMA3 can evaluate DNA integrity in human spermatozoa though
at low concentration yielding green does permit a distinction to be fixation, and number of sperm or duration of exposure to CMA3
madebetweenspermatozoawithpoorlyandhighlyfunctionalmito- mightaffectefficacyofFCoutcome.99
chondria.84 Martinez-Pastor et al.85 observed some relationship Thespermchromatinstructureassay(SCSA;Figure7)isatech-
betweenJC-1stainingandmotility,althoughcorrelationwithmotility niqueoriginallydevelopedbyEvensonetal.100inwhichtheextentof
isregulatedbymanyfactors.Inhighlyfunctionalmitochondria,the DNA denaturation following acid treatment is determined by mea-
concentrationofJC-1insidethemitochondriaincreasesandthestain suring the metachromatic shift from green fluorescence to red
forms aggregates that fluoresce orange. When human spermatozoa fluorescenceforacridineorange,thusdeterminingtheratiobetween
were divided into high, moderate and low mitochondrial potential single- and double-stranded DNA in each spermatozoon. Con-
groups,basedonJC-1fluorescence,theinvitrofertilizationrateswere sequentlythenativespermDNAisstainedgreen,whilethedenatured
higherinthehighpotentialgroupthaninthelowpotentialgroup.86 (becauseitwasalreadyeitherdenaturedbythetimetheacidexposure
JC-1hasalsobeenusedsuccessfullytomeasuremitochondrialfunc- wasissuedorbrokenbytheprocedure)DNAisstainedwithorange.In
tion using fluorometry.87 The main weakness of JC-1 is that two FC,spermcontaininggreaterredtogreenratiosareexhibitingmore
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Figure6 MitochondrialmembranepotentialevaluationbyMitotrackerDeep
Figure4 Simultaneousevaluationofviabilityandacrosomeintegrity(SYBR14/
Red.Histogramshowsspermatozoawithhigh(markerM1)andlow(marker
PE-PNA/PIlabeling).Colordotplotshowsviable,acrosome-intact(lowerleft
M2)mitochondrialmembranepotential.
quadrant),viable,acrosome-ruptured(lowerrightquadrant),dead,acrosome
intact(upperleftquadrant)anddead,acrosome-rupturedspermatozoa(upper
rightquadrant).Greencolorindicatesviablecells,redindicatesdeadones,blue thousandsofspermcanbeevaluatedobjectively.TheSCSAhasalso
colorindicatesdyingspermatozoa(evaluatedasdead).PE,phycoerythrin;PI, been successfully used to check gamete quality after cell manipula-
propidiumiodide;PNA,peanutagglutinin. tion.102ThemostimportantparameterrevealedbySCSAistheDNA
fragmentation index, which assesses the percentage of spermatozoa
DNA denaturation than that of spermatozoa exhibiting lesser red showing susceptibility of DNA to the acid induced denaturation in
togreenratios.101OnebenefitofSCSAoverothermethodsthatevalu- situ. Unfortunately, the method does not really measure the pure
ateDNAintegrityisthattheprocedureisrelativelyeasy,quick,and populationofspermatozoareallyhavingdamagedDNA,whichisto
Figure5 Mitochondrialmembranepotential,evaluatedbyJC-1.Colordotplot
showsarawsemensamplebefore(red)andafter(black)freezekilling(plunging Figure7 Spermchromatinanalysisbyacridineorangestaining.Intactspermato-
the semen aliquot into liquid nitrogen). List mode files were merged with zoaareindicatedbyblackcolor,cellswithincreasedamountofsingle-stranded
FCSExpresssoftware(version3.0;DeNovoSoftware,LosAngeles,CA,USA). DNA(indicatingDNAdamage)areindicatedbyredcolorandspermatozoawith
JC-1,5,59,6,69-tetrachloro-1,19,3,39-tetraethylbenzimidazolyl-carbocyanine immaturechromatinareindicatedbygreencolor.Celldebrisisindicatedby
iodide. orangecolorandgatedoutduringanalysis.
