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LocalizationofAKAP4andtubulinproteinsinspermwith
reducedmotility
ElenaMoretti,GiacomoScapigliati,NicolaAntonioPascarelli,BaccioBaccetti,GiuliaCollodel
DepartmentofGeneralSurgery,BiologySection,UniversityofSiena,RegionalReferralCenterforMaleInfertility,
Siena53100,Italy
Abstract
Aim:Toperformscreening,relatedtoA-kinaseanchoringproteins4(AKAP4)andtubulinproteins,inspermatozoa
withabsentorseverelyreducedmotilityinordertodetectthestatusofthefibroussheathandtheaxonemalstructure.
Methods:Animmunocytochemicalstudyoftubulin,usedasapositivecontrol,andAKAP4wascarriedouttodetect
thepresenceandthedistributionoftheseproteinsindifferentspermsamples.Themorphologicalcharacteristicsof
spermwerestudiedbytransmissionelectronmicroscope(TEM)andtheresultswereelaboratedusingaformula
reportedinpreviousstudies.PCRwascarriedoutonDNAextractedfromperipheralbloodlymphocytestoanalyse
partialsequencesoftheAkap4andAkap3genes.Results:ImmunolabellingoftubulinandAKAP4showeddifferent
patterns,whichledustodividethepatientsintogroups.IngroupI,theabsenceofAKAP4andtubulinwasrevealed,
althoughthesepatientsdidnotshowalterationsintheAkap4/Akap3bindingsite.TEMevaluationhighlightedthata
highpresenceofnecrosiswasassociatedwithtotalspermimmotility.IngroupII,aregularAKAP4andtubulinsignal
waspresent,althoughmotilitywasreducedandTEManalysisrevealedthepresenceofimmaturity.IngroupIII,in
whichaweakAKAP4labelassociatedwithnormaltubulinstainingandreducedmotilitywasobserved,asevere
disorganizationofthefibroussheathwashighlightedbyTEM.Conclusion:WhiletheroleofAKAP4insperm
motilityisunclear,absentorweakAKAP4-labellingseemstobeassociatedwithabsentorweakspermmotility.
(AsianJAndrol2007Sep;9:641–649)
Keywords:AKAP4;immunocytochemistry;motility,sperm;transmissionelectronmicroscope
.
OriginalArticle
.
DOI:10.1111/j.1745-7262.2007.00267.x
www.asiaandro.com
?2007,AsianJournalofAndrology,ShanghaiInstituteofMateriaMedica,ChineseAcademyofSciences.Allrightsreserved.
Correspondenceto:DrGiuliaCollodel,DepartmentofGeneral
Surgery,BiologySection,UniversityofSiena,PoliclinicoS.Maria
alleScotte,Siena53100,Italy.
Tel:+39-0577-2335-39Fax:+39-0577-2335-27
E-mail:collodel@unisi.it
Received2006-06-07Accepted2007-01-22
1Introduction
Maleinfertilityisasignificantprobleminhumans
andmaybecausedbydifferentpathologies,suchasana-
tomicalproblems,infections,hormonalimbalances,chro-
mosomalalterationsandgeneanomalies.However,30%
ofinfertilemenareaffectedbyidiopathicoligoastheno-
teratozoospermiaandthecauseofinfertilityisstillun-
knownasreportedbyCavallini[1].Theanalysisofsperm
motilityplaysacentralroleintheevaluationofmalefer-
tilitybecauseitisknownthatahighpercentageofpoorly
motileorimmotilespermwillnotbeabletofertilize.The
clinicalrelevanceofspermmotilityisevident,butthe
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molecularmechanismsinvolvedinthisprocesshavenot
beenfullyunderstoodyet.
