oup_humrep_dex269 1995..2006 ++

12 Pages·2017·1.17 MB·English

HumanReproduction,Vol.32,No.10pp.1995–2006,2017
AdvancedAccesspublicationonAugust28,2017 doi:10.1093/humrep/dex269
ORIGINAL ARTICLE Andrology
Complex CatSper-dependent
+
2
and independent [Ca ] signalling
i
in human spermatozoa induced
fl
by follicular uid D
o
w
n
lo
a
d
Sean G.Brown1,†, Sarah Costello2,†, Mark C. Kelly3,†, ed
Mythili Ramalingam3,4,†, Ellen Drew4, Stephen J. Publicover2, from
h
Christopher L.R. Barratt3,4,*,and Sarah Martins DaSilva3,4 ttp
s
://a
1SchoolofScience,EngineeringandTechnology,AbertayUniversity,DundeeDD11HG,UK2SchoolofBiosciences,TheUniversityof ca
Birmingham,BirminghamB152TT,UK3ReproductiveandDevelopmentalBiology,SchoolofMedicine,NinewellsHospitalandMedical de
School,UniversityofDundee,DundeeDD19SY,UK4AssistedConceptionUnit,NinewellsHospitalDundee,DundeeDD19SY,UK m
ic
.o
*Correspondenceaddress.SchoolofMedicine,NinewellsHospitalandMedicalSchool,UniversityofDundee,DundeeDD19SY,UK; up
E-mail:[email protected] .c
o
m
/h
u
SubmittedonSeptember20,2016;resubmittedonJuly15,2017;acceptedonJuly31,2017 m
re
p
/a
STUDYQUESTION: Doesprogesteroneinhumanfollicularfluid(hFF)activateCatSperanddoothercomponentsofhFFmodulatethis rtic
effectand/orcontributeseparatelytohFF-inducedCa2+signaling? le-a
b
SotUheMrMcoAmRpYonAenNtsSoWfhEFRF:alhsoFFcoponttreinbtulytesttiom[uClaat2e+s]iCsaigtnSapleinrga,nindcliundcrinegasmesod[Culaa2t+io]in,porfimCaatrSilypedrucehtaonnheiglhacctoivnictyenatnrdatiinohnisbiotifopnroofge[Cstae2r+o]nieo,shciollawteiovnesr., stract/3
WHATISKNOWNALREADY:CatSper,theprincipalCa2+channelinspermatozoa,isprogesterone-sensitiveandessentialforfertility. 2/1
BothhFFandprogesterone,whichispresentinhFF,influencespermfunctionandincreasetheir[Ca2+]i. 0/19
STUDY DESIGN, SIZE, DURATION: This basic medical research study used semen samples from >40 donors and hFF from >50 95
/4
patientswhowereundergoingsurgicaloocyteretrievalforIVF/ICSI. 0
9
6
PARTICIPANTS/MATERIALS, SETTING, METHODS: Semen donors and patients were recruited in accordance with local ethics 42
9
approval (13/ES/0091)fromtheEastofScotlandResearchEthicsService REC1.ActivitiesofCatSperandKSperwereassessedbypatch b
clampelectrophysiology.Sperm[Ca2+] responseswereexaminedinspermpopulationsandsinglecells.Computer-assistedspermanalysis y g
i u
(CASA)parametersandpenetrationintoviscousmediawereusedtoassessfunctionaleffects. es
MAIN RESULTS AND THE ROLE OF CHANCE: hFF and progesterone significantly potentiated CatSper currents. Under quasi- t on
physiologicalconditions,hFF(upto50%)failedtoaltermembraneK+conductanceorcurrentreversalpotential.hFFandprogesterone(atan 23
N
equivalentconcentration)stimulatedsimilarbiphasic[Ca2+] signalsbothinspermpopulationsandsinglecells.AtahighhFFconcentration o
i v
(10%),thesustained(plateau)componentofthe[Ca2+] signalwasconsistentlygreaterthanthatinducedbyprogesteronealone.Insinglecell em
recordings,1%hFF-induced[Ca2+] oscillationssimilarlyitoprogesteronebutwith10%hFFgenerationof[Ca2+] oscillationswassuppressed. be
Aftertreatmentto‘strip’lipid-deriivedmediators,hFFfailedtosignificantlystimulateCatSpercurrentsbutinduicedsmall[Ca2+]iresponses r 201
that were greater than those induced by the equivalent concentration of progesterone after stripping. Similar [Ca2+] responses were 8
i
observed when sperm pretreated with 3μM progesterone (to desensitize progesterone responses) were stimulated with hFF or stripped
hFF.hFFstimulatedviscousmediapenetrationandwasmoreeffectivethantheequivalentdoesofprogesterone.
LARGESCALEDATA:N/A.
LIMITATIONS,REASONSFORCAUTION:Thiswasaninvitrostudy.Cautionmustbetakenwhenextrapolatingtheseresultsinvivo.
†Contributedequallyasfirstauthor.
©TheAuthor2017.PublishedbyOxfordUniversityPressonbehalfoftheEuropeanSocietyofHumanReproductionandEmbryology.
ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense(http://creativecommons.org/licenses/by/4.0/),whichpermitsunrestrictedreuse,
distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.
1996 Brownetal.
WIDERIMPLICATIONSOFTHEFINDINGS:ThisstudydirectlydemonstratesthathFFactivatesCatSperandestablishesthatthebio-
logicallyimportanteffectsofhFFreflect,atleastinpart,actiononthischannel,primarilyviaprogesterone.However,theseexperimentsalso
demonstrate that other components of hFF both contribute to the [Ca2+] signal and modulate the activation of CatSper. Simple in vitro
i
experimentsperformedoutofthecontextofthecomplexinvivoenvironmentneedtobeinterpretedwithcaution.
STUDY FUNDING/COMPETING INTEREST(S): Funding was provided by MRC (MR/K013343/1, MR/012492/1) (S.G.B., S.J.P.,
C.L.R.B.) and University of Abertay(sabbatical for S.G.B.). Additional fundingwasprovided byTENOVUS SCOTLAND (S.M.D.S.), Chief
ScientistOffice/NHSResearchScotland(S.M.D.S).C.L.R.B.isEICofMHRandChairoftheWHOESGonDiagnosisofMaleinfertility.The
remainingauthorshavenoconlictsofinterest.