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beconsideredarelevantdrawback.Inhumans,andonlyundercertain TheFCwillquantitatesubpopulationswithinsamplesandestimate
conditions of assessment of fertility (assisted reproductive techno- globalphosphorylationintheentirespermatozoon,whichmightbet-
logy procedures), males with semen samples with more that 30% tercorrelatewiththecapacitationprocess,sinceFCmostlikelysolely
DNA fragmentation index were considered unable of establishing evaluatestheprocessinthesurfaceoflivespermatozoa.117
pregnancy.103Inanimals,whereSCSAisthemostwidespreadmethod However,whenusingFCinspermplasmamembranephysiology
toassesschromatinintegrityamongdomesticanimals,104–106particu- studies,especiallytoinvestigateearlymembranechanges,oneshould
larlyrelatedtoprocessedsemen(includingstorageandcryopreserva- becarefultoavoidintroducinglabelingartifacts.118
tion),thereisarelationshipbetweenchromatindamageandfertility,
butthresholdsarefarfromestablishedwithcertainty.11Anotherpara- Apoptotic-likechanges
meterindicatedbySCSAistheratioofspermatozoawithhighgreen Apoptosis, ‘programmed cell death’, delimits a sequence of events,
fluorescence.Thesespermatozoahavelesscompactedchromatinand whichleadstothedestructionofcellswithoutreleasingharmfulsub-
consequently higher greenfluorescence and ought tobe considered stancesintoitssurroundingarea.Apoptosisplaysadistinctroleinthe
immature.107 In animals not bred for fertility, e.g. horses, there is developmentandmaintenanceofhealthbyeliminatingoldandunne-
considerablevariationinDNAfragmentationindex,106whilethevari- cessarycells,aswellasunhealthycells.Apoptosismarkershavebeen
ationisminimalinbreedingpigs.108 detectedinspermatozoaofmanyspecies,buttheroleofthesemarkers
isprobablynotyetelucidatedcompletely.49,119–122Changesofsperm
FCTOASSESSSPERMFUNCTIONALITY membrane permeability have been considered as a typical event of
Changesinducedduringcapacitation apoptosis in many studies. Since cryopreservation, freezing and
Capacitation is an important, but rather incompletely understood thawing cause cryodamage, and presumably even apoptotic-like
phenomenon that a spermatozoon undergoes before it can fertilize changes,123apoptoticmarkerscanbegoodtoolsforforecastingsemen
the oocyte. It primarily takes place in the oviduct57 when various freezabilityandcellulardamageoccurduringcryopreservation,albeit
cellularchangessuchasanincreaseinmembranefluidityduetolipid notnecessarilyindictingapoptosisreallyoccurs.124Thecombination
modifications,aninfluxofcalciumtothespermperinuclearandneck ofAnnexinVwithPIisabletosimultaneouslydetermineapoptosis-
regionsandflagellum,thegenerationofcontrolledamountsofreact- likechangesandspermatozoawithcompromisedplasmamembrane.
iveoxygenspecies,aswellasthephosphorylationofproteinresidues, This combination can detect four categories of sperm populations:
occur in a concerted manner.109–111 Therefore, there is no general live,liveearly‘apoptotic’,deadandlate‘apoptotic’,andlate‘necrotic’
agreement regarding which assay is most appropriate to discern cells.125TheAPO-BRDUkittoconductTUNELassayfordetermina-
betweencapacitatedanduncapacitatedspermatozoa.Oneofthemost tion of DNA fragmentation was found more effective than the
widelyusedmethodstomapthesedifferencesisthechlortetracycline Annexin/PIinbullspermatozoa,althoughdetectionofnecroticsper-
assayusingfluorescencemicroscopy.Thisfluorescentantibioticwill matozoa cannot be performed by theAPO-BRDU kit. The TUNEL
detect enhanced fluorescence over the segments of the membrane assaymeasureschangesatthelaterstage,whileAnnexinV/PImeasure
where Ca21 accumulates, and depict different binding patterns on earlyspermapoptosis-likechanges.Poly(ADP-ribose)polymeraseisa
thespermhead,believedtoreflectdifferentstagesofthecapacitation nuclearproteinrecentlybeingusedasaneffectiveapoptosisdetector.