Flagellarspermstructurehasbeenextensivelyde-
scribedinthepublishedliterature,andcorrectorganiza-
tionoftheaxonemalpatternandofperiaxonemalstruc-
turesispivotalforensuringnormalmotility.Duringthe
pastfewyears,attentionhasbeenpaidonthefibrous
sheath,acytoskeletalstructuresurroundingtheaxoneme
andouterdensefibersthatdefinestheextentofthere-
gionoftheprincipalpieceofspermflagellum.Itcon-
sistsoftwolongitudinalcolumnsconnectedbyclosely
arrayedsemicircularribsthatassemblefromthedistalto
theproximalpartofthetailthroughoutthespermiogenetic
process.
Itisgenerallyacceptedthatthefibroussheathplays
aroleasamechanicalsupportofspermflagellum.Fi-
broussheathisabletomodulateflagellarbendingandto
definetheshapeofflagellarbeatsasdescribedbyFawcett
[2].
Recently,extensivestudieshavebeencarriedoutto
identifyproteinsthatconstitutethefibroussheathand
thatareinsomewayactivelyinvolvedinspermmotility.
Eddyetal.[3]foundthatinthehumanfibrous
sheath,A-kinaseanchoringproteins3and4(AKAP3,
AKAP4)arethemostabundantstructuralproteins,an-
choringcyclic-AMP(cAMP)-dependentproteinkinase
Atothefibroussheaththroughtheregulatorysubunitof
kinase.cAMP-dependentphosphorylationofflagellar
proteinsisinvolvedinthebeginningandmaintenanceof
spermmotility.ThesecondmessengercAMPmediates
itsintracellulareffectsinspermatozoathroughcAMP-
dependentkinase(PKA).Theintracellularorganization
ofPKAiscontrolledbyitsassociationwithAKAPs.
Inparticular,AKAP3issynthesizedinround
spermatids,incorporatedintothefibroussheathconcur-
rentlywiththeformationofribprecursorsandisre-
portedtobeinvolvedinorganizingthebasicstructureof
thefibroussheath[4].AKAP4issynthesizedandincor-
poratedintoanascentfibroussheathlateinspermatid
developmentanditplaysamajorroleincompletingfi-
broussheathassemblyasreportedbyBrownetal.[4].
AtargeteddisruptionoftheAkap4genecausingdefects
inmicespermflagellumandmotilityhasalsobeende-
monstratedbyMikietal.[5].Recently,Baccettietal.
[6]usedtransmissionelectronmicroscope(TEM)tode-
terminethegeneticdefect“dysplasiaofthefibrous
sheath”(DFS)inspermfromagroupofinfertilemen.
Inthesecases,immunolabellingoftubulinconfirmedthe
presenceoftypicalshortandthicktailswhereasAKAP4
proteinstainingshowedaweaksignal,revealingadisor-
ganizedandincompletelyassembledfibroussheath.
Moreover,polymerasechainreaction(PCR)fordetect-
ingthepresenceofapartialsequenceofAkap4/Akap3
bindingregionsproducedpositiveresultsaccordingto
Turneretal.[7].
Basedonourdatashowingalterationofthefibrous
sheathinimmotilespermcontaininggeneticdefects,the
presentstudywasundertakentodetermineifsimilaral-
terationswerepresentinspermwithreducedmotility
butwithoutgeneticdefects.
Aspreviouslydescribed,AKAP4labelingwasper-
formedtodetectthetypicalspatialorganizationoffi-
broussheathcomponentsandtubulinstainingwascar-
riedoutinordertocheckaxomeneassembly.Morpho-
logicalcharacteristicswerestudiedbyTEM,avaluable
toolforamoredetailedevaluationofspermultrastructure,
andtheresultswereelaboratedusingtheformulaby
Baccettietal.[8].TEManalysiswasperformedtotry
toexplainthestructuralcausesofabsentorreducedmo-
tilityinthisgroupofpatients.PCRwascarriedouton
DNAextractedfromperipheralbloodlymphocytesto
analysepartialsequencesoftheAkap4andAkap3genes.
