Thiols of flagellar proteins are essential for progressive motility in human spermatozoa

12 Pages·2016·0.88 MB·English

CSIROPUBLISHING
Reproduction,FertilityandDevelopment
http://dx.doi.org/10.1071/RD16225
Thiols of flagellar proteins are essential for progressive
motility in human spermatozoa
Mar´ıaEugeniaCabrillanaA,B,Mar´ıadelosA´ngelesMonclusA,B,
TaniaEstefaniaSa´ezLancellottiA,B,PaolaVaninaBoarelliA,B,
A C
AmandaEdithVincenti ,MiguelMatiasForne´s ,
EduardoAlfredoSanabriaDandMiguelWalterForne´sA,B,E
AAndrologicResearchLaboratoryofMendoza(LIAM),HistologyandEmbryologyInstituteofMendoza
(IHEM),HistologyandEmbryologyArea,DepartmentofMorphologyandPhysiology,Schoolof
Medicine,NationalUniversityofCuyoandCCT-Mendoza,CONICET,Mendoza,Argentina.
BResearchInstitute,SchoolofMedicine,UniversityofAconcagua,Mendoza,Argentina.
CEngineeringSchool,UniversityofCuyo,Mendoza,Argentina.
DBasicScienceInstitute,FacultaddeCienciasExactas,F´ısicasyNaturales,Av.Ignaciodela
Roza590(O),ComplejoUniversitario‘‘IslasMalvinas’’,Rivadavia,NationalUniversity
ofSanJuanandCCT-CONICET,SanJuan,Argentina.
ECorrespondingauthor.Email:[email protected]
Abstract. Male infertility is a disorder of the reproductive system defined by the failure to achieve a clinical pregnancy
after 12 months or more of regular unprotected sexual intercourse. The presence of low-motile or immotile spermatozoa is
one of many causes of infertility; however, this observation provides little or no information regarding the pathogenesis of
the malfunction. Good sperm motility depends on correct assembly of the sperm tail in the testis and efficient maturation
during epididymal transit. Thiols of flagellar proteins, such as outer dense fibre protein 1 (ODF1), are oxidised to form
disulfides during epididymal transit and the spermatozoa become motile. This study was designed to determine how
oxidative changes in protein thiol status affect progressive motility in human spermatozoa. Monobromobimane (mBBr)
was used as a specific thiol marker and disruptor of sperm progressive motility. When mBBr was blocked by dithiothreitol
it did not promote motility changes. The analysis of mBBr-treated spermatozoa revealed a reduction of progressive
motility and an increased number of spermatozoa with non-progressive motility without affecting ATP production. Laser
confocal microscopy and western blot analysis showed that one of the mBBr-positive proteins reacted with an antibody to
ODF1. Monobromobimane fluorescence intensity of the sperm tail was lower in normozoospermic than asthenozoos-
permic men, suggesting that thiol oxidation in spermatozoa of asthenozoospermic men is incomplete. Our findings
indicate that mBBr affects the thiol status of ODF1 in human spermatozoa and interferes with progressive motility.
Additional keywords: cytoskeleton, epididymus, fertility.
Received 27 November 2015, accepted 4 June 2016, published online 1 July 2016
Introduction
themitochondrialsheath,outerdensefibres(ODFs)andfibrous
Goodspermmotilityisanessentialfactorinnormalmalefer- sheath.Thesestructuresareassembledduringspermatogenesis
tility.Menwithlow-motileorimmotilespermatozoaaretypi- throughaprecisearrangementofmicrotubules,mitochondrial
cally infertile or sterile. However, assigning the classification migration and Golgi transformation (Kierszenbaum and Tres
‘subfertile’or‘infertile’inaclinicalsettingonthebasisoflow 2004;XiaoandYang2007).
sperm motility provides little or no information regarding the Proteinsrichinthiolshavebeenfoundinthenucleus,ODFs,
pathogenesisoftheparticularcase(Turner2006). connectingpiece,fibroussheathandoutermitochondrialmem-
Goodmotilitydependsoncorrectassemblyofthespermtail brane(Veraetal.1984;Oko1988;Seligmanetal.1994).ODFs
in the testis in combination with efficient maturation during areacomponentoftheprominentcytoskeletonofthespermcell
epididymaltransit(Cooper2011).Thespermtailcontains the (OlsonandSammons1980;Oko1988).Previousstudieshave
axoneme,whichishighlyconservedinallciliatedandflagel- suggested that the function of ODFs is related to the bending
lated eukaryotic cells, and certain accessory structures found torque of the tail (Lindemann 1996) because of the elastic
onlyinmammalianspermflagella(Turner2006),inparticular structureofthespermtail(Baltzetal.1990).ODFsmayplay
Journalcompilation(cid:1) CSIRO2016 www.publish.csiro.au/journals/rfd
B Reproduction,FertilityandDevelopment M.E.Cabrillanaetal.
aprotectiveroleagainstshearforcesduringepididymaltrans- reagents (analytical grade) were from local companies as
portandejaculation(Baltzetal.1990).Theymayalsosupport LaboratorioBiofarmaS.A.indicated.Monobromobimanestock
axonemal beating through force transmission to the flagellar solution was prepared at 150mM in acetonitrile and stored in
base(Lindemann1996).ImpaireddevelopmentoftheODFshas aliquotsat(cid:1)188C.
beendescribedasamajorcauseoftailabnormalitiesininfertile
men(Haidletal.1991). Samplecollection
Some infertility cases with a high number of decapitated
Semensampleswerecollectedbetweenearly2011throughto
seminalspermatozoamayinvolvelossofODFfunction(Haidl
late2012attheInstituteofReproductiveMedicine(Mendoza,
et al. 1991). Sperm motility is reduced in heterozygous mice Argentina). The study protocol was approved by the Ethics
withimpairmentoftheODF1N-terminalfragment,suggestinga
CommitteeofthelocalSchoolofMedicine(FacultaddeCien-
similar cause for certain cases of reduced fertility in humans
ciasMe´dicas,UniversidadNacionaldeCuyo,resolution#7982/
(Yangetal.2012).Ontheotherhand,changesinrepeatC-X-P
2010,http://fcm.uncuyo.edu.ar/bioetica;verified8June2016).
frequenciesinODF1donotaffectfertilityinhumans(Hoffer- Malepatientswereinformedofthepurposeofthestudy.Vol-
bertetal.1993).
unteer subjects (n¼46; age range 27–38 years) were divided
During epididymal transit, sperm proteins undergo sulfhy-
into a control group (normozoospermic men, n¼31) and an
dryl oxidation and spermatozoa concurrently acquire progres- asthenozoospermic group (patients with idiopathic astheno-
sivemotility(Cornwalletal.1988).Changesofflagellarbeat
zoospermia, n¼15). Each subject provided a single semen
and motility pattern in spermatozoa (Bedford et al. 1973) are
sample.
associatedwithashiftfromflexibletomorerigidflagelladuring
epididymaltransit(CornwallandChang1990).