process.53 Though chlortetracycline is empirically accepted, it is During apoptosis, poly(ADP-ribose) polymerase cleavage occurs
laborioustouseanditsworkingmechanismremainsstillscientifically which is the classical characteristic of apoptosis,126 and it has been
unexplained. detected in human spermatozoa.127 The sequence of sperm death
Early stages of sperm capacitation can be measured by loading might be loss of mitochondrial membrane potential, membrane
spermatozoawiththelipiddyeMerocyanine540.112Thefluorescence change (YO-PRO-11 and PI2) and membrane damage (PI1).
observed by the FC is depending on the degree of lipid disorder.40 However,itwassuggestedthatapoptoticmarkerslikecaspaseactiva-
Merocyanine540isahydrophobicdye,whichcanmonitormembrane tionandYO-PRO-1stainingmighthappenonlyinaspecificsubpo-
integrity especially scrambling of phospholipids whencoupled with pulation of spermatozoa in red deer,128 and perhaps even reflect
YO-PRO-1 and Hoechst 33342 (Figure 850). Bicarbonate has the methodologicaldeviations.
potential to increase Merocyanine fluorescence rapidly, which ulti- Caspases, a specific group of cysteine proteases with strong pref-
matelyindicatesthatphospholipidpackingoftheplasmamembrane erenceforaspartylresidues,isconsideredtobeinvolvedinapoptotic
becomesdisorderedbythision.Likewise,calcium-dependentbinding degenerativeprocessesasexecutionagentscommontoalmostallcell
ofAnnexinVisabletodetectcapacitation-relatedchangesinmem- types investigated.129 Still the question is: does apoptosis occur in
branearchitecture,especiallythedislocationofPSinthelipidmem- spermatozoa or only in those species where remnants of cytoplasm
brane leaflet, thus indicating early subtle changes in membrane arelargelyavailable(usuallythecaseforhumanspermatozoaandfor
integrity during capacitation.113 One study, however, showed that stallionspermatozoaandsomewhatfordogspermatozoa,wherethe
both Merocyanine 540 and Annexin V indicate early plasma mem- amount of cytoplasmic droplets is significant. Nonetheless, Weil
branedegenerationandnotcapacitation—atleastinhumansperm.114 etal.130didnotfindanyevidencefortheinvolvementofcaspasesin
Calciuminfluxiscrucialfortheonsetofproteintyrosinephosphor- mousespermnormaldeath.Furthermore,itwasstatedthatthereisno
ylation. Displacement of calcium in the head plasmalemma occurs reason of occurring apoptosis in spermatozoa.122 However, it was
duringthelatterpartofcapacitation.13Indo-1acetoxymethylester115 evidentthatcaspaseswerepresentintherestrictedsiteforapoptosis
andFluo116arebeingusedforFCdeterminationofcalcium.Recently, inspermatozoa,whichresultedinpoorspermquality.131
aFCdeterminationofdynamicquantificationoftheproteintyrosine
phosphorylationinthespermmembraneproteinduringthecapacita- Spermheaddecondensationduringfertilization
tionwasascertained.116Theantiphosphotyrosineantibodycanquant- Duringmammalianspermiogenesis,thespermchromatinundergoes
ify tyrosine phosphorylation in boar spermatozoa using FC, thus astep-by-step condensation andpackagingmainlycharacterized by
waiving the tiresome traditional evaluations with western blotting. replacementofhistoneswithprotaminesandtheformationofS–Sand
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S–Zn–S bonds between cysteine residues,132 a process that is rein- abnormalspermatozoaandseminalleukocytesarethemainsources
forced during epididymal maturation. Once spermatozoa enter the ofROSinejaculates.144NumerousdirectandindirectmethodsofROS
oocyteatfertilization,itsspermheaddecondensestoformthemale determinationareavailable;however,directinvivodeterminationis
pronucleus,anessentialeventtowardstheformationofthezygote.133 difficultasreactiveoxidantsareveryunstableandgenerallyhavevery
Methodsforassessingdecondensationmustthereforebeconsidered short-lived intermediates. Determination of ROS directly in vivo