Keywords:follicularfluid/patchclampelectrophysiology/CatSper/potassiumchannel/spermatozoa
D
o
w
n
lo
a
d
e
Introduction oofniiststicpreifnfecciptaslocnomCaptoSnpeenrtuPp4o,noerxaproestuhreerteostyhneesregicstoicmoprleexvmenixatunrtaegs-? d from
Human follicular fluid (hFF) affects various important functions of (ii) Do other components of hFF contribute significantly, but separ- h
humanspermatozoa,includinghyperactivatedmotility,chemotaxisand ately,tohFF-inducedCa2+signalling? ttps
acrosome reaction (Baldi et al., 1998). Almost 30 years ago Thomas ://a
c
etal.demonstratedthathFFstimulatedarapidinfluxofCa2+inhuman ad
e
spermatozoa(ThomasandMeizel,1988).Subsequently,progesterone Materials and Methods m
ic
(P4)wasshowntohaveeffectsonspermfunctionsimilartothoseof .o
u
p
hFF and was found to be the component of hFF that was primarily Experimentalsolutions .c
responsibleforinductionofCa2+-influx(Osmanetal.,1989;Thomas om
Details for HEPES buffered saline, bicarbonate buffered capacitating /h
andMeizel,1989).In2011,LishkoandStrunkerindependentlyshowed medium, supplemented Earle’s balanced salt solution (sEBSS), standard um
thatinductionofCa2+influxbyP4wasviathesperm-specificchannel bathsolution(patchsealsandquasi-physiologicalrecording),standardpip- re
p
CatSper(Lishkoetal.,2011;Strunkeretal.,2011),whichisnowknown ettesolution (quasi-physiological recording), Cs+-based pipette andbath /a
etot bale.,s2ti0m1u2l)a.tePd4,byatahwigidhecroanncgeenotrfastimonalslo(~rgμaMnicrmanoglee)c,ulaelsso(Birnehnibkietsr stioolnustiofonrsC(matoSnpoervataleilnctuCrraetSnptsearrceugrriveenntsi)naSnudppblaetmhe(Bnata2r+y)FainledSp1ip.ettesolu- rticle-a
b
KSper channels (Mannowetz et al., 2013). It has been proposed that s
tra
highconcentrationsofP4encounteredinthevicinityoftheoocyteand c
Selectionandpreparationofspermatozoa t/3
itsvestmentsachievefullactivationofCatSperthroughacombination 2
/1
ofCatSperactivationanddepolarizationofmembranepotentialdueto Semensampleswerefromdonorswithnormalspermconcentrationand 0
/1
KSperinhibition(Mannowetzetal.,2013). motility(WHO2010).Sampleswereobtainedbymasturbationafter2–3 9
9
5
As P4 is a primary component of hFF, a logical assumption is that dayssexualabstinence.Afterliquefaction,spermwereisolatedbyeither /4
exposure of human spermatozoa to hFF in vivo activates CatSper. swim-up or density gradient centrifugation (electrophysiological studies) 09
6
However, the ‘clean’ stimuli that are used for in vitro investigations, andlefttocapacitate(37°C,6%CO2)for3–5h(Alasmarietal.,2013a). 42
suchasthosebywhichtheactionofP4onCatSperwasestablished, Samples were obtained and analysed in line with suggested guidance for 9 b
differgreatlyfromthecomplexenvironmentofthereproductivetract humansemenstudiesandvariationsidentified(Bjorndahletal.,2016). y g
u
(Mortimer et al. 2013; Sakkas et al., 2015). hFF is a complex fluid es
(Revelli et al., 2009; O’Gorman et al., 2013) and, in its presence, Humanfollicularfluid t on 2
spermaresimultaneouslyexposedtomultipleligands,potentiallylead- 3
ingtomultipleseparateeffectsand/orinteractions.Significantly,pre- Oocytes were retrieved by transvaginal aspiration 36h after injection of No
treatment with oestrogen (17βE2), which elevates [Ca2+]i in sperm- rb-lhoCodG.coMnotastm(in9a0t%io)noffrothmesteheoolacrygteesstwfoelrliecleinsmofeetaapchhasoevaIrI.yhwFFaswciethnotrui-t vemb
atozoa apparently by a mechanism independent of CatSper (Luconi e
etal.,1999;Lishkoetal.,2011;Mannowetzetal.,2017),reducedthe fnuagteadnta(t02.25200μmgfofirlte1r0emd)inwtaosseeipthaerartuesceedlluolnartchoemdpayonfoenrtesxapnedritmheenstuaptieorn- r 20
Ca2+responsetosubsequentstimulationwithP4(Luconietal.,1999). orstored(at−20°C)untiluse(<1week).hFFprogesterone(inwholeand 18
Consequently, two fundamental questions are (i) Does hFF act on dextran-coated charcoal-stripped samples) was assayed before use
CatSperinamannerconsistentwiththepreviouslydescribedeffects (SiemensADVIACentaur®XPcompetitiveImmunoassaySystem).
+
TableI EffectofhFFonmonovalent(Cs )CatSpercurrentamplitude.
−80mV 80mV
.................................................................. .....................................................................
Stimulus n Control(pA) Treated(pA) P Control(pA) Treated(pA) P
.............................................................................................................................................................................................
1%hFF 13 −89.4±8.3 −199±33.6 0.01 193.3±18.4 507.3±37.7 0.001
CatSperactivationbyhumanfollicularfluid 1997
Strippingofsteroids,prostaglandinsand P4then,afteradelayof300s,asecond‘test’stimuluswasappliedinthe
otherlipid-derivedcomponentsfromhFF continuedpresenceofthedesensitizingP4.
Steroids and prostaglandins were removed from hFF by adapting the
Singlecellrecordings
dextran-coatedactivatedcharcoalmethodforremovalofsteroidsfromser-
Recordings were made as described previously (Nash et al., 2010) but
um(productinformationsheetC9157;Sigma-Aldrich,UK;Supplementary
usingFluo-4.Allexperimentswereperformedat25±0.5°Cinacontinu-
FileS1).
ous flow of medium. Images were captured at 0.2Hz using a 40× oil
objectiveandAndorIxon897EMCCDcameracontrolledbyiQsoftware
Electrophysiology (AndorTechnology,Belfast,UK).Fluorescencefromthespermposterior
head/neckwasbackground-correctedandnormalizedtogive%changein
Currents were recorded from sperm isolated by density gradient using
whole-cell patch clamp (Mansell et al., 2014). To investigate K+ channel intensity(Nashetal.,2010).