Semenanalysis
ODF1 (Cabrillana et al. 2011; Dias et al. 2014) and other
Semen samples were obtained by masturbation (following
ODFproteins(CornwallandChang1990)undergomaturational
$3dayswithoutejaculation),allowedtoliquefyfor30minand
changes (thiol oxidation) during epididymal transit in various
analysed promptly according to WHO criteria (World Health
mammalianspecies.Thesechangesinvolveareductioninthe
Organisation 2010). The results were expressed as median
availabilityofthiolgroupsandofsolubilityinsodiumdodecyl
(5th–95thpercentile).Thecontrolgrouppresentedthefollow-
sulfate(SDS).Wedemonstratedthatthethiol-reactivefluores-
ing characteristics: 70 (34–147.6)(cid:3)106 spermatozoa mL(cid:1)1,
centprobemonobromobimane(mBBr)reducesratspermmotil-
progressivemotility53(31–73.4)%,livecells83(67–95.6)%,
ity in a dose- and time-dependent manner (Cabrillana et al.
normalspermmorphology15(10–40.5)%androundcellplus
2011).
leukocytes ,1(cid:3)106 cells mL(cid:1)1. On the other hand, the
Fluorescent labelling with bimanes provides a convenient,
asthenozoospermic group had a sperm concentration of 31
sensitive method for assessing protein thiol status under
(2–110)(cid:3)106 spermatozoa mL(cid:1)1, progressive motility 28.5
physiological conditions. Because of its lipophilic character
(25–31)%,livecells75(65–92)%,normalspermmorphology
(Kosower et al. 1979; Kosower and Kosower 1987) mBBr
7(4–16)%(strictcriteria,Menkveldetal.1990)androundcell
readily penetrates intact living cells and selectively modifies
andleukocytes,1(cid:3)106cellsmL(cid:1)1.
free cysteine (Cys) residues in proteins under physiological
conditions.Thereactionofbimaneswithsulfhydrylgroupsof
Motilespermisolationtechnique
freeCysresultsinahighlyfluorescentderivative(Petrotchenko
et al. 2006). We demonstrated by matrix-assisted laser Motilespermatozoawereisolatedbythedirectswim-uptech-
niqueofMortimer(1994).Inbrief,1mLofliquefiedsemenwas
desorption/ionization (MALDI) mass spectrometer (MS) that
layeredunder1mLHam’sF-10withoutalbumin.Thetubewas
mBBrlabelsCysintheN-terminaldomainofODF1(Cabrillana
etal.2011). oriented at ,458 and kept in an incubator (Napco) under 5%
CO atmospherefor1hat378C.Thetubewasslowlyreturnedto
Inthepresentstudy,wetestedthehypothesisthatchangesin 2
anuprightposition,theupper500mLofmediumwasremoved
thiol status of flagellar proteins affect progressive motility in
humanspermatozoa,usingmBBrasaspecificthiolmarkerand and sperm concentration was adjusted to 1(cid:3)107 cells mL(cid:1)1
sperm motility disruptor without affecting sperm vitality and withfreshmedium.
ATP production.ODF1was characterised as a mBBr-positive
proteininhumanspermatozoa.Samplesofasthenozoospermic EffectsofmBBronspermmotility
menwereusedasacellularmodelthatlostspermprogressive We have previously demonstrated that mBBr inhibits sperm
motilitywithoutchemicaltreatment.Thethiolstatusofsperma- motilityinrats(Cabrillanaetal.2011).TheeffectofmBBron
tozoafromasthenozoospermicmenwascomparedwiththatof humanspermmotilityhasnotbeenstudiedpreviously.
normozoospermicmentoclarifytherelationshipbetweenthiol Three samples of normozoospermic men were combined
statusandprogressivemotility. with 0.3mM mBBr and sperm motility was assessed during a
60-minperiod.Threesamplesofasthenozoospermicmenwere
processed similarly, except that the assessment period was
Materialsandmethods
30minbecausespermmotilitydecreasedquickly.
Chemicals TodeterminetheminimalconcentrationofmBBrthataltered
Ham’s F-10 medium was from Laboratorio Microvet. Mono- spermmotility,aliquotsofspermsuspensionofnormozoosper-
bromobimane and DTT were from Sigma Aldrich. All other micmenwereincubatedfor60minat378Cunder5%CO with
2
Thiolstatus,ODF1andprogressivemotility Reproduction,FertilityandDevelopment C
Table1. EffectsofmBBronspermprogressivemotility
Thepercentageofprogressivespermmotilitywasmeasuredevery10minduring1hinsampleswithoutmBBr(Control)ortreatedwithvariousconcentrations
ofmBBrasshown.Meansfortheexperimentalconditionswerecomparedwithcontrolvalueattimezero(means(cid:4)s.e.m.);*P,0.01,***P,0.001
Time(min) Spermprogressivemotility(%)
Control mBBr(mM)
0.003 0.03 0.3 3
0 83.66(cid:4)9.81 70.33(cid:4)15.04 67(cid:4)12.76 64.43(cid:4)6.5 64.33(cid:4)13.5
10 78.33(cid:4)11.50 66(cid:4)16.97 69(cid:4)12.72 28(cid:4)2.64* 14.3(cid:4)4.04*
20 78(cid:4)12.52 66.5(cid:4)16.2 66(cid:4)16.97 25(cid:4)3.46* 4(cid:4)1*
30 78(cid:4)16.29 68.5(cid:4)12.02 61(cid:4)9.19 11.66(cid:4)2.08* 0(cid:4)0*
60 73(cid:4)22.5 29.5(cid:4)20.50 30(cid:4)28.28 0(cid:4)0*** 0(cid:4)0***
Ham’s F-10 alone (control) or Ham’s F-10 plus various con- manner(Cabrillanaetal.2011).Hereweexaminedthepossi-
centrationsofmBBr(0.003,0.03,0.3and3mM;Table1). bility that thiol groups are similarly involved in motility of
humanspermcells.