useful formalefertility. Several groups havemeasuredspermchro- might be done by the electron paramagnetic resonance spectro-
matindecondensationbyobservingchangesinsizeandshapeofthe scopy,145butduetothelimitationoftemperatureandtime,indirect
spermheadbymicroscopicexaminationswhenexposedtodetergents methodsarepreferred.Thechemiluminescenceassay146isoneofthe
(mostly sodium dodecylsulphate) and S–S bond reduction (using widespreadusedindirectmethodsofoxidativestressevaluation.And
dithiothreitol),134,135,136 methods that can predict fertility in vitro. inthissystem,thetwomaindyesluminolandlucigeninintwodif-
FC offers an excellent possibility to quantity events associated with ferentsignalingprocessesarebeingused.Luminolfollowsanintracel-
chromatindecondensation.Zuckeretal.137detectedheaddeconden- lulardeoxygenationreaction,whichismediatedbyaheterogeneous
sationofratandhamsterspermatozoapriortoassessmentbyincreases groupofspermperoxidases,whilelucigeninisoxidizedattheextra-
inlightscattering.Samocha-Boneetal.138evaluateddecondensation cellularlevelbythesuperoxideanion.147,148Continuousdiscoveryof
withthehelpofacridineorangedyeinhumanspermatozoa.Useofthe newfluorochromesmadeitpossibletoanalyzeoxidativestressbyFC.
dyePIwithethylenediaminetetraaceticacidandsodiumdodecylsul- The 29,79-dichlorodihydrofluorescein diacetate is a nonspecific dye
phate is able to determine decondensation in boar spermatozoa.139 which could assess intracellular ROS in dog spermatozoa.149 This
Thus,evaluationofdecondensationofspermheadsbyFCisanemer-
dye can be used together with viability dyes for removing the dead
ging but non-established technique for assessing male fertilizing spermatozoa.150Dihydroethidium(hydroethidine)maybeusedasa
potential.
specificprobetodetectsuperoxideanion(Figure9);itisfreelyper-
meableandoxidizedwhenboundtoDNA,thusbecomingvaluableto
Detectionofoxidativestressandlipidperoxidation detectsuperoxide anionsinspermatozoa;151however,otheroxygen
Oxidativestressmaybedefinedasanimbalancebetweenproduction speciesmightalsobedetected,dependingonthefiltersusedfordetec-
ofreactiveoxygenspecies(ROS)andthepotentialityofantioxidants tion.150TheMitoSOX(Invitrogen,Carlsbad,CA,USA)isanothernew
toscavengethese.AcertainlevelofROSisessentialforspermfunc-
probeabletodetectmitochondrialsuperoxideinthelive-cellpopu-
tions such as capacitation, acrosome reaction, sperm–oocyte fusion lation of human spermatozoa.152 The probe is readily oxidized by
and proteintyrosine phosphorylation.140However, it isamatter of
superoxide,whilepreventedbysuperoxidedismutase,thusenabling
concernthathighlevelsofROShaveadverseeffectonspermfunc-
highlyfluorescentoxidationproductstobedetectableuponbinding
tionalityleadingtohighrate(20%–40%)ofinfertility.141Fromstudies
to nucleic acid. However, most of these probes only measure
of different groups, it was found that ROS hamper the integrity of
the amount of lipid peroxidation but cannot locate where this
plasma membrane and nuclear DNA, leading to breakage of DNA
peroxidationoccurs.Morepreciseandspecificprobes(4,5-diamino-
andchromatincrosslinking.142Whenthelevelofthesebreaksishigh,
fluorescein diacetate, BODIPY and 5-iodoacetamidofluorescein;
it might lead to lowered fertility.143 Immature, morphologically
Figure8 Plasmamembraneasymmetryinabullspermsamplemeasuredby Figure9 ROSanalysisbyhydroethidineandHoechst33258.Cellsinthelower
Merocyanine540/YO-PRO-1labeling.Contourplotshowsviablecellswithstable leftquadrantaremembrane-intact,negativeforROSproduction.Cellsinthe
plasmamembrane(lowerleftquadrant),viablecellswithdestabilizedplasma lower-rightquadrantaremembrane-intact,positiveforROSproduction.Cells
membrane(lowerrightquadrant)anddeadspermatozoa(upperleftandright intheupper-rightquadranthaveadamagedmembraneandarepositivefor
quadrants). ROSproduction.ROS,reactiveoxygenspecies.