2Materialsandmethods
2.1Patients
Semensampleswereobtainedfrom16patients(aged
24to33yearsold)withidiopathicinfertilityreferredto
theRegionalReferralCenterforMaleInfertilityforse-
menanalysisafter3yearsofsexualintercoursewithout
conception.Thelymphocytekaryotypeswerenormal
inallcases.Writtenconsentwasobtainedfromallpa-
tientsdonatingsamples,bothinfertilemenandcontrols.
2.2Semenanalysis
2.2.1Lightandelectronmicroscopy
Semensampleswerecollectedbymasturbationaf-
ter3–4daysofsexualabstinenceandexaminedafter
liquefactionfor30minat37oC.Anydelaybetweenejacu-
lationandsampleprocessingwasrecorded.Volume,
pH,concentrationandmotilitywereevaluatedaccording
toWHOguidelines[9].Supravitaleosinstainingwas
usedforevaluatingspermviability.
ForTEM,semenwasfixedincoldKarnovskyfixa-
tiveandmaintainedat4oCfor2h.Fixedsemenwas
washedin0.1mol/Lcacodylatebuffer(pH7.2)for12h,
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postfixedin1%bufferedosmiumtetroxidefor1hat
4oCanddehydratedandembeddedinEponAraldite.Ul-
tra-thinsectionswerecutwithaSupernovaultramicro-
tome(ReickertJung,Vienna,Austria),mountedoncop-
pergrids,stainedwithuranylacetateandleadcitrateand
thenobservedandphotographedwithaPhilipsCM10
transmissionelectronmicroscope(TEM;Philips
Scientifics,Eindhoven,TheNetherlands).Threehun-
dredultra-thinspermsections(approximately50%heads
and50%tails)wereanalyzedforeachpatient.Major
submicroscopiccharacteristicswererecorded,applying
thesameevaluationcriteria,byhighlytrainedexaminers
whowereblindtotheexperiment.TEMdatawereelabo-
ratedusingthemathematicalformulabyBaccettietal.
[8],basedonBayesan’stechnique.Thisformulacon-
siders16selectedsubmicroscopiccharacteristicsof
spermorganellestodefinespermfunctionandcalcu-
latesthenumberofspermatozoafreeofstructuralde-
fects(“healthy”)andthepercentagesofthreemainphe-
notypicspermpathologies:immaturity,necrosisand
apoptosisasdemonstratedbyBaccettietal.[10].More-
overBaccettietal.[8]observedthatthelowestnumber
ofspermatozoafreeofdefectsandassuringnormalfer-
tilitywasslightlyovertwomillion.
Thecontrolswerefivemenwithnormalkaryotype
whohadfatheredachildduringtheprevious1to2years.
2.2.2Immunofluorescence
Semensampleswerewashedtwiceinphosphate
bufferedsaline(PBS),smearedonglassslides,airdried,
rinsedinPBSandfixedfor15mininmethanolat–20oC.
Slideswerethentreatedwithblockingsolution(PBS,
1%bovineserumalbumin,5%normalgoatserum)for
20minatroomtemperature(RT)andincubatedover-
nightat4oCwithmousemonoclonalanti-tubulin(Sigma
Chemical,St.Louis,MO,USA)andmousemonoclonal
anti-AKAP82(BDBiosciences,Erembodegem,Belgium)
specificforhumanAKAP4protein,dilutedat1:100and
1:50respectivelyinPBS,0.1%BSA,1%NGS.After
threewashesinPBS,thesamplesweretreatedwithgoat
anti-mouseIgG-TexasRedconjugatedantibody(Southern
Biotechnology,Birmingham,AL,USA).Finally,the
sampleswerewashedthreetimesinPBSandmounted
withVectashield(VectorLabs,Burlingame,CA,USA).
Incubationinprimaryantibodieswasomittedincontrol
samples.Observationsandphotographsweremadewith
aLeitzAristoplanlightmicroscope(Leica,Wetzlar,
Germany)equippedwithafluorescenceapparatus.A
totalof200spermatozoafromeachsamplewerecounted
andscoredaseitherlabelledornotlabelledwiththere-
spectiveantibody.FortheAKAP4experiment,onlythose
spermatozoastainedthroughoutthelengthoftheprinci-
palpiecewerecounted.Thesamefivesamplesfrom
healthymenofprovenfertilitywereexaminedandused
ascontrols.