DTTblockstheeffectofmBBronsperm Sperm motility in the presence of mBBr was assessed in a
progressivemotility Makler counting chamber (Sefi-Medical Instruments) using
Possibleeffectsonspermmotilitybyreactivegroupspresenton threesamplesofnormozoospermicmen.Movementsofcontrol
mBBrwerealsoinvestigated.Monobromobimaneformsasta- and mBBr-treated cells were recorded with a Hamamatsu
bleandirreversiblethioetherlinkagewithfreethiolgroupsof C4742–95camera(HamamatsuPhotonics)connectedtoaPC
Cys residues (Kosower et al. 1979; Kosower and Kosower (Dell Argentina) with the MetaMorph software program
1987).Anothercontrolwasperformedbypre-blockingofmBBr (MolecularDevices),attachedtoaninvertedmicroscope(Nikon
reactive groups by DTT to prevent sulfhydryl (SH)-related Eclipse T2000; Nikon) in a constant-temperature chamber at
inhibition of sperm motility. Dithiothreitol (3mM) was co- ,378C.
incubated(1:1)with0.3mMmBBrfor1hatroomtemperature Motilitywasmonitoredfor1minat5-minintervalsduringa
(RT) in the dark to ensure reaction. Aliquots of spermatozoa total period of 30min. The images were compiled using the
from normozoospermic and asthenozoospermic men were MetaMorphprogramtoproduceavideo(1min;800frames)of
addedwithHam’sF-10plusblockedmBBr.TheeffectofDTT spermmotility.Spermtrackinginthevideowasanalysedusing
on motility was assessed using Ham’s F-10 plus 3mM DTT. Image Jsoftware (Version 1.46; NIH) withthe ParticleTrack-
Finallytheeffectofacetonitrileonspermmotilitywaschecked er3Dmodular_ java 210 plugins. This is a well-established
(5mLofacetonitrilewasaddedto250mLofspermsuspension, program that provides reliable data (Image J 1.46j; https://
withoutmBBr). imagej.nih.gov/ij/download.html;Meijeringetal.2012).
At10-minintervals,a20-mLsamplefromeachoftheabove Trajectoriesofindividualspermcellswereprojectedontox
conditionswasplacedonawarmedslide(378C),coveredwitha andyaxesbytheprogram,withx/yvaluesexpressedinpixels.
22-mm coverslip and examined under a microscope. Progres- DatawereconvertedtommusingaVisualBasicmacrothatwas
sivespermmotilitywasdeterminedbycountingatrandomthe designedbyMatiasForne´s.Theprogramusesamathematical
numberofprogressivelymotilespermatozoainapopulationof technique(followingsection)tocalculatevaluesofstraight-line
200 cells. For each condition, the percentage of viable cells displacement(generallycalledSL),curvilinearpath(CPortotal
(mean(cid:4)s.e.m.)wascalculated. trajectory) and curvilinear velocity (VCL) of a sperm popula-
tion.Foreachcondition,trajectoriesof300cellsweremeasured
Analysisofspermvitality in three separate experiments and results were expressed as
Spermvitalitywasdeterminedbythehiposmotic(HOS)test.In mean(cid:4)s.e.m. This approach provides a detailed analysis of
briefa5-mLspermsamplewasmixedwith45mLhypo-osmotic spermmovement.
medium(0.735gsodiumcitratedihydrateand1.351gD-fructose
in 100mL of purified water; 150 mOsm kg(cid:1)1 confirmed by a Mathematicaltechniqueforanalysisofsperm
Wescor 5500 cryoscopic vapour pressure osmometer) and progressivemotility
incubatedfor5minat378C.A20-mLaliquotofthemixturewas The distance between grid lines in the Makler chamber is
placedonawarmedslide,coverslippedandspermatozoa(.200) 100mm,sothereisaconversionfactorof0.7mmperpixelwhen
wereexaminedunderamicroscope.Thenumbersofspermato- using the 25(cid:3) microscope objective. SL, CP and VCL were
zoa with a curled tail (viable) and non-curled tail (non-viable; calculatedusingthefollowingequations:
Jeyendran et al. 1984) were recorded. For each condition, the Equation 1: straight-line displacement (SL) is the vector
percentageofviablecells(mean(cid:4)s.e.m.)wascalculated. distance from point A (start) to point B (end) described by a
spermatozoon.
Analysisofspermmovement Equation 2: curvilinear path or total trajectory (CP) is the
OurpreviousstudyshowedthatmBBrreactswithCysthiolsand totaldistancetravelledbyaspermatozoonalongacurvedpath
reduces rat sperm motility in a dose- and time-dependent frompointA(start)topointB(end).
D Reproduction,FertilityandDevelopment M.E.Cabrillanaetal.
Equation3:curvilinearvelocity(VCL)istherateofchange Kosower and Kosower 1987; Shalgi et al. 1989). For each
ofposition(curvilinearpath)during1min. experimental condition, fluorescence intensity of 200 sperm
cells was estimated using the Image J program (‘Analyze’
Dx¼ðx (cid:1)x Þ(cid:3)scale option, histogram function applied to a limited area (middle
end start
ð1Þ
and principal piece) of sperm tail). This measurement was
Dy¼ðy (cid:1)y Þ(cid:3)scale
end start
termedrelativefluorescenceintensity(RFI).RFIwascalculated
X for each condition in three separate experiments and mean,
X ¼ Dx (cid:3):::scale
i minimumandmaximumvalueswererecorded.