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Invitrogen) to detect oxidative stress in spermatozoa are emerging. spermcellislabeledwithaDNA-specificfluorochrome,whileother
Among these, BODIPY became popular in andrology.153–157 sperm-sizedparticles(likelipiddroplets)arenot.However,insome
BODIPY-basedprobeshavehigh-quantumyieldandhighsensitivity cases, certain sperm subpopulations remain unlabeled (like in sim-
offluorescence,andtheappearanceofBODIPY-labeledspermatozoa ultaneous viability and acrosome integrity studies with FITC-PNA
canberegardedasanindicationofphysiologicallyrelevantexposure andPI)andcannotbeseparatedfromthesignalsofnon-spermpart-
ofphospholipidstoROS.83However,itisalsoassumedthatFC-sperm iclesduetotheirsimilarscatterproperties,resultingintheoverestima-
sorting generates lipid pre-oxidation leading to oxidative stress, tion of the unlabeled sperm subpopulation and a consequent
althoughthereisnodirectevidenceonthisprediction.PriortoFC- underestimationoftheothers.Inothercases,theappliedfluorescent
sperm sorting, semen samples are highly extended beforehand probesmayhaveanonspecificbindingtothenon-spermparticles—
disturbingthenaturaldefenseagainstoxidation.158Levelsof8-hydro- resultinginfalse-positivelabeling.TheFCseemstobeoverestimated
deoxyguanosinehaveinhumanspermatozoabeenshowntobehighly byupto10%theproportionofviableunstainedspermatozoawhile
correlatedwithDNAdamage.159,160 underestimating the damaged subpopulation.22 This, together with
the variation that pipetting, might introduce when assessing sperm
APPLICATIONOFFCFORTHESORTINGOFSPERMATOZOABY suspensionswithlownumbersneedsfurtherstudies.
CHROMOSOMALSEX Oneapproachtosolvethis‘damageunderestimation’problemisto
Thepracticalapplicationofsexingspermatozoa,synergisticallywith usepositivelabelingoneveryspermcell.Thomasetal.,64forexample,
otherreproductivetechniques,couldimprovetheefficiencyofanimal added SYTO 17, a DNA-specific, membrane permeable dye to the
productionbothinbiologicalandeconomicterms.Selectionofsex FITC-PNA/PI combination, to add a positive fluorescent signal to
hasimportantimplicationsforpopulationsinwhichonesexhasmore the otherwise unlabeled sperm subpopulation with intact plasma
intrinsicvalue(e.g.,rareanimalgeneticresources),forinstance;stud membraneandacrosome.Nagyetal.39developedthisapproachfur-
operationsandfemaledairyreplacements,ortheavoidanceofmales thertouseasimilardyecombinationwhichcanbeappliedtoFCswith
forpigproduction,etc.SexpreselectionbasedonFCworksonsperm asingle,488-nmlaserline(theexcitationandemissionpeaksofSYTO
DNA content measurement to facilitate sorting of X chromosome 17are621and634nm,respectively;therefore,itisnotsuitablefor
fromtheYchromosome-bearingsperm.TheXchromosomecarries conventionalbench-topinstruments).Inourlaboratory,whereweuse
moreDNAthanthatoftheYchromosomeandtheprincipleofFCsex aBDLSRFCequippedwiththreelaserlines,weusuallyapplyaDNA-
sortingisbasedonthisdifference.InFC-basedsexsorting,spermato- specific,membranepermeabledyetolabeleveryspermcell(aswith
zoaarefirstlyloadedwithafluorochrome.Thenon-intercalatingvital the exception of pathological cases we do not expect to have other
fluorescent probe Hoechst 33342 is being used in FC-based sexing particleswithDNAinthespermsamples)andaddafluorescentgateto
where it penetrates the sperm membrane and binds to the DNA. the scatter gate to narrow our analysis to spermatozoa only, for
Dropletscontainingspermatozoaemittinganappropriatefluorescent example, Merocyanine 540/YO-PRO-1/Hoechst 3334250 where the
signalintheFCacquireelectricalchargeandaresortedintocollection Hoechst signal was used as fluorescent gate, or MitoTracker Deep
