Beruflich Dokumente
Kultur Dokumente
spermatozoaisolatedfromStaphylococcusaureuswhencharacterizedusing
LCMS (Liquidchromatographymassspectrometry) showedthatthis 20
kDa protein had peptide sequence similarity with hsp70 protein. It was
foundtocompletely(100%)inhibitMg++ATPaseactivityofspermatozoa
at concentration of 100gmL1. Sperm samples treated with SIF also
showed reduction in calcium ionophoreinduced acrosome reaction as
compared to control samples (treated with calcium ionophore alone).
BindingstudiesofFITClabelledSIFwithspermatozoausingfluorescent
microscopyshowedbindingofSIFtothesurfaceofspermatozoaindicating
thepresenceofSIFbindingreceptor.Thereceptorwasextractedby3M
NaClandpurifiedbygelpermeationchromatography.Characterizationof
thereceptorbyMALDITOF(Matrixassistedlaserdesorptionionization
timeofflight)indicatedthatthereceptorsharedsequencesimilaritywith
MHCclassIIantigen.Acalorimetricstudyshowedthatthereceptormoiety
on spermatozoa was specific for the purified ligand as binding of the
receptor to ligand was enthalpically (11.9kJmole1) as well as
entropically (21.53Jmole1K1) favored resulting in the Gibb's free
energyof18.57kJmole1.
1.Introduction
Staphylococcusaureusisamongstthemostversatileandsuccessfulofthe
human pathogens. It has the ability to cause a variety of infections in
numerousecologicalnicheswithinthehost.Itcolonizesthenares,axillae,
vagina, pharynx, or damaged skin surfaces and causes a variety of
suppurative(pusforming)infectionsandtoxinosesinhumans.Besidesthis,
S. aureus is arguably the dominant organism implicated in primary
infertility,amongmalesandfemalesalike[1].S.aureushasbeenobserved
ascausativeorganismaccountingfor68.2%ofseminalfluidinfections[2].
ThisisconsistentwiththatreportedbyOkonetal.,whereS.aureuswas
isolated from 62.5% of the seminal fluids [3]. S. aureus has also been
reportedtobecommonlyisolatedmicroorganismfromcervicalsamples[4].
Huweetal.studiedtheinfluenceofdifferenturopathogenicmicroorganisms
onhumanspermmotilityparametersbymeansofCASAandreportedthat
S.aureusretardsthespermmotility[5].SimilarstudiesweredonebyLiuet
al.ontheeffectofcertainuropathogenicmicroorganismsonhumansperm
motilityparametersandfoundsignificantdecreaseinspermmotilitywhen
spermatozoa were coincubated with S. aureus [6]. Some authors have
suggested that direct interaction between bacteria and spermatozoa
facilitatesimmobilizationofspermatozoa[79],whileothershavereported
evidenceforsolublespermicidalfactorproducedandsecretedbybacteriain
theextracellularmedium[10].
Inanearlierworkdoneinourlaboratory,wehavealsobeenabletoisolatea
strain of S. aureus from cervix of an infertile woman, causing 100%
immobilizationofspermatozoawithoutagglutination.Thefactorresponsible
forspermimmobilizationwasisolatedandpurified[11].Further,itseffect
onvariousspermparameterswasstudied.Motilityofspermatozoarequires
energyintheformofATP[12]thatisprovidedbyspermmitochondriato
powertheflagellarmotionthatpropelsthespermtothesiteoffertilization
[13]. It thus seems possible that the inhibition of sperm motility after
treatment with SIF may be due to decrease in the ATPase activity or
imbalanceinthemoleculesofbiologicalsignificance.Further,theanalysis
of acrosome reaction in human sperm is recommended to estimate the
fertilizationabilityofhumanspermintheclinicalfield[14].Hence,the
effect of SIF on Mg++ ATPase activity and acrosome reaction was
evaluated.Thenanattemptwasmadetoisolatethecorrespondingreceptor
forSIF[15].FurthercharacterizationofbothSIFandSIFbindingreceptor
willhelpinbetterunderstandingofSIFandreceptorinteractionswhichcan
beaimedasapotentialcandidatefortreatmentofunexplainedinfertility.
2.MaterialsandMethods
2.1.SemenSamples
Semen samples were obtained from males attending infertility clinic at
Government Multispeciality Hospital, Sector 16, Chandigarh, by
masturbation into sterile wide mouth container. Only ejaculates showing
normal semen parameters according to WHO criteria [16] were used.