Toassess[Ca2+] oscillations,pairedexperimentswereconductedusing D
function,cellswerestudiedunderquasi-physiologicalconditions(standard i ow
(2pB5ipr0oe0twtmensa)en.tdaMlb.e,am2th0b1rsa6on)leu.tRiopenovste)ernsuatsilainlpgowtaeansrtaimahlespld(pEraott—o−ct9oo2l e(m−sVt9im2batetoetw6ree8esmntinVgraoVmvmpesr) [cbCeylales2y+fer]oiftomrrantthhseeiensoatc.mcuerrseanmcpeleofecxypcolisceadl[tCoa2h+F]FioosrcPill4a.tiTornascfeosllowwerinegetxhaemininiteiadl nloaded
andmembraneconductance(Gm)werecalcurelavtedaspreviouslydescribed fro
Assessmentofspermfunction m
(Brownetal.,2016).MonovalentCatSpercurrentswererecordedusing h
Cs+-baseddivalent-freepipetteandbathsolutions.Currentswereevoked Viscous media penetration test and Computer-assisted sperm analysis ttp
s
by a ramp protocol (−80 to 80mV over 1s). Membrane potential was (CASA)werecarriedoutaspreviouslydescribed(Alasmarietal.,2013a; ://a
heldat0mVbetweenramps.Divalent(Ba2+)CatSpertailcurrents(Lishko Williamsetal.,2015). ca
d
et al., 2011) were evoked by 400ms pulses followed by stepping to e
m
−150mV (200ms). Vm was held at −70mV between sweeps (Lishko Ethicalapproval ic
etal.,2011).Tailcurrentamplitudeswereusedtoplotvoltageactivation .ou
(G–V)curves.Dataweresampledat2kHz,filteredat1kHz.Tailcurrent WrittenconsentwasobtainedfromeachIVFpatientinaccordancewith p.c
the Human Fertilization and Embryology Authority (HFEA) Code of o
data were leak subtracted using pClamp P/4 protocol to minimize the m
Practice(V8)underlocalethicsapproval(13/ES/0091)fromtheEastof /h
impactofmembraneresistance(PClamp10software,Axoninstruments). u
ScotlandResearchEthicsServiceREC1.Similarly,volunteerspermdonors m
Assessmentof[Ca2+]isignals wapeprreovraelcnruuimtebderunEdReNr-1th2e-0s5a7m0ReaetththicealUanpivperrosvitayloinfBDirmunindgeheaman.d ethical rep/artic
Populationrecordings le
Dataanalysis -a
Followingswim-up,sperm(≈6million/ml)werecapacitated(3–5h)then b
s
loadedwith4.5μMFluo-4for30min,washedtwice(700gfor10min)and DatawereanalyzedusingMicrosoftExcel™orGraphPadPrism™(version5, tra
resuspendedinsEBSS.[Ca2+]iwasassessedusingaFLUOstarmicroplate GraphPad Software Inc.). Statistical significance was determined using ct/3
reader(BMGLabtechOffenburg,Germany)with488nm(excitation)and Student’s paired/unpaired t-test or analysis of variance (ANOVA) and 2/1
520nm (emission) filters. After a control period, (30–60s) stimuli were adjustedusingtheHolm–Bonferronicorrection(Gaetano,2013)asappro- 0/1
9
addedusingamultichannelpipetterasdescribedbyStrunkeretal.(2011). priate. Percentage data were ArcSine converted before testing. Data are 9
To compare [Ca2+]i responses to hFF and equivalent [P4] aliquots from presentedasmean±SEMwithP<0.05indicativeofstatisticalsignificance. 5/40
thesamefluo-4loadedsample,testswereperformedinparallel.Emission AllsetsofexperimentalrepeatsincludespermandhFFsamplesfrommore 9
6
was background-corrected and normalized to the control (pre-stimulus) than one donor. Values of ‘n’ for patch clamp experiments are given in 42
9
amplitude. To compare duration of P4 and hFF-induced transients, the TablesI–VIandshowthenumberofcellspatched.Unlessstatedotherwise, b
half-duration(midpointoftherisingphasetomidpointofdecay)wascalcu- thevaluesof‘n’for[Ca2+]iandmotilityassessmentsprovidedintextandfig- y gu
lated.Indesensitizationexperiments,cellswerefirststimulationwith3μM urelegendsshowthenumberofexperimentsusedforstatisticalanalysis. e
s
t o
n
2
Results 3
TableII EffectofhFFonCatSperV50. N
o
v
Stimulus n Control(mV) Treated(mV) P hFFandionchannelcurrents em
........................................................................................ b
e
1%hFF 12 61.8±5.2 25.1±2.7 <0.001 EffectsofhFFonCatSpercurrent r 2
500nMP4 4 71.7±8.0 15.1±6.1 <0.01 SinceP4isanactivatorofCatSper,wefirstusedwhole-cellpatchclamp 018
electrophysiology to examine the effect of hFF on CatSper currents
+
TableIII EffectofstrippedhFF(ShFF)onmonovalent(Cs )CatSpercurrentamplitude.
−80mV 80mV
................................................................... .....................................................................
Stimulus n Control(pA) Treated(pA) P Control(pA) Treated(pA) P
.............................................................................................................................................................................................