tubes by an electromagnetic field. The development of high-speed Red 633 in combination with SYBR14, where the later was used to
sortingisoneofthemajordevelopmentsofsortingtechnology,since labeleveryDNA-containingevent.78Obviously,thisapproachneedsa
the unique, asymmetric shape of mammalian spermatozoa makes larger,moreexpensiveinstrument,andcannotalwaysbefollowedin
conventionalflowsorterslesseffectiveinsexing(Figure10).Garner laboratoriesequippedwithsmaller,single-laserFCs.Asanalternative,
and Seidel161 have written a good historical overview. Offspring of theuseofpretreatmentsandamathematicalmodelofalienparticles
predetermined sex using FC have been successfully produced using can reduce particle misidentification,173 and can be performed in
fresh and frozen–thawed spermatozoa in several mammalian parallelwiththefunctionalassessmentinthesameFC.Anotherprob-
species: pigs,162 cattle,163 sheep164 bottlenose dolphins,165 goat166 lem that may arise is that dyes label non-sperm elements, leading
and humans.167 Further refinements to standard FC technology insteadtooverestimationofthepopulationofdeadcells.174
includedreplacementofthebeveledneedlebyanorientatingnozzle
withaceramictipgivingspermlesstimetoloseorientation,which FUTUREPERSPECTIVESFORFCINSPERMATOLOGY
improvedcorrectorientationofspermto70%.168Conversiontohigh- The main direction where the positive power of FC can be further
speedmodifiedFCoperatingunderincreasedpressurehadimproved expanded is the development of domain-specific multicolor assays
the accuracy and efficiency (20 000–25 000 events per second) of forbasicandappliedspermatology.FCofferstheopportunitytomea-
sperm sorting and purity of sorted population.169 Although it was sure several sperm attributes on every single spermatozoon and on
recently reported by some groups that stressful process of sorting thousandsofspermatozoaperspermsample;therefore,itcanimprove
mightweakenstatusoflivestockspermatozoa,strategiesforrestoring semenqualitycontrolextremely.However,itisimportanttoremem-
and strengthening their potentials were also discussed.170 Besides bertomeasureattributesthataredifferentandhavelowcorrelation
evaluating sex chromosome content of individual spermatozoa, FC withanother.Itisimportanttopointoutthatduetodifferencesin
offers a precise tool to do quick cytogenetic analyses on sperm: light sources, optical set-up and other adjustable parameters of the
aneuploidycanbedetectedbasedonDNAcontentofindividualsperm instruments,theanalysisresultsatcellularlevel(theplots)arenoteasy
cells.171,172 tocomparebetweenlaboratories.Thus,thereisaneedforvalidating
eachassayintheusersownlabwiththeuseofpositiveandnegative
THE‘DAMAGEUNDERESTIMATION’PROBLEM controls.MultiparameterspermstudiesbyFCarenotnew:Graham
Asitwasindicatedthroughoutthepresentreview,oneofthebiggest et al.175 measured viability, acrosome integrity and mitochondrial
challengesinapplyingFCinspermatologyisthecorrectrecognitionof membranepotentialsimultaneously.Newerfluorochromeswithnar-
spermatozoaandtheclearseparationfromsignalsofotherparticles roweremissionspectraandnovelFCswithseverallaserlineswidenthe
present in the actual samples. With assays like the SYBR14/PI dye possibilitiesfurther.Werecentlystartedtotestafluorescentprobeto
combinationforviabilitymeasurements,itisrelativelyeasyasevery evaluate plasma membrane integrity by FC. The probe (Live/Dead
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Figure10 SeparationofX-andY-bearingspermatozoawithaconventionalsorterflowcytometer,withoutorientinghead.Themajorityofspermatozoaarenotproperly
oriented,thereforeexcludedfromanalysis(spermeventsoutsideoftheyellowrectangularregionontheleftcontourplot).Spermatozoaintheyellowregionaregatedto
thehistogramontheright.