ð2Þ
X
Y ¼ Dy (cid:3):::scale
i
IdentificationofmBBr-labelledproteinsandwesternblot
pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
V ¼ Dx2i þDy2i ð3Þ analysisforODF1
i t
Two semen samples of normo- and asthenozoospermic men
were washed twice with 1(cid:3) PBS and incubated with 0.3mM
mBBr (final concentration) for 20min. After that the samples
ColocalisationofmBBrstainingwithODF1
werewashedandthepelletwassubjectedtoosmoticshockby
Aliquots (1mL) of semen from three normozoospermic men
5minincubationin0.5(cid:3)Ham’sF-10andthenincubated10min
were centrifuged at 500g for 15min at room temperature and
with 10mM ethylenediamine tetraacetic acid (EDTA) and
sperm cells were resuspended in culture medium at a final
10mM DTT. Each sample was sonicated at 10W in an ultra-
concentration1(cid:3)107cellsmL(cid:1)1.Twoaliquotswereobtained
sonicator(Kontes)forfivecyclesof30satRT.Osmolaritywas
and0.2mLmBBrstocksolutionwasaddedtooneofthem(final
adjustedbyadditionof10(cid:3)PBS,thensampleswerecombined
concentration0.3mM)andincubatedfor20min.Bothaliquots
with Laemmli sample buffer (0.0625M Trizma Tris-HCl, pH
werewashedtwicebycentrifugation,resuspendedinphosphate-
6.8, 2% (w/v) SDS, 10% (v/v) glycerol, 0.01% (w/v) bromo-
bufferedsaline(PBS;0.01Mphosphatebuffer,0.0027MKCl,
phenol blue) and DTT concentration was adjusted to 20mM
0.137MNaCl,pH7.4),fixedwith2%(w/v)paraformaldehyde
(Lishkoetal.2010).Samplesweresonicatedforanother5min
inPBSfor20min,washedtwicewithPBS,incubatedfor1hin
andboiledfor5min.Totalcelllysatesandthemolecular-weight
0.1%(v/v)TritonX-100and5%(w/v)bovineserumalbumin
standard(wide-range,dualcolour;Bio-Rad)wereloadedontoa
(BSA) in PBS, incubated with primary antibody anti-ODF1
12.5% (w/v) acrylamide gel with 4% (w/v) stacking gel
(sc-27907, 1:200; Santa Cruz Biotechnology) for 1h at RT,
(Laemmli 1970). Monobromobimane fluorescence intensity
washed extensively with PBS and secondary antibody added
observedintheacrylamidegelwasrecordedbyacamera(model
(B7014, 1:500; Sigma Aldrich) for 15min. Then cells were
LAS 4.000; Fujifilm). Proteins were transferred to a nitrocel-
incubated withfluorescein (FITC)–streptavidin (Vector Labo-
lulosemembrane(Towbinetal.1979).Forimmunoblotting,the
ratories Inc.) for 15min. Cells were mounted with anti-fade
membranewasincubatedovernightinblockingbuffer(3%(v/v)
Fluoroshield(SigmaAldrich).Picturesweretakenbyconfocal
TeleosteangelatinfishinTowbinbuffer(TBS)-T,1.92mMTris
microscopy(modelFV10-ASW;Olympus).Otheraliquotswith
base, 0.1% (v/v) Tween 20) at 48C, incubated 1h at RT with
mBBr were processed as above but primary antibody was
primary antibody anti-ODF1 (1:5000), washed twice with
omitted.
TBS-T and incubated with biotin-conjugated anti-goat IgG
antibody(B7014,1:3000;SigmaAldrich)for1hatRT.Bound
Monobromobimanefluorescenceintensityofspermtail
antibodywasvisualisedbyenhancedchemiluminescence(1M
fromnormozoospermicandasthenozoospermicmen
Tris base pH 8.5, 250mM luminol, 90mM cumaric acid, 3%
Differences in signal intensity generated by mBBr were only (v/v) H O ). The membrane was stained with Amido Black
2 2
observedintheheadofmatureversusimmatureratspermatozoa (Harper and Speicher 2001) and a chemiluminescence image
measuredbyflowcytometry(Shalgietal.1989).Wepreviously (ODF1 signal) and Amido Black colour image (membrane
reporteddifferencesinfluorescencesignalsinheadandtailof staining proteins) were captured by the Fujifilm camera as
mature and immature rat spermatozoa by measuring the fluo- above. Two western blots of anti-ODF1 and the fluorescent
rescencesignalbymicroscope(Cabrillanaetal.2011). signal of mBBr obtained from two acrylamide gels were nor-
Forthepresentexperiments,1mLsemenfromnormo-and malised by densitometry analysis using the Image J program
asthenozoospermicmenwascentrifugedal500gfor15minat (‘Analyze’option,gelfunctionapplied,areaundercurvemea-
room temperature. For each of the two types of samples, two sured;Zarellietal.2009).Thenormalisationwasexpressedas
aliquots were obtained and one was incubated with 30mM theratiobetweenthemBBrsignalversusAmidoBlackoptical
DTT (to reduce disulfide bonding to sulfhydryl groups; density and ODF1 blot versus Amido Black optical density.
Cleland1964)for20minat378C.Pelletsfromnormozoosper- Molecular weight was calculated by the method of Ferguson
mic,normozoospermicþDTT,asthenozoospermicandastheno- (1964).
zoospermicþDTT aliquots were washed twice with PBS and
resuspended in culture medium to a final concentration of
1(cid:3)107cellmL(cid:1)1.Monobromobimane(0.2mLstocksolution) EffectofmBBronATPproduction
was added to100mL of eachspermsuspension (finalconcen- ToassesstheeffectofmBBronATPlevelsmotilespermatozoa
tration0.3mM)andincubatedfor20min(Kosoweretal.1979; selectedbyswimupfromthreenormozoospermicsampleswere
Thiolstatus,ODF1andprogressivemotility Reproduction,FertilityandDevelopment E
used. The sperm suspension was separated into five aliquots. 0.3mMmBBr(two-wayANOVAresultsforcomparisonwith
Thesewerewashedtwiceandincubatedfor20minat378Cwith controlattimezero:t¼3.818;P,0.01andt¼5.25;P,0.001
either:(1)Ham’sF10,(2)Ham’sF10withoutglucosebutwith respectively; Table 1). For the 3mM mBBr group only, the
2.52mMof2-desoxi-D-glucose(DOG;glucosewasreplacedby percentageoflivespermatozoawasnotablyreducedat60min
an equal concentration of DOG, a competitive inhibitor of (HOS test, two-way ANOVA: t¼7187; P,0.001; Fig. 1b).
glucosetoblockglycolysis(Glyc);MukaiandOkuno2004)and ThesefindingsindicatethatmBBrprimarilyaffectsmotilitybut
40mM rotenone (this reagent was added to inhibit oxidative notspermvitality.
phosphorylation(OxPhos);Nascimentoetal.2008),(3)Ham’s
F10 plus 0.3mM mBBr co-incubated with 3mM DTT, (4)
DTTblockstheeffectofmBBr
Ham’s F10plus0.3mMmBBror (5)Ham’sF10plus30mM
DTT. Two aliquots from each of the five conditions detailed ThereactivegroupofmBBrwasblockedbyDTTtodetermine
above,with5(cid:3)106spermatozoamL(cid:1)1ofmedium,wereusedto whether this groupwas responsible forthe effect of mBBr on
determineATPlevel. sperm progressive motility. The reduction in progressive
ATP production was assessed using the ENLITEN ATP motility was abrogated or blunted when mBBr was incubated
assaykit(PromegaCorporation),aluminescencemethodwith withDTTbeforeaddingittospermatozoa.Whenspermatozoa
the enzyme luciferase, which catalyses the formation of light wereincubatedwithDTTonly(3mM)adecreaseinprogressive
from ATP,luciferinand oxygen.Thegeneratedluminescence motilitywasobserved,butthedifferencewasnotsignificantin
intensityisproportionaltotheamountofATPinthesample.For comparison with control (two-way ANOVA: t¼0.8737;
this, 50mL of sperm suspension at 5(cid:3)106 cells mL(cid:1)1 was P.0.05;Fig.1a).Thevehiclehadnosignificanteffectonthe
placed into a white-walled 96-well luminometer plate. Then, sperm motility during the time frame of the experiment. The
5mLof0.5%oftrichloroaceticacid(TCA,finalconcentration) numberofnon-viablespermatozoawasnotincreasedbyanyof
wasaddedasATP-extractingreagent.ImmediatelythepHofthe thesetreatments(Fig.1b).
suspension was neutralised with 15mL of Tris-acetate buffer
(1MTrisma,pHwasadjustedto7.7withglacialaceticacid).