Samples underwent liquefaction at room temperature for 30mins.
Experimentswereperformedwithin1hofobtainingsamples.Apreparation
of washed sperm samples was done, in which the sperm cell pellet was
retained after centrifugation at 500rpm for 10min and thereafter it was
washedtwicewithsterilephosphatebuffersaline(PBS)(50mM,pH7.2).
2.2.Microorganism
Cervical swabs were taken from 16 women below the age of 35 years,
sufferingfromunexplainedinfertilityattendingtheDepartmentofObstetrics
and Gynaecology at Government Multispeciality Hospital, Sector 16,
Chandigarh.Sampleswerestreakedonbloodagarplatesandtheplateswere
incubatedaerobicallyat37Cfor2448h.Isolateswereidentifiedaccording
to the Bergeys Manual of Determinative Bacteriology. Microorganisms
wereidentifieduptogenuslevelonly.Intotal,27isolateswereobtained.
Amongst the various isolates, Staphylococcus (51.85%) was the
predominant organism present. When the effect of 24 and 48h old cell
culture and cell free supernatant of cervical isolates on human sperm
motilitywasstudiedinvitro,theresultsshowedthat17outof27isolates
(63%)significantlydecreasedspermmotility[17].Oneoftheisolatecausing
100%immobilizationofspermatozoawasselectedforfurtherstudies.Only
thisstrainwasfurtheridentifiedbybiochemicaltestsandwasfoundtobe
coagulasepositiveStaphylococcusaureus.Thisstrainhasbeendesignatedas
S.aureusVP2.
2.3.IsolationandPurificationofSIFfromBacteria
SIFwasextractedandpurifiedfrom72holdcellcultureofS.aureusbythe
method previously standardized in the laboratory [11]. Briefly, the cell
cultureofS.aureusgrowninbrainheartinfusion(BHI)broth,undershake
conditions(220rpm)at37Cfor72h,wascentrifugedat5,000rpmfor15
minat4C.SIFwaspurifiedfromthesupernatantbyammoniumsulphate
precipitation, gel permeation chromatography, and ion exchange
chromatography. To check the purification status, polyacrylamide gel
electrophoresis(PAGE)wascarriedout.
2.4.LiquidChromatographyMassSpectrometry(LCMS)ofPurifiedSIF
2.4.1.ProcessingofBandsandTrypticDigestion
FollowingPAGE,proteinbandwasexcisedandgelpiecewaswashedonce
with100LHPLCgradewaterfor10minandthendestainedin100Lof
destaining solution 1 (50% 100mM ammonium bicarbonate in 50%
acetonitrile) by incubating at 37C for 30min. Destaining solution was
discardedandgelpiecewasdehydratedin100Lofacetonitrile(ACN)at
37Cfor5minandthenairdriedfor5minat37C.Thegelpiecewas
reducedin150Lof10mMofDithiothreitol(DTT)/100mMofammonium
bicarbonatebyincubatingat56Cfor30min.Itwasthenalkylatedin100
Lof50mMiodoacetamide/100mMammoniumbicarbonateindarkfor30
minandalkylationbufferwasdiscarded.Gelpiecewaswashedwith100L
destainingsolution,removed,andagaindehydratedwith100LACNfor5
minandthenairdriedat37Cfor5min.Gelpiecewasrehydratedin~5L
trypsinsolution(20ngL1)for10minat37C.Gelpiecewasincubatedat
37Cfor16h.
2.4.2.PeptideExtraction
To extract the peptides after the overnight incubation, ~20L peptide
extractionsolution(1%Trifluoroaceticacid,TFA)wasaddedandsonicated
inawaterbathsonicatorfor5min.15Loftheextractedpeptideswere
usedforsubsequentMSanalysis.
2.4.3.LCMS
A6LsolutionwasinjectedforanalysiswithLCMS/MS(Agilent,Palo
Alto, CA, USA). Data was analyzed using Agilent Ion Trap Analysis
softwareversion5.2andproteinswereidentifiedbydatabasesearchagainst
theMASCOTdatabase.