FixableRedDeadCellStainKit,InvitrogenL-23102)hasbeenapplied toFCforspermatologicalanalysis.Forinstance,FCdeterminationof
inmultiparameterfluorescentmicroscopy(Figure11176)successfully. cytosoliccalcium,proteintyrosinephosphorylationandcyclicaden-
IthasseveraladvantagesoverthecurrentlyusedFCviabilityassays:the osine monophosphate are being developed at our laboratory. To
probelabelsboththeviableanddeadcellswiththesamecolor,but evaluatecomplexcellularsystem,multicolorFCcanmeasureintra-
with different intensities (viable cells seem to be unlabeled when cellularfunctionalmarkersandcellsignaling.Multiplexedbeadassays
checked with fluorescent microscopy, but the dim fluorescence can which consists of a series of spectrally discrete particles, defined by
bedetectedwithFC).Therefore,onlyonedetectorisusedfordetecting eitherone-colorfluorescenceintensityorbyatwo-colorfluorescence
viable and dead spermatozoa. Since the probe labels intracellular address, which can be used for capturing and quantitating soluble
amides, it indicates the integrity of the plasma membrane over the analytes,aresuitableformeasuringmultipleDNAandRNAsequences
wholecellsurface,notjustovertheheaddomainasdoestheSYBR14/ ormultipleproteins.
PIcombination.Finally,itisafixablekit—fixingsamplesbeforeFC Anewapproachofstatisticalmodem-basedmultiparametricclus-
analyseswouldofferamoreflexibleworkscheduleintheflowlabor- teringtoidentifycellsubpopulationhasbeenproposed.179Thisrobust
atory.Ourfirstpilotexperimentsarepromising(Figure12). clusteringmethodisflexibleandgivesreproducibleresults.Thenovel
Spermfunctiontestingcanbeexpectedtoshedlightontheunder- ImageStreamtechnologyiscombiningFCwithcellimagingparallelto
lyingreasonsbehindmaleinfertility.177,178Itisexpectedthatwiththe quantification and visualization of spermatid-specific thioredoxin-
helpofnewprobesalargenumberofnewtechniqueswillbeadopted 3 protein in defective spermatozoa.180 Cytometry imaging with
Figure11 MergedfluorescentandDICphotosofbullspermatozoalabeledwith
Live/DeadFixableRedDeadCellStainKit(InvitrogenL-23102)andAlexaFluor
488PNA(L21409,green).Spermatozoawithintactplasmamembraneand
intactacrosomeshownofluorescence,cellswithdisruptedplasmamembrane Figure12 ContourplotshowsspermatozoalabeledwithLive/DeadFixableRed
showredtailand/orheadandrupturedacrosomesaregreen.PNA,peanut DeadCellStainKit(InvitrogenL-23102).Intactcellsareinlowerleftquadrantand
agglutinin. deadspermatozoaareinlowerrightquadrant.
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