EffectofmBBronsamplesofasthenozoospermicmen
Finally100mLofATPreagentwasaddedandincubatedfor60s
Whenspermatozoaofasthenozoospermicmenwereincubated
at 258C. The relative light units were measured with a lumin-
with 0.3mM mBBr, a reduction in the percentage of motile
ometer (Fluoroskan; Ascent FL Thermo Scientific) and then
spermatozoa was observed at 10min, but the difference in
convertedtoconcentrationvaluesusingacalibrationfactorof
comparisonwithcontrolwasnotsignificant(two-wayANOVA:
thestandardsolutionandexpressedasmM(mmolL(cid:1)1).
t¼0.8838;P.0.05;Fig.1c).
Treatments with vehicle,DTT or mBBrþDTT alsohad no
Statisticalanalysis significanteffectonspermmotilityorspermvitality(Fig.1d).
Data were analysed using the statistical packages GraphPad
Prism4software(http://www.graphpad.com/scientific-software/ EffectsofmBBronspermprogressivemotility:mBBr
prism; verified 8 June 2016). Unless noted otherwise, results affectsSLPandVCLbutnotCP
presented in the textand figures are mean(cid:4)s.e.m.from three
In the 0.3mM mBBr group, a reduction in progressive sperm
separate experiments performed in duplicate. Data for mBBr
motility and corresponding increase in non-progressive sper-
effectonspermmotilitywereanalysedbytwo-wayanalysisof
matozoawereobservedat10min.Videotrackingrevealedno
variance (ANOVA)for comparisonof meanswiththecontrol
differenceinthepercentageofimmotilespermatozoa(Fig.2a;
value. The Bonferroni post hoc test was used for all compar-
seeFigsS1andS2,availableasSupplementaryMaterialtothis
isons. Data were analysed by one-way ANOVA (Kruskal–
paper). During incubation with mBBr, some of the motile
Wallistest)andDunn’sposthoctestwasusedforallcompar-
spermatozoabeganmovinginthesameplace,buttherewasno
isons,tocomparemeansbetweeneachtimeandinitialtime.RFI
change in the number of hyperactivated spermatozoa. This
valuesandratiosofopticaldensitymeasurementswereanalysed
motility change may be related to the significant decreases
bytheKruskal–WallistestandDunn’sposthoctestwasusedfor
observed inVCL (Kruskal–Wallis:P,0.001;Fig.2b)andin
allcomparisons.Differencesbetweenmeanswereconsideredto
SL (Kruskal–Wallis: P,0.001; Fig. 2c). No change was
be significant when P,0.05. All the data were analysed by
observed in CP (Kruskal–Wallis: P¼0.6791; Fig. 2d). The
Kolmogorov–Smirnov normality test before application of
meanofeachmeasurementobtainedfromspermrecords(Fig.2)
ANOVA and data found to be non-normal were subjected to
wascomparedwithvalueattimezero.Amorphologyevaluation
non-parametrictest.
wasperformedafterrecordanalysis.
Results ColocalisationofmBBrstainingwithODF1
EffectsofmBBronspermmotilityandvitality Samples of normozoospermic men were subjected to mBBr
Asignificantdeclineinthepercentageofprogressivelymotile stainingandimmunodetectionofODF1.Spermnucleiandtails
spermatozoainsamplesofnormozoospermicmenbeganafter werelabelledbymBBr(Fig.3b1,c1).Thespermtail,neckand
10minofincubationandwascompletedby30minforthegroup perinuclearzoneshowedpositiveimmunoreactivitywithFITC-
treatedwith3mMmBBrand60minforthegrouptreatedwith labelledanti-ODF1(Fig.3a2,c2).ColocalisationofmBBrwith
F Reproduction,FertilityandDevelopment M.E.Cabrillanaetal.
(a) (b) 100 ∗
%) 100
y ( 80
Normozoospermic m progressive motilit 86420000 ∗ ∗ ∗∗∗ ∗∗∗ Live sperm (%) 642000
er
Sp 0 0
0 10 20 30 60 0 10 20 30 60
Time (min)
Veh Control DTT (3 mM) Control mBBr (0.3 mM) mBBr (3 mM)
mBBr (0.3 mM) mBBr (0.3 mM) (cid:2) DTT (3 mM) DTT (3 mM) mBBr (0.3 mM) (cid:2) DTT (3 mM)
(c) (d)100
100
%)
y ( 80
oospermic sive motilit 8600 perm (%) 60
Asthenoz m progres 4200 Live s 4200
er
p
S 0 0
0 10 20 30 0 10 20 30
Time (min)
Veh Control DTT (3 mM) Control mBBr (0.3 mM)
mBBr (0.3 mM) mBBr (0.3 mM) (cid:2) DTT (3 mM) DTT (3 mM) mBBr (0.3 mM) (cid:2) DTT (3 mM)
Fig.1. EffectsofmBBronspermmotilityandvitality.(a,c)Progressivespermmotilitywasexpressedasapercentageof200spermatozoa
counted.Spermsuspensionswereplacedinfourtubescontainingeitheracetonitrile(Veh),Ham’sF-10(Control)orvariouscombinationsof
mBBrandDTTasindicatedinthefigureandexaminedat0,10,20,30and60min.Eachpointrepresentsthepercentage(mean(cid:4)s.e.m.)of
threeseparateexperimentsforsamplesfrom(a)normozoospermicand(c)asthenozoospermicmen.(b,d)Spermvitalitywasmeasuredunder
thesameexperimentalconditionsdescribedabove.Barsrepresentthepercentage(mean(cid:4)s.e.m.)oflivespermatozoaforvarioustreatments
insamplesfrom(b)normozoospermicand(d)asthenozoospermicmen.Two-wayANOVAwasperformedtocomparedatapointswith
controlvalueattimezero.*P,0.01,***P,0.001.