2.5.InVitroEffectofSIFonSpermMg++ATPaseActivity
Mg++ATPaseactivityofspermatozoawasstudiedaccordingtoKielley[18]
andChappel[19]withslightmodifications.TrisHCl(0.2M,pH7.6)washed
spermatozoa(1108/mL)weresonicatedat50Hzfor10min(10cyclesof
30swith1mininterval)at4C.ThereactionmixtureforATPaseconsisted
of0.2mLTrisHClbuffer(0.2M,pH7.6),0.2mLofMgCl2(5mM),0.2mL
ofATP(6mgmL1),and0.2mLofsonicatedspermsuspension.Different
concentrationsofSIF(12.5g,25g,50g,and100g)wereaddedtothe
reaction mixture. The mixture was incubated at 37C for 1h. After this
periodofincubation,thereactionwasstoppedbyadding1mLofcold10%
Trichloroaceticacid(TCA)andthenincubatedat4Covernightforprotein
precipitation.Thecontroltubescontainedallthecomponentsofthereaction
mixturebutTCAwasaddedinthebeginningtostoptheATPaseactivity.
Inorganicphosphorus(Pi)releasedwasdeterminedaccordingtothemethod
ofBoyceetal.[20].OneunitofATPaseisexpressedasgofthePireleased
after1hofincubation.Theexperimentwasrepeatedthrice.
2.6.InVitroEffectofSIFonAcrosomeReactionofSpermatozoa
Thesemen sample after liquefaction was washed twice(500rpm, 5min)
with human tubal fluid medium (HTFM) containing 1% human serum
albumin(HSA)[21].Spermconcentrationwasadjustedto40106mL1
andspermsuspensionwasdividedintotwoaliquots(testandcontrol).The
test sample was incubated for 3h at 37C with equal volumes of SIF,
whereasthecontrolwaspreparedbyaddingequalvolumeofPBS(50mM,
pH7.2)undersameconditions.After3h,spermatozoafrombothtestand
control were treated with either 0.1% DMSO (spontaneous acrosome
reaction)or10McalciumionophoreA23187(inducedacrosomereaction)
for1hat37C.Subsequently,spermatozoawerewashedinHTFMwithout
HSA(500rpm,10min).Thepelletwasresuspendedin3%glutaraldehyde
and incubated for 20min at 37C. After further washing the pellet was
resuspendedin1050LHTFMandsmearedontoaslide.After drying,
spermatozoawerestainedinBismarkbrown(0.8%indeionizedwater,pH
1.8)for5min,washedthreetimeswithwater,andstainedwithRoseBengal
(0.8% in 0.1M Tris buffer, pH 5.6) for 25min. After second washing
procedure,spermatozoaweredehydratedin50%,70%,and96% ethanol
rinsed with water, and examined at 1000x magnification under light
microscope.
2.7.BindingStudiesofSIFtoSpermatozoabyFluorescentMicroscopy
Purified ligand was conjugated to FITC using FITC labeling kit (GeNei
FITC Labelling Kit Bangalore Genei (India) Pvt. Ltd.). 1mg of purified
ligandwasmixedwithFITCaccordingtoF/Pratioaspertheinstructions
giveninthekit.
LiquefiedsemensamplewaswashedtwicewithPBSandthepelletwas
finallysuspendedin500LofPBS.To100Lofspermsuspension,200L
ofconjugatedligandwasaddedandincubatedat37Cfor1h.Then150L
of3%formaldehydewasaddedandthereactionmixturewasincubatedat
37Cfor1h.Afterincubation,thereactionmixturewaswashedthricewith
PBS.Thepelletwasfinallydissolvedin50LofPBS(50mM,pH7.2).A
wetmountwaspreparedandobservedunderfluorescentmicroscope1000x
magnification.
2.8.ExtractionofReceptorforSIFfromHumanSperm
SIF receptor was extracted from human spermatozoa by the method
previously standardized in the laboratory [15]. Briefly, the receptor was
extractedbytreatingwashedspermatozoawith3MNaClfor4hat37C
undershakingconditions.3MNaCltreatedspermsamplewascentrifugedat
1500rpmfor15minandthendialyzedextensivelyagainstPBS(50mM,pH
7.2)undercoldconditions.Itwasconcentratedusingpolyethyleneglycol
(PEG6000).Thepurificationof thereceptorwasfurtherdoneusinggel
filtrationchromatographyandthepurityofthispreparationwascheckedby
nativePAGE.
