Insulin-like growth factor 1 increases apical fibronectin in blastocysts to increase blastocyst ...

15 Pages·2014·1.14 MB·English

HumanReproduction,Vol.30,No.2pp.284–298,2015
AdvancedAccesspublicationonNovember28,2014 doi:10.1093/humrep/deu309
ORIGINAL ARTICLE Embryology
Insulin-like growth factor 1 increases
apical fibronectin in blastocysts
to increase blastocyst attachment
to endometrial epithelial cells in vitro
D
o
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CharmaineJ. Green1, Stuart T. Fraser1,2, andMargot L.Day1,* loa
d
e
12DDiisscciipplliinneeooffPAhnyastioolmogyya,nBdosHcihstIonslotigtuy,teS,ySdyndenyeMyMedeicdaiclaSlcShcohoolo,Ul,UninvievresritsyityoofSfSydydnneey,y,KK2255––MMeeddicicaallFFoouunnddaattiioonnBBuuiillddiinngg,,SSyyddnneeyy22000066,,AAuussttrraalliiaa d from
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*Correspondenceaddress.Tel:[phone];Fax:[phone];E-mail:[email protected] ttp
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://a
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SubmittedonJune23,2014;resubmittedonOctober14,2014;acceptedonOctober28,2014 a
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e
m
ic
studyquestion:Doesinsulin-likegrowthfactor1(IGF1)increaseadhesioncompetencyofblastocyststoincreaseattachmenttouterine .ou
p
epithelialcellsinvitro? .c
o
summaryanswer: m
IGF1increasesapicalfibronectinonblastocyststoincreaseattachmentandinvasioninaninvitromodelofimplantation. /h
what is known already: Fibronectinintegrininteractionsareimportantinattachmentofblastocyststouterineepithelialcellsat um
re
implantation. p
/a
studydesign,size,duration:Mouseblastocysts(hatchedornearcompletionofhatching)wereculturedinserumstarved(SS) rtic
le
mediumwithvaryingtreatmentsfor24,48or72h.Treatmentsincluded10ng/mlIGF1inthepresenceorabsenceofthePI3kinaseinhibitor -a
b
LY294002,anIGF1receptor(IGF1R)neutralizingantibodyorfibronectin.Effectsoftreatmentsonblastocystsweremeasuredbyattachment s
ofblastocyststoIshikawacells,blastocystoutgrowthandfibronectinandfocaladhesionkinase(FAK)localizationandexpression.Blastocysts trac
wererandomlyallocatedintocontrolandtreatmentgroupsandexperimentswererepeatedaminimumofthreetimeswithvaryingnumbers t/30
ofblastocystsusedineachexperiment.FAKandintegrinproteinexpressiononIshikawacellswasquantifiedinthepresenceorabsenceofIGF1. /2/2
participants/materials, setting, methods: Fibronectinexpressionandlocalizationinblastocystswasstudiedusingim- 84/7
munofluorescenceandconfocalmicroscopy.Globalsurfaceexpressionofintegrinavb3,b3andb1wasmeasuredinIshikawacellsusingflow 27
0
cytometry. Expression levels of phosphorylated FAK and total FAK were measured in Ishikawa cells and blastocysts by western blot and 59
imageJanalysis.BlastocystoutgrowthwasquantifiedusingimageJanalysis. by
g
mainresultsandtheroleofchance:ThepresenceofIGF1significantlyincreasedmouseblastocystattachmenttoIshikawa ue
s
cellscomparedwithSSconditions(P,0.01).IGF1treatmentresultedindistinctapicalfibronectinstainingonblastocysts,whichwasreducedby t o
n
thePI3kinaseinhibitorLY294002.ThiscoincidedwithasignificantincreaseinblastocystoutgrowthinthepresenceofIGF1(P,0.01)orfibro- 1
2
nectin(P,0.001),whichwasabolishedbyLY294002(P,0.001).Apicalexpressionofintegrinavb3,b3andb1inIshikawacellswasunaltered A
p
byIGF1.However,IGF1increasedphosphorylatedFAK(P,0.05)andtotalFAKexpressioninIshikawacells.FAKsignallingislinkedtointegrin ril 2
activationandcanaffecttheintegrins’abilitytobindandrecognizeextracellularmatrixproteinssuchasfibronectin.Treatmentofblastocystswith 0
1
9
IGF1beforeco-culturewithIshikawacellsincreasedtheirattachment(P,0.05).ThiseffectwasabolishedinthepresenceofLY294002(P,
0.001)oranIGF1Rneutralizingantibody(P,0.05).