1%ShFF 8 −130.3±28.9 −105.6±32.2 0.013 300.8±68.6 258.7±74.9 0.07
1%hFF 8 −130.3±28.9 −189.9±52.0 0.05 300.8±68.6 431.5±85.8 0.008
1998 Brownetal.
(I ).hFF(diluted1%) potently potentiated bothinward andout- (Fig. 2b; Table IV; P < 0.01). The concentration of P4 present in 1%
CatSper
ward monovalent CatSper currents (Fig. 1a,b; Table I; P < 0.01). P4 ShFFis2–3nM,whichhasbeenreportedtoincreaseCatSpercurrents
potentiatesCatSpercurrentsprimarilybyshiftingchannelactivationto (Lishkoetal.,2011).Wethereforeassessedwhetherwecoulddetect
+
morenegativevoltages(Lishkoetal.,2011).Assessmentofvoltagesen- this effect under our recording conditions. Both using standard Cs
sitivityofCatSperactivation(usingBa2+tailcurrents)showedthat1% saline recording (P4 added directly to Cs+ saline before perfusion of
hFFshifted the G–V curve to morenegative voltages(Fig. 1c), signifi- the recording chamber) and also when progesterone was first dis-
cantly changing the V (Table II; P < 0.001). Similarly, 500nM P4 solvedinamixtureof1%standardbathsolution(containing2mMCa2+,
50
causedanegativeshiftoftheCatSperG–Vcurve(Fig.1d,TableII;P< 0.7mMMg2+)and99%Cs+saline(tomimicionicconditionsinShFF
0.01)asdemonstratedpreviously(Lishkoetal.,2011). experiments), superfusion of sperm with 2nM P4 significantly
increased both outward and inward currents (Table V). Finally, we
increased the concentrations of divalent chelators (EGTA, EDTA) in D
EffectsofsteroidstrippingonhFF-stimulationofCatSpercurrents ourCs+recordingsalineto9mMofeachtochelateanyresidualCa2+ ow
n
hFFcontains,inadditiontoP4,prostaglandins(Lishkoetal.,2011)and and Mg2+ from the hFF. Under these conditions, we observed a lo
other ligands that may influence [Ca2+] signalling. To examine the ad
i response to ShFF in some cells (Supplementary Fig. S2) and mean e
d
esaffmecptleosfodfeFpFlewtinegreli‘psitdr-ipdpeeridv’eudsainggodniesxtstr(asnt-ecrooaidtesdancdhaprcroosatla.gTlahnisdpinrso)-, isnigwnaifircdanantd(ToaubtwlearVd;cPur>ren0t.s1)w.eErxeaimncinreaatisoend,obfu[tPt4h]isceoffneccetnwtraastionnost from
cedurereduced[P4]by98.6±0.13%(n=31;SupplementaryFig.S1). showedthatdetectableeffectsofShFFoccurredonlywithhFFsamples http
tShpeenrmtoat1o%zohaFwFefrroemexthpeosseadmfierssatmtopl1e%inccuhbaarcteodal-ssimtriiplaprleydbhuFtFw(iSthhoFuF)t wherethe[P4]wasunusuallyhigh(SupplementaryFig.S2). s://ac
a
dextran-coatedcharcoal.ShFFfailedtostimulateICatSper,(bothinward EffectofhFFonmembranepotentialandK+current de
m
asunbdseoquutwenatrdapcpulricreatnitosnwoefrheFsFmpaollteern;tFiaigt.ed2ab;oTtahbilnewIIaI;rdPa<nd0.o0u5t)w, barudt ToinvestigatethepossibleeffectsofhFFonmembranepotential,cells ic.ou
were challenged with hFF (1, 10 and 50% dilution) under quasi- p
currentsamplitude(Fig.2a;TableIII;P=0.05;P<0.01,respectively). .c
physiological conditions (see Materials and Methods section). hFF did o
Similarly, when tail currents were used to assess CatSper activation, m
notalterrestingmembranepotentialoroutwardmembraneconduct- /h
hFF but not ShFF shifted voltage sensitivity to less positive potentials anceindicatingthathFFdidnotmodulate/suppressK+channelfunction um
at these dilutions (Fig. 3; Table VI). Stimulation with P4 significantly rep
/a
TableIV EffectofstrippedhFF(ShFF)onCatSperV50. bdueptoatla1ri0zeμdMmefefemcbtsrawneerepontoetnstiiganliafincadntre(dTuacbeledVcIo).nductance at 30μM rticle
-a
b
..S..t.i.m...u.l.u..s........n.........C..o..n.t..r.o..l.(.m...V..)........T..r..e.a..t.e..d..(.m...V..).........P...... hFFandsperm[Ca2+] stra
i c
11%%ShhFFFF 44 5544..00±±1100..88 591..30±±84..80 0N.0S1 hFF-induced[Ca2+]isignalsinspermpopulations t/32/1
In agreement with previous reports hFF, similarly to P4, caused a 0
/1
dose-dependent,biphasicelevationof[Ca2+]iconsistingofatransient 99
5
/4
0
9
6
TableV Isfailureof1%ShFFtopotentiateCatSpercurrentsduetocontaminationwithdivalentcations? 4
2
9
b
.−..8.0...m..V................................................. 8..0..m...V..................................................... y g
u
Stimulus n Control(pA) Treated(pA) P Control(pA) Treated(pA) P e
s
............................................................................................................................................................................................. t o
2nMP4 4 −60.3±13.5 −90.0±18.9 0.02 193.4±23.7 237.4±36.7 0.046 n 2
2nMP4withCa/Mg 5 −62.1±16.7 −111.9±21.7 0.002 156.6±22.1 213.2±16.0 0.012 3 N
ShFFwith9mMEGTA,9mMEDTA 17 −98.9±14.4 −125.6±21.7 0.12 214.6±24.7 223.9±31.7 0.62 ov
e
m
b
e
r 2
0
1
+ 8
TableVI EffectofhFFonK currentreversalpotentialandconductance.
Erev(mV) Gm(ns/pF)
.................................................................... .....................................................................
Stimumlus n Control(pA) Treated(pA) P Control(pA) Treated(pA) P
.............................................................................................................................................................................................
1%hFF 6 −34.6±4.4 −36.5±6.6 >0.05 1.02±0.17 1.12±0.21 >0.05
10%hFF 3 −22.0±9.0 −22.8±9.1 >0.05 0.79±0.20 0.72±0.25 >0.05