2.9.MatrixAssistedLaserDesorption/IonizationTimeofFlight(MALDI
TOF)ofReceptor
Processingofproteinbands,trypticdigestion,andpeptideextractionwas
doneinthesamewayasdescribedearlierforLCMSofSIF.samplefor
MALDITOF analysis was prepared using dried droplet method. 1L
peptide solution (peptide extracts after tryptic digestion) and 1L of a
suitablematrix,forexample,alphacyanohydroxycinnamicacid(HCCA)in
1:2v/vofacetonitrile(ACN):0.1%TFA,weremixednicely.1Lofthis
mixturewasspottedonaMALDItargetplateandallowedtoairdryatroom
temperature.Peptidecalibrationstandard(BRUKER)wasalsopreparedin
thesameway.MALDItargetplatewasloadedintoUltraflexMALDITOF
forsubsequentpeptidespectraacquisitionandanalysis.ALASERpowerof
337nmwavelengthwasusedforionizationofthesamplesspottedonthe
target plate. Peptide peaks were calibrated withpeaks obtainedfromthe
peptide calibration standard. After peptide spectra were obtained, MS
analysiswascarriedoutusingFlexanalysissoftware(v2.2,BRUKER).
SubsequentMSdataanalysiswascarriedoutusingBiotoolssoftware(v2.2,
BRUKER)andMASCOTsearchengine(MatrixScience)againsttheNCBI
database.
2.10.CalorimetricAnalysisofReceptorLigandInteraction
TheenthalpyofbindingofligandwiththereceptorwasdeterminedinPBS
usingamicroreactioncalorimeter.Anamountof1.5mLofreceptor(200g
mL1)inbufferwasplacedineachofthecalorimetricvialsand250L
syringe(speed3Lsec1)wasloadedwiththeligand(500gmL1).The
experimentwasconductedusingthetitrationmode.Thebindingconstant,
K,andenthalpyofbinding,werecomputedbyusingiterativenonlinearleast
squareregressionmethod.
3.Results
S.aureusisolatedfromthecervixofawomanwithunexplainedinfertility
inhibitedspermmotilitybysecretingthefactorextracellularly.Further,the
factorwasisolatedandpurifiedaspreviouslyreported.PurifiedSIFwasa
proteinofabout20kDawithremarkablespermimmobilizationactivity.
3.1.LCMSofPurifiedSIF
AccordingtoLCMSmeasurementsofthe20kDaband,theproteinshowed
the peptide sequence similarity with hsp70 protein when matched with
proteins in the NCBInr database, using the Mascot search program. The
searchyieldedatopscoreof143forhsp70likeproteinasshownbythe
histogramofthescoredistributionforthe20bestmatchingproteins.Protein
scoresgreaterthan57()wereconsideredtobesignificant.
3.2.InVitroEffectofSIFonSpermMg++ATPaseActivity
The effect of purified SIF from S. aureus on the Mg++ adenosine
triphosphataseactivityofpooledandsonicatedspermatozoafromhuman
wasstudied.Fromtheresults(Table 1),itcouldbeobservedthattheSIF
inhibitedMg++ATPaseactivityofspermatozoaindosedependentmanner.
Ataconcentrationof100gmL1ofSIF,therewasnodetectableMg++
ATPase activity, whereas at lower concentration of 50gmL1 of SIF,
ATPaseactivitydecreasedfrom931.71.3units(control)to195.232.3
units.
Table1:EffectofSIFonMg++ATPaseactivity.
3.3.InVitroEffectofSIFonSpermAcrosomeReaction
WhentheeffectofSIFonhumanspermacrosomereactionwasstudiedtwo
patternsofstainingwereobservedunderlightmicroscope(1000x).Redor
pinkstainingoftheacrosomalregionindicatedintactacrosomes,whereas
white,brown,oryellowishacrosomeswereinterpretedasacrosomereacted.
The percentages of spontaneously acrosome reacted spermatozoa in SIF
treated samples were comparable to control (DMSO). On treatment with
calcium ionophore, majority (approx. 100%) of spermatozoa underwent
acrosome reaction. However, spermatozoa incubated with SIF in HTFM
(with1%HSA)failedtoundergoacrosomereactioninresponsetocalcium
ionophorechallenge(Figure1).