2.3PCRanalysis
DNAwasextractedfromperipheralbloodlympho-
cytesusingtheQIAampDNABloodKit(QIAGEN,
Valencia,CA,USA).
PCRproductscorrespondingtoaregionofhAkap4
involvedinbindingtoAkap3(site1)andtoaregionof
Akap3involvedinbindingtohAkap4(site2)weream-
plifiedaccordingtoTurneretal.[7].Oligonucleotide
primersflankingtherespectivebindingsiteswereused:
site1:senseprimer5''-TCAGTGCCCTTATAGG-
TGAG-3'',antisenseprimer5''-GCAGAGCTTCATCAC-
AGATTC-3'';
site2:senseprimer5''-TTGAGGAATCTCCACA-
GCG-3'';antisenseprimer5''-CCAACGGTCTTTCACA-
CAACTTC-3'').
ControlDNAwasextractedfromthebloodoffive
fertilemen.
3Results
Sixteensemensamplesfrommenwithidiopathicin-
fertilitywereexaminedbylightandelectronmicroscopy
todeterminespermconcentration,motilityandmorpho-
logy.Inallsamples,immunocytochemistryforthelo-
calizationofAKAP4andtubulinwasperformed.
Amongthegroupofinfertilepatients,spermcon-
centrationwasnormalineightoutof16patientsaccord-
ingtoWHOguidelines[9].Rapid(a)andslow(b)pro-
gressivemotilitywasabsentorseverelyreducedinall
analysedsamples.Theseparametersarereportedin
Tables1,2and3.
ImmunolabelingofAKAP4proteinandtubulin,al-
lowedustoseparatethepatientsintothreegroups(Tables
1–3):
GroupI:patients1–5inwhomthelabelwasnega-
tiveforbothantibodiesandmotilitywastotallyabsent
(Table1);
GroupII:patients6–10inwhomtheAKAP4signal
(Figure1A,1B)andthetubulinlabel(Figure1C,1D)
were100%,exceptinpatient9inwhomAKAP4label-
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Table1.Spermparameters,immunocytochemicalscreeningforA-kinaseanchoringproteins4(AKAP4)andtubulinproteinsandtransmis-
sionelectronmicroscope(TEM)analysisofspermatozoafromfiveinfertilepatients.Thesepatientsshowedabsenceofmotilityand
negativeimmunolabellingforbothproteins.Rapid(a)andslow(b)progressivemotility.
Numberof
Motility(%)
AKAP4Tubulin
Numberof
Casessperm/mLlabellinglabelling
healthysperm
ApoptosisNecrosisImmaturity
(×10
6
)
(a+b)
(%)(%)
(%)(%)(%)
18.0000015.56099.79056.780
257.80005711.88091.97058.440
315.0000322116.38272.04958.359
469.0000015.77996.18657.297
530.800000.69285.22825.575
Mean36.1000655.612.05989.04551.290
SD26.50001434.36.59610.93614.392
Table2.Spermparameters,immunocytochemicalscreeningforA-kinaseanchoringproteins4(AKAP4)andtubulinproteinsandtransmis-
sionelectronmicroscope(TEM)analysisofspermatozoafromfiveinfertilepatients.Thesepatientsshowedreducedmotilityandpositive
immunolabellingforbothproteins.Rapid(a)andslow(b)progressivemotility.