50%hFF 3 −23.95±3.8 −24.0±4.0 >0.05 0.64±0.06 0.57±0.04 >0.05
10μMP4 3 −28.2±2.8 −18.28±4.6 0.09 0.51±0.06 0.41±0.03 0.32
30μMP4 4 −41.4±3.5 −21.0±5.5 0.023 0.68±0.08 0.25±0.06 0.026
CatSperactivationbyhumanfollicularfluid 1999
(a) +80 mV (b)
0 mV 0 mV P = 0.001
600
800 –80 mV
400
600 control
+80 mV
FF (1%)
A) pF) 200
I (p 400 I (pA/ Dow
0 n
200 lo
a
–80 mV de
d
0 –200 fro
m
h
–200 –400 P = 0.01 ttps
://a
c
a
(c) 1.2 (d) 1.2 de
m
ic
1 1 .o
u
p
Gmax 0.8 max 0.8 .com
G/ 0.6 G/G 0.6 /hum
re
0.4 0.4 p/a
0.2 control 0.2 cPo4n (t5ro0l0 nM) rticle-a
hFF (1%) bs
0 0 tra
–120 –60 0 60 120 180 –120 –60 0 60 120 180 ct/3
Vm (mV) Vm (mV) 2
/1
0
Figure1 hFFpotentiatesCatSpercurrentsandshiftsthevoltagesensitivitytolessdepolarizedpotentials.(a)RepresentativeCs+-mediatedCatSper /19
9
currentintheabsence(black)andpresence(red)of1%hFF.Voltageprotocolimposedisshownabove.(b)Meanamplitudes(±SEM)ofCatSpercur- 5/4
rentsrecordedintheabsence(left)andpresence(right)of1%hFF(n=8hFFsamples).Blackbarsshowinwardcurrent(−80mV),whitebarsshow 09
outwardcurrents(80mV;n=13).(candd)Showconductance-voltage(G–V)relationshipsforBa2+-mediatedCatSpertailcurrentsintheabsence 64
2
andpresenceof1%hFF(c,n=12)and500nMP4(d,n=4).hFF,humanfollicularfluid. 9 b
y
g
u
e
s
t o
cfoolnlocwenetdrabtyioanphlaatdeabuee(Fnigd.e4tae,rbm).inUesdinwgehFdFirseacmtlpylecsominpwarheicdh[tChae2P+4]i hSiFmFi-lianrdlyuctoedp[oCpau2la+ti]oinsigmneaalssuinresminegnlets,ceslilnsgle cell imaging of [Ca2+]i at n 23 No
signals induced by hFF (diluted to 10, 1, 0.1 and 0.01%) and by an theposteriorhead/neckshowedtransientresponsesinthevastmajor- ve
m
equivalentconcentrationofP4alone(usingaliquotsof spermfrom ityofcellsexposedtohFF,whichresembledthoseinducedbyP4alone b
e
thesamebatchofFluo-4loadedspermcellsruninparallel).Analysis (Fig.5a,b).InP4-stimulatedcellstheinitialCa2+transientwasoftenfol- r 2
of these data pairs showed that at low concentrations of hFF lowedby[Ca2+] oscillations(notsynchronizedandthereforedetectable 01
i 8
(0.01–1%) the amplitudes of signals induced by hFF and P4 were only in single cell records; Harper et al., 2004; Kirkman-Brown et al.,
similar(Fig.4c,d).However,atthehighesthFFconcentration(10%) 2004; Fig. 5a). In cells stimulated withhFF, oscillationswere observed
the [Ca2+]i plateau induced by hFF (assessed 10min after stimulus buttheiroccurrence wasmarkedlyconcentration dependent. 1% hFF,
application)wasconsistentlygreaterthanthatinducedbyanequivalent similarlyto300nMP4(estimatedequivalent[P4])inducedoscillationsin
concentrationofP4(meanamplituderatiohFF:P4=1.6±0.1;Fig.4d ≈25%ofcells(Fig.5c;P=0.47;n=10).However,whereas3μMP4
redsymbols;P=0.001;n=7;).Incellsstimulatedwith10%hFFthe wassimilarlyeffective(19%ofcells;e.g.Fig.5a),10%hFF-inducedoscil-
[Ca2+]itransientalsoappearedlongerthanincellsfromthesamebatch lationsinonly4%ofcells(Fig.5b,d,e;P=0.002,n=10).
ofFluo-4loadedspermcellsstimulatedwithanequivalentconcentration
ofP4(Fig.4a,b).Assessmentofthetransient‘half-duration’(latencyfrom [Ca2+]iresponsestocharcoal-strippedhFF
midpoint oftherising phase tomidpoint ofdecay) confirmed that this Since the ability of 1% hFF to potentiate CatSper currents was
wasthecase(P=0.0005;n=7). removedbystrippingofsteroids/prostaglandinswithdextran-treated
2000 Brownetal.
(a) 600 (b) 1.2
500
1
400 +80 mV
300 ax 0.8
m
pF) 200 G/G 0.6
A/
p 100
I ( 0.4 control
0 hFF (1%) D
–100 –80 mV 0.2 ShFF (1%) ow
n
–200 0 loa
d
–120 –60 0 60 120 e
–300 d
Vm (mV) fro
m
h
ttp
s
Figure2 Charcoal-strippedhFF(ShFF)doesnotpotentiateCatSpercurrents.(a)Mean±SEMinwardCatSpercurrentsat−80mV(black)andout- ://a
wardcurrentsat80mV(white;n=8cells)undercontrolconditions,inpresenceof1%strippedhFF(ShFF)and1%time-control(hFF;7FFsamples). ca
d
ShFFreducedcurrentamplitude(P<0.05)butsubsequentapplicationofcontrolhFFpotentiatedbothinwardandoutwardcurrents(P<0.01com- e
m
paredtoShFF).(b)1%strippedhFF(ShFF)failedtoalterCatSpervoltagesensitivitybutsubsequentapplicationofcontrolfollicularfluid(hFF)causeda ic
significantleftwardshiftinvoltagesensitivity(V50P<0.01comparedtocontrolandShFF).n=4cells,fourhFF. .oup
.c
o
m
/h
u
m
re
p
/a
rtic
le
-a
(a) 120 pA/pf (b) 80 pA/pf (c) 60 pA/pf bstra
70 c
control 100 control 60 control 45 t/32
hFF (1%) 80 hFF (10%) hFF (50%) /10
50 /1
9
60 40 30 95
/4
30 0
40 9
6
20 15 4
2
20 10 9 b
y
0 0 0 gu
e
–120 –80 –40 0 40 80 –120 –80 –40 0 40 80 –120 –80 –40 0 40 80 s
–20 –10 t o
mV –20 mV –15 mV n 2
3
N
Figure3 hFFdoesnotaffectK+channelactivityrecordedunderquasi-physiologicalconditions.Ineachpanel,blacktraceshowsmean(±SEM)con- ov
e
trolcurrentandredtraceshowsmean(±SEM)ofcurrentsrecordedafterexposuretohFF.(a)1%hFF;n=6cells,fourhFFtested;(b)10%hFF;n=3 m
b
cells,threehFFtested;(c)50%hFF;n=3cells,threehFF. er 2
0
1
8
charcoal (Fig. 2a), we examined whether hFF-induced [Ca2+] signals isolatedbysubtractionoftraces(ShFF-equivalent[P4]),showedacti-
i
were similarly affected. Surprisingly, [Ca2+] responses were always vationlaterthanthe[Ca2+] signalinducedbyP4andpeaked60–100s
i i
detectedincellpopulationsstimulatedwith1%ShFF,withthe[Ca2+] afterstimulation(Fig.6b).