Figure 1: Red or pink staining of the acrosomal region indicated intact
acrosomes,whereaswhite,brown,oryellowishacrosomeswereinterpreted
asacrosomereacted.Inducedspermacrosomereactionontreatmentwith
calciumionophore(1000x)positivecontrol(a).EffectofSIFoncalcium
ionophoreinducedspermacrosomereaction(1000x)(b).Spermacrosome
reactionontreatmentwithDMSO(1000x)negativecontrol(c).Effectof
SIFonspermacrosomereactioninDMSOtreatedsample(1000x)(d).
3.4.BindingStudiesofSIFwithSpermatozoabyFluorescentMicroscopy
WhenspermsamplestreatedwithFITClabelledSIFwereobservedunder
fluorescent microscope, bright green fluorescence was seen over the
spermatozoa which depicts the binding of SIF with spermatozoa. This
indicatesthatsomereceptorsmightbepresentonthesurfaceofspermatozoa
towhichSIFbinds(Figure2).
Figure 2: Fluorescent microscopy of human spermatozoa incubated with
FITClabelledSIF1000x.
3.5.MALDITOFofPurifiedReceptor
AccordingtoMALDITOFanalysisofthepurified62kDaband,theprotein
showedthepeptidesequencesimilaritywithMHCclassIIantigenwhenthe
resultingspectrumwasusedtosearchformatchingproteinsintheNCBInr
database,usingtheMascotsearchprogram.Thesearchyieldedatopscore
of 68 for MHC class II antigen (protein scores greater than 66 are
significant;).
3.6.CalorimetricStudyofReceptorLigandInteraction
The binding constant, K (1350/M), and enthalpy of binding, , (11.9kJ
mole1) were computed from the experimentally calculated enthalpy of
interaction between ligand and receptor, using iterative nonlinear least
squareregressionmethod(Table2).Thevaluesoffreeenergyandentropy
werefoundtobe18.57kJmole1and21.53Jmole1K1,respectively,
calculatedfromthefollowingequations:
Table2:Heatreleasedonreceptorligandinteraction.
4.Discussion
S. aureus is an aggressive, opportunistic Grampositive pathogen that
colonizestheskin,anteriornares,axillae,pharynx,andurogenitaltractof
20%ofhealthyhumans.JiangandLureportedS.aureusasthepredominant
floraininfertilemenwithasignificantdecreaseinspermmotility[22].
Inanearlierworkdoneinourlaboratory,S.aureuscausingimmobilization
of human spermatozoa was isolated from the cervix of a woman with
unexplained infertility, and a sperm immobilization factor (SIF) was
purified.FurthercharacterizationwasdoneusingLCMS.LCMSstudies
indicatedthatSIFshowedpeptidesequencesimilaritywithhsp70protein.
Neuer et al. [23] reported that heat shock proteins (HSP) are
immunodominantantigensofnumerousmicrobialpathogens,forexample,
Chlamydiatrachomatis,whichhavebeenrecognizedasthemaincauseof
infertility[23].
TofurtherelucidatethecontributionofSIFtoreproductivefailure,theeffect
ofpurifiedSIFfromS.aureusontheMg++ATPaseactivityofspermatozoa
wasstudiedandtheresultsshowedthatSIFdecreasedtheATPaseactivity.
TherewasnodetectableMg++ ATPase activitywhenspermatozoa were
incubatedwithSIFatconcentrationof100gmL1.Anyagentaffecting
ATPase activityof sperms affects themotilityof spermatozoa makingit
incapableoffertilization.Forexample,incaseofseaurchinspermatozoa,it
wasobservedthatmotilespermhadanATPaseactivityof0.16MPi(min
mg protein)1, while sperm that had been rendered nonmotile by
homogenizinghadanactivityof0.045MPi(minmgprotein)1[24],
suggesting inhibition of Mg++dependent ATPase could be one of the
possiblemechanismsofspermimmobilization.
Further, the evaluation of acrosome reaction can be used to predict
fertilizationsuccess.Althoughtherateofspontaneousacrosomalreactionof
spermatozoadoesnotcorrelatewiththesuccessrateofinvitrofertilization
(IVF),theindexofinducibility,measuredbythedifferencebetweeninduced
(afterincubationwithcalciumionophoreorexposuretolowtemperature)
and spontaneous acrosomal reaction, is of prognostic value for sperm
fertilizationcapacity[25].WhentheeffectofSIFonhumanspermatozoa
was studied, it was found that the percentages of acrosomereacted
spermatozoaobtainedinteststreatedwithSIFwereconsistentlylowerthan
those obtained in calcium ionophoretreated samples (positive control).