ODF1wasobservedinthespermtail(Fig.3c4,whitearrow), normozoospermic and asthenozoospermic men (Kruskal–
neckandperinuclearzone(Fig.3c4,whitearrowhead). Wallis:P,0.001;Fig.4i).
Monobromobimanefluorescenceintensityofspermtail IdentificationofmBBr-labelledproteinsandwesternblot
fromnormozoospermicandasthenozoospermicmen analysisforODF1
Monobromobimane-stained sperm tails from samples of nor- Western blotting with anti-ODF1 antibody revealed a
mozoospermic men (Fig. 4a) showed lower fluorescence 14.8-kDaproteinbandcoincidingwiththefluorescentmBBr
intensity than those from samples of asthenozoospermic men band in SDS-polyacrylamide gel electrophoresis (PAGE).
(Fig.4b).Thehighestfluorescenceintensitywasobservedfor Monobromobimane fluorescence intensity for this ODF1-
spermatozoa of normozoospermic men treated with 30mM positivebandwaslowerinsamplesofnormozoospermicmen
DTT and then stained with mBBr (Fig. 4e). For spermatozoa thaninsamplesofasthenozoospermicmen(Fig.5a,number4),
from asthenozoospermic men, DTT treatment had no effect but no difference was seen comparing ratios of gel fluores-
on fluorescence intensity (Fig. 4g). Similar results were cenceandODF1blotdensitometry(Kruskal–Wallis:P.0.05;
obtained for comparisons of RFI values of spermatozoa from Fig.5b).
Thiolstatus,ODF1andprogressivemotility Reproduction,FertilityandDevelopment G
(a) (b)
20
80 ∗∗∗ ∗∗∗ ∗∗∗
70 ∗
15
%) 60 ∗∗∗
y ( 50 m/s
otilit 40 ∗ μL 10
m C
m V
30
er
p 5
S 20
10
0 0
0 10 20 30 0 10 15 20 30
(c) (d) 140
10
ment ∗∗∗ ∗ CP) 130
ace 8 ath/m 120
e displm SL) ∗∗∗ near pμctory ( 110
Straight linμ( 64 CurviliTotal traje 10900
80
0 10 15 20 30 0 10 15 20 30
Time (min)
Fig.2. Monobromobimaneeffectsonspermprogressivemotility;mBBraffectsSLandVCL,butnotCP.Effectsof0.3mMmBBronspermmotilityof
normozoospermicmenwereevaluatedbycalculating(a)percentagesofprogressive((cid:5)),non-progressive(’)andimmotile(¢)spermatozoaandthe
parameters(b)curvilinearvelocity(VCL),(c)straight-linedisplacement(SL)and(d)curvilinearpathortotaltrajectory(CP).Spermtrackingdatawere
analysedandexpressedasmean(cid:4)s.e.m.Meansofmeasurementsobtainedfromspermrecordswerecomparedbetweenvarioustimepointsandvalueattime
zero.*P,0.01,***P,0.001.
EffectofmBBronATPproduction blotting analysis and confocal laser microscopy have shown
When spermatozoa were incubated with DOG and rotenone thatoneofthemBBr-fluorescentproteinsisODF1.
((cid:1)Glyc (cid:1)OxPhos CONTROL), a reduction in ATP level was TheabovefindingsindicatethatproteinslabelledbymBBr,
observedasexpected(one-wayANOVA:t¼7.319,P,0.001; including ODF1,are involvedin regulationof spermprogres-
Fig.6).Incontrast,noreductioninATPproductionwasnoted sivemotility.
when the spermatozoa were incubated with mBBr, mBBr Monobromobimane was used both as a chemical agent to
blockedwithDTTorDTTaloneincomparisonwiththecontrol interfere with sperm motility and as a marker of sperm thiol
(one-way ANOVA: t¼1.39, t¼2.259, t¼6.651 respectively, status. It exerts a direct, specific effect on thiol groups and
P.0.05;Fig.6). exhibitsafastreaction(seconds)withsmallthiolsandaslower
reaction with protein thiols (minutes to hours, Kosower and
Kosower 1987). In rat spermatozoa, mBBr reduces sperm
Discussion
motility after 10min of incubation and binds the N-terminal
Thisstudywasdesignedtoevaluatethecontributionofflagellar domain of ODF1 (Cabrillana et al. 2011). These findings
proteinsrichinthiolstospermmotility.WhenCysresidueswere suggest that protein thiols and ODF1 are involved in sperm
blocked by mBBr, progressive motility was reduced without motility.However,toanalysechangesinprogressivemotility,
affecting ATP production or sperm vitality. When mBBr was humanspermatozoaaremoreappropriatethanratspermatozoa.
blockedbyDTT,theeffectonspermmotilitywasavoided. BindingofDTTthiolstothemBBrreactivesiteblocksthe
The mBBr fluorescence signal in sperm tail was lower for effect of mBBr on sperm motility. This finding suggests that
normozoospermic than for asthenozoospermic men. This dif- mBBr binds to protein thiols that are necessary to sperm
ference could indicate that an adequate amount of thiols in motility. As we reported previously, the effect of mBBr on
flagellar proteins is necessary for good motility. Western spermmotilitywasabolishedbyapreviousincubationofmBBr
H Reproduction,FertilityandDevelopment M.E.Cabrillanaetal.
mBBr ODF1-FITC PhC Overlay
A1 A2 A3 A4
(cid:3) mBBr/
(cid:2) ODF1
B1 B2 B3 B4
(cid:2) mBBr/
(cid:3) ODF1
C1 C2 C3 C4
(cid:2) mBBr/
(cid:2) ODF1
Fig.3. ColocalisationofmBBrstainingwithODF1.Confocalfluorescencemicrographsofspermatozoafromnormozoospermic
men.(a1,b1,c1)Bluefluorescence:locationofmBBraffinityproteins.(a2,b2,c2)Greenfluorescence:FITC-labelledanti-ODF1.
(a3,b3,c3)Phasecontrast(PhC)images.(a4,b4,c4)Overlayofblueandgreenfluorescencesignals.(c4)Arrowandarrowhead:
colocalisationofmBBrandFITCsignals.Imagesshownarerepresentativeofthreeseparateexperiments.