i
transient amplitude being 36.8 ± 1.8% of that in the parallel control In single cell imaging experiments where immobilized sperm were
(1%hFF)experiments(Fig.6a;SupplementaryFig.S3;P=3.2×10−12; superfusedwith1%ShFForequivalent[P4],cellsfailedtogeneratethe
n=21).In28experimentswhereparallelrecordingswerecarriedout [Ca2+] transient seen in the equivalent population experiments and
i
withShFFand[P4]equivalenttothatinShFF,[Ca2+] transientampli- insteadweobservedaslow[Ca2+] ramp(Fig.7a).Thisreducedeffi-
i i
tudesweresimilar(P=0.14).However,thesubsequent[Ca2+] ‘plat- cacyofstimulideliveredbyperfusionisduetobindingofprogesterone
i
eau’ was significantly greater with ShFF (43 ± 9% for the period 30- totheperfusiontubing(seeDiscussionsection).Themeanincreasein
240spost-stimulus;P=4.8×10−6;Fig.6b).The‘non-P4’component, [Ca2+] wasgreaterintheShFF-treatedcells,buttheeffectwashighly
i
CatSperactivationbyhumanfollicularfluid 2001
(a) 300 10% hFF (b) 300 2.8 μM P4
1% hFF 280 nM P4
250 250
0.1% hFF 28 nM P4
%) 200 0.01% hFF %) 200 2.8 nM P4
e ( e (
nc 150 nc 150
e e
c c
es 100 es 100
or or
Δ flu 50 Δ flu 50
D
0 0 ow
–50 0 200 400 600 800 –50 0 200 400 600 800 nloa
seconds seconds de
d
fro
(%)c) 400 [Ca2+]i transient (%)d) 250 [Ca2+]i plateau m http
Δ hFF-induced fluorescence ( 123000000 11000%..10%%1 h% F hhF FFhFFFF Δ hFF-induced fluorescence ( 11205050000 11000%..10%%1 h% hF hF FFhFFFF s://academic.oup.com/humrep
0 0 100 200 300 400 0 0 50 100 150 200 250 /artic
le
P4-induced Δ fluorescence (%) P4-induced Δ fluorescence (%) -ab
s
tra
Figure4 [Ca2+]iresponsestohFFandprogesteronearesimilarbutnotidentical.(aandb)Showanexampleof[Ca2+]iresponsesinducedinpaired ct/3
experimentsusing(a)fourdilutionsofhFF(darkblue=0.01%,lightblue=0.1%,green=1%,red=10%)and(b)P4atconcentrationsequivalentto 2/1
thoseinthehFFdilutions(darkblue=2.8nM,lightblue=28nM,green=280nM,red=2.8μM).(candd)Showrelativeamplitudes(Δfluorescence 0/1
(ti%o)n)sooffthhFeF[(C0a.021+%]it=radnasireknbtslu(ec,)0a.n1d%[=Cali2g+h]tibplluaete,a1u%(d=,garseseenss,e1d0%10=mriendp)oasntd-stPim4autlactoionnc)einntdruatcieodnsineqseuviveanlesnettstootfheoxspeeirnimtheenthsF,Feadcilhutuiosninsg.Sfoixurdidffielur-- 995/4
0
enthFFsampleswereused.Lineineachgraphmarkspositionofequalresponseamplitude.AtthehighesthFFconcentrationused(10%;redsymbols), 9
6
plateauresponsesareconsistentlylargerthanthoseofequivalent[P4](P=0.001). 42
9
b
y
g
u
e
variable and the difference was not significant (Fig. 7a,b; P = 0.14). stimulateCatSperbyseparatemechanismsthatdonotcross-desensitize st o
After 5–10min exposure to 1% ShFF or equivalent [P4], oscillations (Schaeferetal.,1998),thiscouldreflectasmallcontributionofprosta- n
2
developedin~20%ofcells(Fig.7c,d),resemblingtheresponsetoP4 glandinstothehFF-induced[Ca2+]itransient.Wethereforeinvestigated 3 N
ramps(Harperetal.,2004). whetherthedesensitization-resistantcomponentofhFFwasremovedby ov
charcoal stripping. In six experiments ShFF always induced a [Ca2+] em
i b
EffectsofP4desensitizationon[Ca2+]i rsetismpuolnusse) (w1h1i.c5h±w2as.0%sigonfificthaanttlyevgorkeeadterby(Pthe=d2es.8e×ns1i0ti−zi5n)gt3haμnMtPh4e er 20
1
responsetohFF responsetoasecondstimulationwith3μMP4;Fig.6e,f. 8
Component(s)ofhFFnotremovedbycharcoalstrippingcontributesig-
nificantly to late/sustained components of hFF-induced [Ca2+] signals
i hFFandspermmotility
(Fig.6b).Tofurtherinvestigatethis,wetestedtheeffectofdesensitiza-
tionoftheP4responseonthe[Ca2+] signalinducedbyhFF.Asprevi- ToassessfunctionaleffectsofhFFonmotility,wemeasuredhyperactiva-
i
ouslydescribed(Aitkenetal.,1996;Schaeferetal.,1998),whensperm tion and penetration intoviscous medium. Both hFF (1 and 10%) and
were pre-stimulated with 3μM P4 complete desensitization occurred equivalent [P4] significantly stimulated penetration (P < 0.005; n = 6)
(Fig.6c).However,whenP4-desensitizedcellswerestimulatedwithhFF but the effect of hFF was significantly greater (Supplementary Fig. S4).
therewasaclearresponse(13.8±0.9%ofthatevokedbythepreced- hFFalsoinducedadose-dependentincreaseinhyperactivation,whereas
ing,desensitizingP4stimulus;P=3.2×10−5comparedtosecondstimu- theeffectofequivalent[P4]wassmallandnotsignificant(P<0.05;n=
lation with 3μM P4; n = 10; Fig. 6d,f). Since P4 and prostaglandins 6;SupplementaryFig.S5a).Analysisofthekinematics(VCL,ALH,LIN)