BacteriaincludingUreaplasmaurealyticumandMycoplasmahominishave
been demonstrated to have a negative effect on ionophoreinduced
acrosomalreactionofhumanspermatozoainvitro[26,27].ElMullaetal.
alsostudiedtheeffectofE.colionacrosomalreactionofspermatozoa[28].
Itwasshownthattheinducibilityoftheacrosomereactionwassignificantly
lowerinsemensamplespretreatedwithE.colithaninthecontrolsamples.
TheresultsdemonstratedthatE.coliaffecttheinducibilityoftheacrosome
reaction in vitro and may impair the fertilizing capacity of human
spermatozoa. Several studies have found that sperm responsiveness to
acrosomereactioninducersisreducedininfertilepatients[29].Therefore,
SIF inhibiting the induction of acrosome reaction may be impairing the
fertilizingcapacityofhumanspermatozoa.
FromtheresultsitappearsthattheeffectofSIFoninhibitionofMg++
ATPase may be separate from ionophore mediated acrosome reaction as
disrupted spermatozoa were used for the estimation of Mg++ ATPase
activitywhileintactspermatozoawereevaluatedforacrosomereaction.
Further,when thefluorescent microscopy of spermatozoaincubatedwith
FITClabelledSIFwasdone,brightgreenfluorescencewasseenoverthe
spermatozoa surface. This binding between SIF and spermatozoa gives
directevidencethatsomekindofreceptorsmightbepresentonthesurface
ofspermatozoatowhichlabelledSIFbinds.Therefore,inthepresentstudy,
anattemptwasmadetoextract,purify,andcharacterizethereceptorfrom
spermatozoathatisresponsibleforitsinteractionwithSIFproducedbyS.
aureus. MALDITOF studies of receptor indicated that receptor shows
sequencesimilaritywithMHCclassIIantigenwhenmatchedwithNCBInr
database.These resultsareinagreementwiththose of earlier studies by
Morimoto et al. who clearly demonstrated the expression of the major
histocompatibilitycomplex(MHC)classIImoleculesonmurinespermcells
bymeansofradioimmunoassayaswellasbyenzymeimmunoassay[30].
The study revealed that the site of sperm for binding foreign DNA was
mediatedbythecomplexstructureoftheMHCclassIImoleculeslocalized
attheposteriorregionofspermhead.
Ligand association with the receptor typically involves changes in the
intramolecularandintermolecularinteractionsanddynamicsofthesystem
components. The changes inbondinginteractions thatoccur uponligand
bindingarereflectedinthereactionenthalpyandentropy,whichinturn
determinethefreeenergyofreceptorligandassociation.Microcalorimetric
instrumentsandmethodsareessentialtoolsforthegeneralunderstandingof
binding thermodynamics of biological macromolecules and specific
biologicalsystems.Inthepresentstudy,whenmicroreactioncalorimeterwas
usedtodeterminethefreeenergy(G),enthalpy(H),andentropy(S)of
binding of SIF with purified receptor it was observed that reaction was
exothermic as heat was produced which indicated specific interaction
betweenreceptorandpurifiedSIF.
From the above preliminary observations, it can be concluded that SIF
isolatedfromS.aureuscouldbindtospermatozoaandspermsurfaceprotein
might act as receptor for binding to SIF. The study also indicates that
receptorligand interaction might be responsible for immobilization of
spermatozoa by SIF. Thus, the future studies of detailed molecular
mechanismsofspermimmobilizationbythesereceptorligandinteractions
couldpossiblyleadtousefulinsightsforimprovementsinthetreatmentof
infertility, and in attempts to create safe as well as effective sperm
immobilizingcontraceptives.