Numberof
Motility(%)
AKAP4Tubulin
Numberof
Casessperm/mLlabellinglabelling
healthysperm
ApoptosisNecrosisImmaturity
(×10
6
)
(a+b)
(%)(%)
(%)(%)(%)
612.52+15(17)100100149810.86025.03078.410
74.51+7(8)1001002810.16330.55381.141
8123.87+9(16)1001007707935.05226.11071.130
941.06+10(16)9510010787094.62031.55078.170
1010.03+8(11)10010010599.15030.09093.320
Mean38.413.699.0100370417.47.96928.66780.434
SD49.83.912.20517613.12.9272.9018.098
Table3.Spermparameters,immunocytochemicalscreeningforA-kinaseanchoringproteins4(AKAP4)andtubulinproteinsandtransmis-
sionelectronmicroscope(TEM)analysisofspermatozoafromsixinfertilepatients.Thesepatientsshowedstronglyreducedmotilityand
positiveimmunolabellingfortubulin,buttheywerenegativeforAKAP4.Rapid(a)andslow(b)progressivemotility.
Numberof
Motility(%)
AKAP4Tubulin
Numberof
Casessperm/mLlabellinglabelling
healthysperm
ApoptosisNecrosisImmaturity
(×10
6
)
(a+b)
(%)(%)
(%)(%)(%)
1110.31+2(3)109006.82032.07474.498
126.01+2(3)2080110.86234.59671.343
134.93+5(8)101001934.19725.31786.584
14102.54+6(10)20100949384.50324.71363.722
1540.53+5(8)201003459875.78628.12357.988
16120.00+4(4)20100150690.74625.87084.210
Mean47.4616.795.876031.35.48628.44973.058
SD51.535.28.0137304.63.3424.03611.194
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Table4.Spermparameters,immunocytochemicalscreeningforA-kinaseanchoringproteins4(AKAP4)andtubulinproteinsandtransmis-
sionelectronmicroscope(TEM)analysisofspermatozoafromfivemenofprovenfertility.Rapid(a)andslow(b)progressivemotility.
Numberof
Motility(%)
AKAP4Tubulin
Numberof
Casessperm/mLlabellinglabelling
healthysperm
ApoptosisNecrosisImmaturity
(×10
6
)
(a+b)
(%)(%)
(%)(%)(%)
A204.7469595706959660.85420.13444.110
B85.0499510030381284.34021.58047.290
C93.050100100537101414.12518.82739.876
D183.77010010064254042.98519.76540.007
E89.076100100458910750.93319.97537.777
Mean131.158.298.099.0359521432.64720.05641.812
SD58.213.82.72.2299016000.9910.9913.826
lingwas95%.Spermmotilitywasreducedinallpa-
tients(Table2);
GroupIII:patients11–16inwhomtheAKAP4signal
(Figure1E,1F)wasweaker(atleast20%),thetubulin
labelrangedfrom80%to100%(Figure1H)andmotility
wasstronglyreduced.
Thefivepatientswithprovenfertility,whowereused
ascontrols(Table4,A–E),showednormalimmunofluo-
rescentstainingfortubulin(Figure1H)andAKAP4
(Figure1G)in95%–100%oftails(Table4).
Allspermsamplesfrominfertileandfertilemenwere
analysedbyTEMandthedataobtainedwereprocessed
usingthemathematicalformulabyBaccettietal.[8].
MathematicallyelaboratedTEManalysis(Tables1–
4)confirmedthat16examinedpatientswereinfertile
(Tables1–3),showinganumberofhealthyspermof
<2000000.Necrosis(Figure2A)wasextremelyhigh
(Table1)ingroupI(89.045±10.936)versustheother
groups(Tables2and3)andcontrols(Table4).This
spermpathologyischaracterisedbyabsentorreacted
acrosomes,misshapennucleiwithdisruptedchromatin,
swollenanddisassembledmitochondriaandalteredax-
onemalandperiaxonemalstructures.Inthesecases,eosin
Ystainingconfirmedthepresenceofdeadspermat>
80%.
GroupIalsoshowedahighpercentageofsperm
withapoptoticcharacteristics(12.059±6.596)suchas
misshapenacrosomes,nucleiwithmarginatedchroma-
tinandswollenmitochondriairregularlyorganizedinto
largecytoplasmicresidueswithtranslucentvacuoles
(Figure2B).