Normozoospermic Normozoospermic(cid:2)DTT Sperm tail RFI
(a) (c) (e) (f)
(i)
sity 600 ∗∗∗ ∗∗∗
n
e
nt
e i ∗∗∗
c 400
1.1 μ m en
c
s
e
or 200
Asthenozoospermic Asthenozoospermic(cid:2)DTT e flu
(b) (d) (g) (h) ativ
el 0
R N A TT TT
(cid:2) D (cid:2) D
N A
DTT mBBr
Normozoospermic (cid:3) (cid:2)
Asthenozoospermic (cid:3) (cid:2)
Normozoospermic (cid:2) (cid:2)
Asthenozoospermic (cid:2) (cid:2)
mBBr PhC mBBr PhC
Fig.4. Monobromobimanefluorescenceintensityofspermtailfromnormozoospermicandasthenozoospermicmen.(a–h)Fluorescencemicroscopyof
mBBr-labelledspermatozoafrom(a,c)normozoospermic,(b,d)asthenozoospermic,(e,f)normozoospermicþDTTand(g,h)asthenozoospermicþDTT
men;magnification630(cid:3)(scalebar¼1.1mm).(i)RFIofmBBrfromspermtailplottedasbox(mean)anderrorbars(minimumtomaximum)forthefour
experimentalconditions.RFIvaluesfortheotherthreeconditionswerecomparedwithnormozoospermicRFI.***P,0.001.
Thiolstatus,ODF1andprogressivemotility Reproduction,FertilityandDevelopment I
(a) Anti ODF1 mBBr Anti ODF1 mBBr MW
1
2
75 kDa
(b)
1.5
o)
ati
y (r 1.0
3 25 kDa sit
n
e
d 0.5
c
pli
4 O
0
Western blot mBBr
Normozoospermic Asthenozoospermic
Normozoospermic Asthenozoospermic
Fig.5. IdentificationofmBBr-labelledproteinsandwesternblotanalysisforODF1.(a)SDS–PAGEofspermproteinslabelledwithmBBr
observedunderUVlightandafterblottingwithanti-ODF1antibody,ofnormozoospermicandasthenozoospermicmen.MW,molecularweight.
Referenceweightmarkers:1,81.28kDa;2,69.18kDa;3,22.9kDa;4,14.79kDa.(b)Ratiosofopticaldensityofanti-ODF1westernblotsignal
andmBBrfluorescence,relativetoopticaldensityofAmidoBlack-stainedmembrane.
60
mBBr.GiventhatmBBrreactswithproteinthiols,ourattention
wasfocussedondeterminingwhichproteinscouldbeinvolved
inthisprocess.
Changes in sperm tail angulation have also been observed
40
M) (datanotshown),suggestingthatproteinthiolsareinvolvedin
forwardmovementofspermatozoa.Changesintailshapehave
μP ( been correlated with thiol stabilisation and sperm maturation
T
A (Cornwall et al. 1988; Yeung et al. 1999) and the protein
20
∗∗∗
adenylatecyclase10(ADCY10)hasbeenshowntobeinvolved
inflagellarspermangulationsinmice(Ijirietal.2014).
Several groups have reported relationships between ODF1
0 and sperm motility. Yang et al. (2012) observed that sperm
(cid:2)Glyc (cid:3)Glyc DTT/ mBBr DTT motilitywasreducedinheterozygousmicewithadefectinthe
(cid:2)OxPhos (cid:3)OxPhos mBBr
N-terminalfragmentofODF1.However,themechanismwhere-
Control Control
byODF1affectsspermmotilityisunknown.
Fig.6. EffectofmBBronATPproduction.TheconcentrationofATPwas In spermatozoa stained with mBBr, both the head and tail
determinedinhumanspermatozoaincubatedwith:(1)Ham’sF10alonewith becomefluorescent.Rat,humanandotherspecieshavedemon-
activeglycolysis(Glyc)andoxidativephosphorylation(OxPhos)pathways strated involvement of protamines in nuclear mBBr fluores-
(þGlycþOxPhosCONTROL),(2)Ham’sF10withoutglucosebutwith cence (Shalgi et al. 1989; Seligman et al. 1994; Aleem et al.
2.52mMDOGand40mMrotenonewithinactiveGlycandOxPhospath- 2008; Ramos et al. 2008; de Mateo et al. 2011). We showed
ways((cid:1)Glyc(cid:1)OxPhosCONTROL),(3)Ham’sF10plus0.3mMmBBrco- previouslythatODF1isrelatedtomBBrsignalsinratspermtail
incubatedwith3mMDTT,(4)Ham’sF10plus0.3mMmBBror(5)Ham’s
(Cabrillanaetal.2011).SeveralauthorshavedescribedODF1in
F10plus30mMDTT.ResultsareexpressedasconcentrationofATPinmM
thespermtail(Oko1988;OkoandClermont1989;Shaoandvan
(mean(cid:4)s.e.m.) of three separate experiments. One-way ANOVA was
derHoorn1996;Schallesetal.1998;Petersenetal.1999;Shao
performedtocomparedatapointswithcontrolvalue.***P,0.001.
et al. 2001) and Kiersenbaun (2002) detected ODF1 during
spermatid differentiation, specifically in the area where the
withDTT.Thismethodologicaltoolefficientlyblockstheeffect future head was forming. In the present study, FITC-labelled
ofmBBronspermmotility. anti-ODF1andmBBrstainingwerecolocalisedinhumansperm
In the present study, VCL and SL were decreased after neckandtailandintheperinuclearzone(Fig.3b3).
10min of incubation with mBBr, but CP was unaffected; this Shalgi et al. (1989) demonstrated differences in mBBr
outcomeindicatesthattheprogressivemotilitywasaffectedby fluorescence intensity between mature and immature rat
J Reproduction,FertilityandDevelopment M.E.Cabrillanaetal.
spermatozoa,bydirectfluorescenceobservationandflowcyto- labelledbymBBr,suchinteractiondoesnotaffecttheproduc-
metry. Our previous study revealed differences in both sperm tionofATP.Inthissense,Costanstinietal.(1995)reportedthat
whole-cellfluorescenceandtailsignals(Cabrillanaetal.2011). mBBr does not inhibit ATP production in liver cells. These
Inflowcytometrytheentirecellfluorescenceismeasured; outcomesreinforcetheimportanceofthethiolstatusofflagellar
however,thismethodwasappliedtoquantifythethiolstatusof proteinsinspermmotility.
onlythespermnucleibecausealowpercentageofthefluores- Takentogether,ourfindingsindicatethatanappropriatethiol
cencesignalcomesfromthespermtail(Seligmanetal.1991; status of flagellar proteins, such as ODF1, is necessary to
Ramos et al. 2008). Based on these previous findings we maintain progressive motility of human spermatozoa. Good
measuredRFIonlyinspermtailfromoursamples.Microscopic motilitydepends,amongothermechanisms,onspermmatura-
observationspermittedadetailedanalysisofmBBrfluorescence tionduringepididymaltransit.Thisprocessinvolvesanequili-
changesatthespermtail. brated thiol oxidation in sperm flagellar proteins and when
InanotherstudySeligmanetal.(1994)usedmBBrlabelling mBBr binds to the remnant thiols sperm progressive motility
toevaluatethiolstatusbyflowcytometricanalysistomeasure isinhibited.