Acknowledgments
ThisworkwassupportedbyfundsfromtheDepartmentofBiotechnology
(DBT),NewDelhi.TheauthorswouldalsoliketothankDrAbhaSarwal,
M. D., Pathology, Government Multispeciality Hospital, Sector 16,
Chandigarh,India,forherassistanceinconductingthestudies.
selama3 hpada37Cdenganvolumeyangsamadari
SIF, sedangkan kontrol dibuat dengan menambahkan
volumeyangsamadariPBS(50 mM,pH7,2)dalam
kondisi yang sama. Setelah 3 h, spermatozoa dari
keduaujidankontroldiperlakukandenganbaik0,1%
DMSO(reaksiakrosomspontan)atau10 pMkalsium
ionoforA23187(reaksiakrosomdiinduksi)untuk1 h
pada37C. Selanjutnya,spermatozoadicucidiHTFM
tanpaHSA(500 rpm,10 min). Peletdisuspensikan
dalam3%glutaraldehiddandiinkubasiselama20 min
pada37C. Setelahmencucilanjutpeletdisuspensikan
dalam1050 uLHTFMdandioleskankeslide. Setelah
kering, spermatozoa yang bernoda di Bismark coklat
(0,8% dalam air deionisasi, pH 1,8) selama 5 min,
dicucitigakalidenganair,dandiwarnaidenganRose
Bengal(0,8%dalam0,1MbufferTris,pH5,6)selama
25 min. Setelahprosedurmencucikedua,spermatozoa
yang dehidrasi di 50%, 70%, dan 96% etanol dibilas
dengan air, dan diperiksa di bawah mikroskop
pembesaran1000xcahaya.
2.7. MengikatStudiSIFuntukSpermatozoaoleh
FluorescentMikroskop
Ligan dimurnikan terkonjugasi untuk FITC
menggunakanlabelFITCkit(GeneiFITCPelabelanKit
Bangalore Genei (India) Pvt. Ltd). 1 mg ligan
dimurnikandicampurdenganFITCmenurutF/Prasio
sesuaipetunjukyangdiberikandalamkit.
CairsampelairmanidicuciduakalidenganPBSdan
sepertiyangdijelaskansebelumnyauntukLCMSdari
SIF. sampeluntukanalisisMALDITOFdibuatdengan
menggunakan metode tetesan kering. 1 uL solusi
peptida(ekstrakpeptidasetelahpencernaantryptic)dan
1 uLmatriksyangcocok,misalnya,alphasianoasam
hydroxycinnamic(HCCA)dalam1 : 2v/vasetonitril
(ACN): 0,1% TFA, dicampur dengan baik. 1 uL
campuran ini terlihat di piring sasaran MALDI dan
dibiarkan udara kering pada suhu kamar. Standar
kalibrasipeptida(BRUKER)jugadisiapkandengancara
yang sama. Pelat target MALDI dimuat kedalam
UltraflexMALDITOFuntukakuisisispektrumpeptida
berikutnyadananalisis. SebuahkekuatanLASERdari
337 nmpanjanggelombangdigunakanuntukionisasi
sampelterlihatdipiringsasaran. Puncakpeptidayang
dikalibrasidenganpuncakyangdiperolehdari kalibrasi
standar peptida. Setelah spektrum peptida diperoleh,
analisisMSdilakukandenganmenggunakanperangkat
lunak analisis Flex (v 2.2, BRUKER). Selanjutnya
analisis data MS dilakukan dengan menggunakan
softwareBiotools(v2.2,BRUKER)danmaskotmesin
pencari(MatrixSains)terhadapdatabaseNCBI.
2.10. KalorimetriAnalisisReseptorLiganInteraksi
Entalpi pengikatan ligan dengan reseptor ditentukan
dalam PBS menggunakan kalorimeter microreaction.
Sejumlah 1,5 mL reseptor (200 pg mL1) dalam
bufferditempatkandimasingmasingbotolkalorimetrik
dan250 uLjarumsuntik(kecepatan3 uL sec1)
daerahakrosomditunjukkanacrosomesutuh,sedangkan
acrosomes putih, coklat, atau kekuningan ditafsirkan
sebagai akrosom bereaksi. Diinduksi sperma akrosom
reaksipadapengobatandengankalsiumionofor(1000x)
controlpositif(a). PengaruhSIFpadakalsiumionofor
sperma yang disebabkan reaksi akrosom (1000x) (b).
Akrosom reaksi sperma pada pengobatan dengan
DMSO(1000x)controlnegatif(c). PengaruhSIFpada
akrosom reaksi sperma dalam DMSOdiobati sampel
(1000x)(d).