GroupIIshowedameanofpercentageofsperm
necrosis(Table2)verysimilartothatofcontrols(Table
4),whichwasconsiderednormal.However,averyhigh
presenceofapoptosisandimmaturity(Figure2C)com-
paredtocontrols(Table4)wasobserved.Immature
spermatozoagenerallyshowedalteredacrosomesand
roundorellipticalnucleiwithuncondensedchromatin.
Inparticular,ahighpercentageofspermatozoawithlarge
cytoplasmicresidues,oftenembeddingcoiledaxonemes
(Figure2C,2D)washighlightedalthoughtheaxonemal
andperiaxonemalstructures,includingthefibroussheath,
weregenerallynormal(Figure2D),asalsorevealedby
immunocytochemicalanalyses.
GroupIIIshowedameanpercentageofspermpa-
thologies(Table3)similartothepreviousgroup(Table
2).Moreover,despitereducedmotility,TEManalysis
showed“9+2”patternaxonemeswithstructuraldefects,
particularlyinvolvingthefibroussheath,thatappeared
poorandbadlyassembled(Figure2E).
PCRproductscorrespondingtoaregionofhAkap4
involvedinbindingtoAkap3(site1)andtoaregionof
Akap3involvedinbindingtohAkap4(site2)werepresent
inallexaminedpatientsandcontrols.
4Discussion
Althoughspermmotilityisoneofthemostimpor-
tantpredictors
offertilizingability,themechanismsun-
derlyingmotilityabnormalities
arestillpoorlyunderstood.
RecentresearchperformedbyLietal.[11]hasfur-
nishednewcluesregardingthekeymolecularmediators
of
spermmotility.However,itiswellknownthatsperm
motilityisregulatedbythecAMP-dependentproteinki-
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Figure1.Ultravioletandlightmicrographsofejaculatedspermatozoa.(A),(B):StainingwithmonoclonalantiA-kinaseanchoringproteins
4(AKAP4)antibodyhighlightedthepresenceofcoiledtailsinspermfrompatients6–10,alsoconfirmedbytheanti-tubulinlabel(C,D).
(E,F):Immunocytochemicallabellingofspermfrompatients11–16performedwithAKAP4monoclonalantibodyhighlightedaweak
signal.NormalstainingofAKAP-4(G)andtubulin(H)wasalsoshown.(A),(B):scalebar=22μm;(C),(D):scalebar=21μm;(E),(F):
scalebar=34μm;(G):scalebar=30μm;(H):scalebar=41μm.
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Figure2.Transmissionelectronmicroscope(TEM)micrographsoflongitudinalandcrosssectionsofsperm.(A):Anecroticspermatozoon
characterizedbyacrosomewithsparsecontent(sA)andamisshapennucleuswithdisruptedchromatin(dCh).Theaxonemeisrolledupand
altered(aAX)andmitochondria(M)areswollenanddispersed.Theplasmamembraneisbroken(arrow).Scalebar=1μm.(B):An
apoptoticspermcharacterizedbymisshapennucleuswithmarginated(mCh)chromatin.Acytoplasmicresidueembeddingswollen
mitochondria(M)andacoiledaxonemeispresent.Scalebar=0.74μm.(C):Animmaturespermcharacterizedbyanirregularnucleuswith
uncondensedchromatin(uCh).Thealteredacrosome(aA)appearsfarawayfromthenucleus.Cytoplasmicresidue(CR)embedsswollen
mitochondria(M)andcoiledaxoneme(aAX),showingaregular“9+2”patternandanalmostassembledfibroussheath(FS).Scalebar=
0.7μm.(D):Arolledupaxonemewithanormalpattern.Scalebar=2.5μm.(E):Acrosssectionoftheprincipalpiecewithnormalpattern
andpoorfibroussheath(pFS).Scalebar=0.17μm.