SH levels and observed increased protamine oxidation in More studies are in progress to elucidate the mechanism
samplesofasthenozoospermicmen.However,flagellarfluores- underlying this effect, including the activity of GAPDH, the
cencewasnotanalysed. rolesofflagellarproteinsotherthanODFs,theoutermitochon-
In another flow cytometric study, de Mateo et al. (2011) drial membrane and fibrous sheath and how the epididymal
characterised mBBr-labelled spermatozoa from subfertile environmentpromotesanadequateproteinthiolstatusandgood
patients with oligoasthenoteratozoospermia and demonstrated spermmotility.
biphasicdistributionsreflectingbothoveroxidationandincom- Thedifferenceinthethiolstatusbetweennormozoospermic
pletethioloxidation.However,littleinformationisavailableon andasthenozoospermicmencouldallowustodevelopatestto
spermtailproteins. evaluatepossiblematurationdefectsinthesepatients.
Inthepresentstudy,thetailfluorescenceintensityofmBBr-
treatedspermatozoaofasthenozoospermicmenwashigherthan
Acknowledgements
thatofmBBr-treatednormozoospermicmen.Whenspermato-
The authors are grateful to Silvana Ortega, Biochemistry and Antonio
zoa of normozoospermic men were preincubated with DTT,
Martinez, M.D. Ph. D. (Institute of Reproductive Medicine, Mendoza
high mBBr incorporation in sperm tail proteins was observed
Argentina)forprovidingaccesstothesemensamplesandDrS.Andersonfor
underfluorescentmicroscopy.Thisfindingpresumablyreflects
Englisheditingofthemanuscript.ThisstudywassupportedbytheResearch
a high level of thiols in comparison with non-DTT-treated InstituteofAconcaguaUniversity(CIUDA-2013–2015),SECTyP-National
samples. Reduction of spermatozoa of asthenozoospermic UniversityofCuyo(2014–2015)andPIP-CONICET(2012–2015).
menwithDTTbeforemBBrincubationresultedinRFIvalues
similartothoseofnon-DTT-treatedspermatozoa.Theseresults
References
suggest that the spermatozoa from these asthenozoospermic
Aleem,M.,Padwal,V.,Choudhari,J.,Balasinor,N.,andGill-Sharma,M.K.
menhaveincompletethioloxidationduringepididymaltransit.
(2008).Spermprotaminelevelsasindicatorsoffertilisingpotentialin
ThemolecularweightofODF1hasbeenreportedas27kDa
sexuallymaturemalerats.Andrologia40,29–37.doi:10.1111/J.1439-
byseveralgroups(ShaoandvanderHoorn1996;Schallesetal.
0272.2008.00805.X
1998;Petersenetal.1999;Shaoetal.2001).Ontheotherhand, Baltz,J.M.,Williams,P.O.,andCone,R.A.(1990).Densefibersprotect
antibodies against the synthetic peptide DSVRRDIKKV- mammalian sperm against damage. Biol. Reprod. 43, 485–491.
DRELRQLRCI, derived from the N-terminal region of the doi:10.1095/BIOLREPROD43.3.485
putativeprotein(raisedinrats)boundtoa32-kDaproteinband Bedford,J.M.,Calvin,H.,andCooper,G.W.(1973).Thematurationof
(Gastmannetal.1993).Weusedagoatpolyclonalanti-ODF1 spermatozoa in the human epididymis. J. Reprod. Fertil. Suppl. 18,
antibody (G-17, sc-27907) raised against a peptide mapped 199–213.
Cabrillana,M.E.,Monclus,M.A.,SaezLanzelotti,T.E.,Boarelli,P.V.,
neartheN-terminusofhumanODF1.Thisantibodyrecognised
Clementi,M.A.,Vincenti,A.E.,Yunes,R.M.F.,andForne´s,M.W.
a14.8-kDaproteinbandthatwasmBBrpositive.Moreaggres-
(2011). Characterization of flagellar cysteine-rich sperm proteins,
sive treatment is required to obtain cytoskeletal proteins of
involved in motility, by the combination of cellular fractionation,
ejaculated spermatozoaforcomparisonwiththoseof epididy-
fluorescence detection and mass spectrometry analysis. Cytoskeleton
mal spermatozoa. This cleavage of proteins could explain the 68,491–500.doi:10.1002/CM.20525
differencesinobservedrunningprofiles. Cleland, W. W. (1964). Dithiothreitol, a new protective reagent for SH
A characterisation of mBBr-labelled proteins by MALDI groups.Biochemistry3,480–482.doi:10.1021/BI00892A002
TOF/TOFhasshownthatproteinssmallerthan33kDaundergo Cooper, T. G. (2011). The epididymis, cytoplasmic droplets and male
thiol oxidation during the sperm maturation process in the fertility.AsianJ.Androl.13,130–138.doi:10.1038/AJA.2010.97
epididymisandtheseproteinsareassociatedwiththecytoskel- Cornwall,G.A.,andChang,T.S.K.(1990).Characterizationofsulphydryl
proteinsinvolvedinthemaintenanceofflagellarstraightnessinhamster
eton,energeticmetabolismandantioxidantdefence(Diasetal.
spermatozoa. J. Androl. 11, 168–181. doi:10.1002/J.1939-4640.1990.
2014).ThisauthoralsosuggestedthatthestabilisationofODF
TB01597.X
and fibrous sheath (FS) is related to the organisation of the
Cornwall,G.A.,Vindivich,D.,Tillman,S.,andChang,T.S.(1988).The
energyproviderapparatus.
effectofsulfhydryloxidationonthemorphologyofimmaturehamster
EventhoughproteinsinvolvedinthegenerationofATP,like epididymal spermatozoa induced to acquire motility in vitro. Biol.
glyceraldehyde-3-phosphate dehydrogenase (GAPDH), are Reprod.39,141–155.doi:10.1095/BIOLREPROD39.1.141