3.4. MengikatStudiSIFdenganSpermatozoaoleh
FluorescentMikroskop
KetikasampelspermadiobatidenganFITCberlabelSIF
diamati di bawah mikroskop fluorescent, fluoresensi
hijau terang terlihat di atas spermatozoa yang
menggambarkan pengikatan SIF dengan spermatozoa.
Halinimenunjukkanbahwabeberapareseptormungkin
hadirpadapermukaanspermatozoayangSIFmengikat
(Gambar2).
Gambar2:mikroskopFluorescentspermatozoamanusia
diinkubasidenganFITCberlabelSIF1000x.
3.5. MALDITOFdimurnikanReceptor
Menurut analisis MALDITOF dari dimurnikan 62
kDa Band, protein menunjukkan peptida kesamaan
urutan dengan MHC kelas II antigen ketika spektrum
yang dihasilkan digunakanuntukmencari yangcocok
proteindalamdatabaseNCBInr,menggunakanprogram
pencarianMascot. Pencarianmenghasilkantopskor68
untukMHCkelasIIantigen(proteinskorlebihbesar
dari66yangsignifikan;).
3.6. KalorimetriStudiReceptorLiganInteraksi
Mengikatkonstan,K(1350/M),danentalpimengikat,,
(11,9 kJ mol1) dihitung dari entalpi eksperimen
dihitung dari interaksi antara ligan dan reseptor,
menggunakan nonlinear berulang setidaknya metode
regresikuadrat(Tabel 2). Nilainilaienergibebasdan
entropiyangditemukan18,57 kJ mol1dan21,53 J
mol1K1, masingmasing, dihitung dari persamaan
berikut:
Tabel2:Panasdirilispadainteraksiliganreseptor.
4.Diskusi
S. aureus adalah agresif, oportunistik Grampositif
patogenyangberkolonisasikulit,naresanterior,aksila,
faring, dan saluran urogenital dari 20% dari manusia
sehat. JiangdanLumelaporkanS.aureussebagaiflora
dominan pada pria infertil dengan penurunan yang
signifikandalammotilitassperma[22].
Dalam karya sebelumnya dilakukan di laboratorium
kami,S.aureusmenyebabkanimobilisasispermatozoa
manusia diisolasi dari leher rahim seorang wanita
dengan infertilitas dijelaskan, dan faktor imobilisasi
sperma (SIF) adalah dimurnikan. Karakterisasi lebih
ionofordimediasireaksiakrosomspermatozoasebagai
terganggu digunakan untuk estimasi aktivitas Mg ++
ATPase sementara spermatozoa utuh dievaluasi untuk
reaksiakrosom.
Selanjutnya,ketikamikroskopfluorescentspermatozoa
diinkubasi dengan FITC berlabel SIF dilakukan,
fluoresensi hijau terang terlihat di atas permukaan
spermatozoa. InimengikatantaraSIFdanspermatozoa
memberikan bukti langsung bahwa beberapa jenis
reseptor mungkin hadir pada permukaan spermatozoa
yang berlabel SIF mengikat. Oleh karena itu, dalam
penelitianini,upayayangdilakukanuntukmengekstrak,
memurnikan, dan mengkarakterisasi reseptor dari
spermatozoa yang bertanggung jawab untuk interaksi
denganSIFdiproduksiolehS.aureus. StudiMALDI
TOF dari reseptor menunjukkan bahwa reseptor
menunjukkan kesamaan urutan dengan MHC kelas II
antigen ketika dicocokkan dengan database NCBInr.
Hasil ini sesuai dengan penelitian sebelumnya oleh
Morimotoetal. yangjelasmenunjukkanekspresiutama
histocompatibility complex (MHC) kelas II molekul
padaselspermamurinedengancararadioimmunoassay
serta oleh enzim immunoassay [30]. Hasil penelitian
menunjukkanbahwasitusspermauntukmengikatDNA
asingdimediasiolehstrukturkomplekskelasIImolekul
MHClokaldidaerahposteriorkepalasperma.
Asosiasi ligan dengan reseptor biasanya melibatkan
perubahan dalam interaksi intramolekul dan
KaryainididukungolehdanadariDepartemen
Bioteknologi(DBT),NewDelhi. Penulisjugaingin
mengucapkanterimakasihDrAbhaSarwal,MD,
Patologi,RumahSakitMultispecialityPemerintah,
Sektor16,Chandigarh,India,untukbantuannyadalam
melakukanpenelitian.