2002 Brownetal.
(a) (b)
350 3 μM P4 350 10% hFF
300 300
%) 250 %) 250
e ( 200 e ( 200
c c
n n
ce 150 ce 150
s s
e e
or 100 or 100
u u
Δ fl 50 Δ fl 50 D
o
0 0 wn
0 5 10 15 0 5 10 15 lo
–50 minutes –50 minutes ad
e
d
(c) P = 0.47 (d) P = 0.002 (e)60 P = 0.006 from
35 35 http
50 s
30 30 ng ://a
ells oscillating 122505 ells oscillating 122505 % cells oscillati 234000 cademic.oup.c
% c 10 % c 10 10 om
/h
5 5 0 um
0 0 M) %) rep/a
μ3 10 rtic
P4 ( FF ( le-a
b
s
sFaimguplreew5ereSeinxgpleosceedllt[oC3a2μ+M]irPe4sp(ao)nasnedst1o0%hFhFF.F(a(ba)n.dPabn)elSchoswhoewxsammpealens±ofS[ECMa2p+e]ricreensptaognesoesfcinellaspinawirehdichex[pCear2im+]eontscinillawtihoinchsoccecllusrferdomafttehrestsiammue- tract/32
lationofsperm(fromthesamesample)with300nMP4(black)or1%hFF(red);n=10pairedexperiments.Paneldshiowsresultsfromasimilarseries /10
of10pairedassessmentsusing3μMP4(black)and10%hFF(red;P<0.01).(e)Showsdatafromthe3μMP4/10%hFFexperimentssummarizedin /19
9
paneldwithpairedexperimentsjoinedandshowninsamecolour. 5
/4
0
9
6
4
2
9
indicatedthiseffectofhFFwasprimarilyduetoincreasedcurvilinearvel- depolarizingthemembranepotentialandpotentiallyaugmentingacti- b
y
ocity(P<0.01;SupplementaryFig.S5b). vationofCatSper.WecoulddetectnoeffectofhFFonconductance gu
e
orrestingVmevenwith50%hFF(containing10–15μMprogesterone; s
Fig.3).InpositivecontrolexperimentswithP4,wesawnosignificant t on
Discussion effect with 10μM but clear inhibition of conductance with 30μM P4 23
N
(equivalent[P4]to100%hFF;TableVI).ThuseffectsofhFFonKSper o
OurfindingsclearlyshowthatCatSperisactivatedbyhFFandthatthis ve
istheprimarycontributiontohFF-induced[Ca2+]isignallinginhuman mpoatyenotciaclluyrinatvehriyghcleorsecopnrocexnimtriatytiotonsthtehoanoctyhtoe.se used in this study, mbe
sperm. However, by direct comparison of responses to hFF and to r 2
0
equivalent[P4],charcoalstrippingofhFFanddesensitizationoftheP4 [Ca2+] signalsinducedbyhFF 18
response, we identified clear differences between the responses to i
hFFandtoP4whichindicatethatregulationof[Ca2+]ibyhFFisconsid- [Ca2+]itransientsinducedbytreatmentofhumanspermsuspensions
erablymorecomplexthansimpleactivationofCatSper. withhFFweresimilarinamplitudetothoseinducedbyanequivalent
[P4]andactivationofCatSperbyP4isapparentlytheprimarydeter-
minantofthisresponse.However,whenspermwerestimulatedwith
Modulationofionchannelactivity
and[Ca2+] byhFF 10% hFF, the sustained [Ca2+]i signal was >60% greater than that
i induced by an equivalent [P4]. Recently, Mannowetz et al. reported
The electrophysiological data clearly show that hFF, similarly to P4, that endogenous steroids other than P4 also modulate activity of
enhances CatSper currents and shifts CatSper voltage sensitivity to CatSperinhumansperm.17beta-estradiolandhydrocortisone,both
lesspositivepotentials(Fig.1;TablesIandII).Mannowetzetal.(2013) presentinhFF,inhibit thestimulatoryactionof1μM P4(IC =833
50
reportedthathighconcentrationsofP4alsoinhibitKSper(I ≈7μM), and 153nM, respectively) and their actions might be expected to
50
CatSperactivationbyhumanfollicularfluid 2003
(a) (b) 70
175
hFF(1%) 60 ShFF(1%)
150
e (%) 125 ShFF(1%) e (%) 50 Pdi4ff e[ereqnucivealent]
nc 100 nc 40
e e
c c
es 75 es 30
or or
Δ flu 50 Δ flu 20 D
25 10 ow
n
0 0 loa
0 150 300 450 0 150 300 450 de
–25 seconds –10 seconds d fro
m
(c) 350 (d) 350 http
s
Δ fluorescence (%) 11223050505000000 1asdtdition (P4) ad(2dPni4dti)on Δ fluorescence (%) 11223050505000000 1asdtdition (P4) a(2hdnFddFit)ion ://academic.oup.com/hum
re
0 0 p/a
–50 0 150seco3n0ds0 450 –50 0 150 300 450 rticle
seconds -a
b
(e) 350 (f) 100 stra
c
ce (%) 235000 1asdtdition (P4) 4 addition) 6800 t/32/10/199
n 200 P 5
Δ fluoresce 110500 2a(SdndhdFitFio)n stude (% 1 40 P < 5 × 10–5 /4096429 b
50 plit 20 y g
m ue
0 0 150 300 450 A 0 st on
–50 seconds 23
N
o
v
e
m
2F1igeuxpreeri6meCntosm(5podnifefenrtesnotfhtFhFeuhsFeFd-)inidnuwcehdich[Caali2q+u]oitssigfnraolmarteheressaismtaenstpteormP4sdaemspelnesittrizeaattieodnwanitdhc1h%arhcFoFal(rsetrdip)painndg.1(%a)SMhFeFan(b[lCuea)2.+(]bi)reMspeoanns[eCfar2o+m]i ber 2
0
responsefrom28pairedexperiments(9differenthFFused)inwhichaliquotsfromthesamespermsampleweretreatedwith1%ShFF(blue)orthe 1
equivalentconcentrationofP4(black).Greenshowsthe‘non-P4’componentobtainedbysubtractionoftraces.(c–e)Examplesof[Ca2+] responses 8
i
inthreeparallelrecordingswherespermwerefirststimulatedwith3μMP4(firstaddition-blacktraces)then,afteranintervalof5min,exposedto
either a second 3μM P4 stimulus (6μM P4 total; c, second addition-black trace), 1% hFF (d, second addition-red trace) or 1% ShFF (e, second
addition-bluetrace).Ineachpaneltheresponsestothefirst(3μMP4)stimulusandtothesecondstimulusareoverlaid(arrowattopleftshowstime
ofadditions).When3μMP4wasfollowedbyasecondP4stimulusthesecondresponsewasnegligible(desensitization).However,wheneither1%
hFFor1%ShFFwasaddedasthesecondstimulustherewasasmalltransientfollowedbyaplateau.(f)Meanamplitude(±SEM)of[Ca2+] transients
i
evokedbythefirst3μMP4stimulus(P4(1)black)andbyasecondadditionofP4(P4(2);n=7;black),hFF(hFF(2);n=10;red)orstrippedhFF(ShFF
(2);n=6;blue).AllamplitudesarenormalizedtothatinducedbythefirstP4additioninthatexperiment.