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nase(proteinkinaseA)-mediatedphosphorylationof
groupsofflagellarproteins.Inmouse,humanandbull
spermatozoa,twomajorfibroussheathproteins,AKAP4
(alsocalledAKAP82orfibroussheathcomponent1)and
itsprecursorproAKAP4,havebeenidentifiedasmem-
bersoftheA-kinaseanchorprotein(AKAP)byCarrera
etal.[12,13].Thehypothesisisthat,byanchoringthe
activityofPKAinthefibroussheath,AKAPsplaycentral
rolesintheregulationofnormalspermmotility.Turner
etal.[14]didnotfindevidenceofanassociationbe-
tweenthedegreeofprocessingofpro-hAKAP4andin-
creasesordecreasesinmotilityinspermatozoafrom
normalmen.
Recently,Brownetal.[4]reportedthatAKAP4an-
chorsAKAP3andtwonovelspermatogeneticcellspe-
cificproteins,Fibroussheathinteractingproteins1and
2(FSIP1;FSIP2).
Mikietal.[5]demonstratedthattargeteddisruption
oftheAkap4genecausestheabsenceofspermmotility
togetherwithatotallackoffibroussheathontheprinci-
palpieceofmaturemicesperm.
Baccettietal.[15]describedararespermtaildefect
characterizedbyabsenceofthefibroussheathinhumans.
AKAP4labellingwaspresentatthetesticularlevelin
cytoplasmicresiduesandresidualbodies,yetitwasto-
tallyabsentinejaculatespermatozoa.Moreover,ina
caseofdisorganizedandincompletelyassembledfibrous
sheath,suchasfibroussheathsperm,Baccettietal.[6]
foundmoderateanddiffusedimmunofluorescentstain-
ingofAKAP4.
Theaimofthisstudywastoassessthestatusofthe
fibroussheathandtheaxonemalstructurebyperform-
ingscreening,relatedtoAKAP4andtubulinproteins,in
spermatozoawithabsentorseverelyreducedmotility.
ImmunolabellingoftubulinandAKAP4inspermfla-
gellashoweddifferentpatterns,leadingustodividethe
patientsintogroups.IngroupI,inwhichspermmotility
was0%,noAKAP4ortubulinlabellingwasdetected.
Whenspermmotilitywasgreaterthan0%,avariable
patternofAKAP4andtubulinstainingwasobserved
(groupsIIandIII).
IngroupI,TEMevaluationhighlightedthatahigh
presenceofnecrosiswasassociatedwithcasesoftotal
immotilityandtheabsenceofAKAP4andtubulin,indi-
catingalossofantigenicitycausedbypost-necroticpro-
teindegradation.Inordertoexcludeageneticoriginof
theabsenceofAKAP4,PCRanalysiswasperformedto
detectthepresenceofapartialsequenceofAkap4/Akap3
bindingregionsanditproducednormalresults.
IngroupII,despitereducedmotility,regularAKAP4
andtubulinsignalswereobserved.Thisapparentincon-
sistencywasjustifiedbyTEManalysisthatrevealedthe
presenceofspermimmaturity.Weobservednumerous
cytoplasmicresidues,typicalmarkersofthispathology
andresponsibleforthedecrementofmotility.Theax-
onemalandperiaxonemalstructuresembeddedinthese
cytoplasmicresidues,includingthefibroussheath,were
generallynormalasalsorevealedbyimmunocytochemi-
calanalyses.
IngroupIIIinwhichaweakAKAP4labelwas
observed,associatedwithgoodtubulinstaining,TEM
analysisshowedaseveredisorganizationofthefibrous
sheathandanormal“9+2”axonemalpattern.This
patternwasquitesimilartothatobservedincasesof
dysplasiaofthefibroussheath(DFS)asalreadydescribed
byBaccettietal.[6].However,noneofthesepatients
wereaffectedbythisgeneticspermdefect,characterized
byatypicalultrastructuralfeatureashighlightedbyChemes
etal.[16].
Inconclusion,whiletheroleofAKAP4insperm
motilityisunclear,absentorweakAKAP4labelingseems
tobeassociatedwithabsentorweakspermmotility.
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EditedbyDrAleksanderGiwercman
AsianJournalofAndrology
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