2004 Brownetal.
[P4], (after a latency of 5–10min) were similarly effective. However,
whenchallengedwith10%hFF,just4%ofspermgeneratedoscillations
(a) (b) comparedto19%with3μM(equivalent)P4(Figs5d,e),againsuggesting
50 that substances within hFF modulate human sperm Ca2+ signalling by
ShFF/progesterone %) 60 P = 0.14 mechanismsotherthanCatSperactivation.Darszonetal.assessed[Ca2
e (%) 40 mulus ( 50 +th]eianadcraocsroomsoemraelascttaiotunsa(Snádnccohenzc-luCdáerddetnhaastceatlcaiul.m, 2o0s1c4ill)a.tiIofntshseupspperermss
Δ fluorescenc 123000 0 min post-sti 234000 eohniobccitoy’tuaenctrceoorssmomphlieeghxr,ectahocintsicomenn.atyraitniohinbsito[Cf ah2F+F]iocnalcaiupmprooascchilliantgiotnhseancdum‘duilsuins-- D
1 o
e w
0 0 5 10 plitud 10 Cofhaanrcaocatilvsetr‘icpopcikntgaailn’idnehvFidFenceforpresence nload
–10 minutes Am 0 P4 ShFF TofurtherinvestigatetherelativecontributionsofP4andothercom- ed fro
ponents to the observed effects of hFF, samples were treated with m
(c)500 ShFF (d)35 P = 0.54 dperoxstrtaagnl-acnodaintesd), crehmarocovianlgtaolm‘sotsrtip9’9l%ipido-fdePr4iv.eInd flaugoorniimstestr(iscteerxopidesr/i- https://a
ce (%) 340000 s (%) 2350 mrneaeslpnwotsnasstheestigo[nCtifihace2a+rn]eitlstyirdagunraselieaPtn4et,rsbe(uFvtiogt.khee6dbs)ub.byFsSeuhqrFtuhFeenwrtmesoruersetca,oinwneshdiset[neCnawt2e+w]iiptshriega-- cademic
cen cell 20 treated sperm with P4 to desensitize the P4-induced [Ca2+] signal .ou
uores 200 ating 15 (Aitkenetal.,1996;Schaeferetal.,1998),wefoundthatasmaill,sus- p.com
Δ fl 100 Oscill 10 t6aci–nfe)d.TrehsepseonosbeseprevrastisiotendsiwndhiectahteerthstaimthuFlaFtiinngclwuditehshfaFcFtoorrsSthhFaFtc(oFing-. /hum
0 5 tributetoand/orregulateCa2+-signallingthatareresistanttostripping rep
–100 0 5minutes10 15 0 P4 ShFF woritphrdoestxatgrlaann-dcionast.edcharcoalandarethereforeunlikelytobesteroids /article
Figure7 Singlecell[Ca2+]iresponsesto1%ShFF.(a)Showsmean maTrhilyouaghrethspeo[nCsae2+to]itrreasnisdiueanltPin4du(cseedebaybo1v%e)S,hwFFhaepnpweaereidnvteostbigeapterid- -abstra
lreenstpo[Pn4se]s(btolac1k%; nSh=FF6(reexdp;enri=me1n0tse;x4p4e7rimceelnlst)s,;a8r2r6owcemlls)araknsdsetiqmuuivluas- effects on patch-clamped sperm we observed no stimulation of ct/32
addition.Bothstimuliinduceda[Ca2+] rampratherthanthebiphasic CatSper currents, suggesting that othercomponents of hFFmodulate /10
response seen in fluorimetric experimeints. (b) Shows mean (±SEM) theresponsetoP4.Twofactorsshouldbetakenintoaccountininter- /19
aremsppolitnusdeeso(Δf1fl2uiondreivsicdeunaclec)el9lsmstiinmuaflateterdstwimituhluSshFaFp,palricraotwionm.a(rck)sSshtiomwus- ppreertfuinsgiotnhetusbeindgat(aa.sFeivrisdte,nPc4edapbpyliereddubcyepdeerfffiucsaiocnyobfinPd4sitnootuhreimpalagsitnicg 95/409
lusaddition.Red,yellowandblackcellsdevelopedoscillations5–10min experimentsandalsoobservedbyothers;TStrunkerpersonalcommu- 64
laafttieornsstiamftuelratisotinm.u(dla)tisohnowwsithpr1o%poSrthioFFns(orefdc;elnls=gen1e0raetxinpger[Cimae2n+t]si;o8sc2i6l- ndiicreactitoand),dtithiousncoofmShpFaFritsoonthweiwthefllluinodriumceetdriacs[iCgnai2fi+c]ainatss[Cesas2m+]einrte,swpohnesree 29 by g
cells)orequivalent[P4](black;n=6experiments;447cells). (Fig.6),ismisleading.Thisisparticularlysignificantsincetheinhibitory ue
s
effect of hFF was masked at higher [P4] (Supplementary Fig. S2). t o
n
Second,divalentcationsinhFF(2.2mMCa,0.68mMMg;Chongetal., 2
3
result in a response to hFF smaller than that of an equivalent [P4] 1977;Ngetal.,1987)maybeinadequatelybuffered,maskinganystimu- N
(Mannowetzetal.2017).TheconcentrationofP4inhFF(typically> latoryeffect(IC forCa2+~100nM;Lishkoetal.,2011).However,(i) ov
50 e
30μM)maybehighenoughfortheseinhibitoryeffectstobeoutcom- in‘supplemented’controlexperimentswhereCa2+/Mg2+waspresent mb
e
peted(Mannowetzetal.,2017),butthestimulatoryeffectsobserved at equivalent levels to that in ShFF, responses to 2nM P4 resembled r 2
with10%hFFindicatethatothercomponentsofhFF,whenpresentat thoseseenin‘divalent-free’controls(TableV)and(ii)increaseddiva- 01
8
sufficient concentration, either activate (or suppress inactivation of) lent cation buffering (calculated [Ca2+] + [Mg2+] with 1% ShFF =
CatSperoractivateother[Ca2+] signallingcomponentsthatcontrib- 2.14nM) failed to rescue stimulation of CatSper currents by ShFF
i
utetothesustained[Ca2+] signal(seebelow). (Table V; Supplementary Fig. S2). We conclude that residual P4 in 1%
i
Single cell [Ca2+] responses to P4 resemble population responses ShFF(a[P4]sufficienttoactivateCatSperindivalentcation-supplemented
i
(transientandplateauphase;Kirkman-Brownetal.,2000)butsomecells controlrecordings(TableV)),whendeliveredbyperfusiontubing,failed
then generate repetitive oscillations (Fig. 5a; Harper et al., 2004; significantlytopotentiateCatSpercurrentandproposethatothersub-
Kirkman-Brownetal.,2004)thatmayregulatemotilityand/oracrosome stances present in hFF, resistant to charcoal stripping, partially inhibit
reaction(Harperetal.,2004;Bedu-Addoetal.,2007;Sánchez-Cárdenas the response of the channel to low (nM) concentrations of P4. Thus,
etal.,2014;Alasmarietal.,2013a,b).Inpairedexperiments,1%hFFand the slowly-developing ShFF-induced [Ca2+] ramp seen in imaging
i
300nMprogesterone(equivalentconcentration)bothinducedrepetitive experiments(Fig.7a,c)isapparentlyinducedindependentlyofCatSper
[Ca2+] oscillationsin~20%ofcells(Fig.5c),while1%ShFFandmatched activation.ThecomplexityofhFF,evenaftercharcoalstripping,issuch
i