limitations,reasonsforcaution:
Thisstudyusesaninvitromodelofattachmentthatusesmouseblastocystsandhumanendo-
metrialcells.Thisinvolvesaspeciescrossoverandalthoughthisusehasbeenwelldocumentedasamodelforattachment(ashumanembryo
numbersarelimited)theresultsshouldbeinterpretedcarefully.
widerimplicationsofthefindings:
ThisstudypresentsmechanismsbywhichIGF1improvesattachmentofblastocyststoIshi-
kawacellsanddocumentsforthefirsttimehowIGF1canincreaseadhesioncompetencyinblastocysts.Failureoftheblastocysttoimplantisthe
majorcauseofhumanassistedreproductivetechnology(ART)failure.Asgrowthfactorsareabsentduringembryoculture,theiradditionto
embryoculturemediumisapotentialavenuetoimproveIVFsuccess.Inparticular,IGF1couldprovetobeapotentialtreatmentforblastocysts
beforetransfertotheuterusinanARTsetting.
&TheAuthor2014.PublishedbyOxfordUniversityPressonbehalfoftheEuropeanSocietyofHumanReproductionandEmbryology.Allrightsreserved.
ForPermissions,pleaseemail:[email protected]
IGF1Increasesmouseblastocystattachmentinvitro 285
study funding/competing interest(s):
ThisworkwassupportedbyinternalfundsfromtheUniversityofSydneyandthe
BoschInstitute.Noneoftheauthorshasanyconflictofinteresttodeclare.
trialregistrationnumber:
N/A.
Keywords:PI3kinase/fibronectin/implantation/insulin-likegrowthfactor1/integrin
Introduction ThecurrentstudyinvestigatedtheroleofIGF1inattachment,usinga
well-characterizedinvitromodelofattachmentthatinvolvescultureof
Unsuccessfulembryoimplantationisthemajorcauseofhumanassisted blastocystsonIshikawacells.Ishikawacellsareawelldifferentiatedendo-
reproductivetechnology(ART)failure(Milleretal.,2012).Implantation metrial adenocarcinoma cell line (Nishida et al., 1985; Hannan et al.,
D
is a highly coordinated event in which the receptive endometrium is 2010)thatdisplaysapicaladhesiveness(Heneweeretal.,2005)anda o
w
primedtoreceiveadhesion-competentblastocysts.Growthfactorspro- similarintegrinexpressionprofiletoareceptiveendometrium,under nlo
ducedbyboththeembryoandthefemalereproductivetractarethought thecontrolofestrogenandprogesterone(Lesseyetal.,1996;Castel- ad
e
tsotatseu,pwphoircthdisesvyenlochprmoenniztedofwtihtheubtlearsitnoecryesctetpotiavintya,dtoheesnisounr-ecoblmaspteotceynstt banadumhuemtaanl.,(S1i9n9gh7)e.tEaml.,b2ry0o1s0;oKfadnifefekroenettaspl.,e2ci0e1s1ian,c2lu0d1in2g,2m0o1u3s;eK,arnagt d from
implantationability(Wangetal.,2000;Armant,2005).Therefore,an etal.,2014)areknowntoattachtoIshikawacells.Thesecharacteristics http
understandingofhowgrowthfactorsinfluenceimplantationisessential makeIshikawacellsaninvaluablemodelforthestudyofimplantationin s
toimproveART. vitro, especiallyasthe use of human embryosis limited byavailability ://ac
a
Insulin-likegrowthfactor1(IGF1)isoneparticulargrowthfactorthatis (Kang et al., 2014). In the present study we demonstrate that IGF1 d
e
presentatthematernal–embryointerfaceinanumberofmammalian increasedapicalfibronectinexpressiononblastocystsandattachment mic
species(Murphyet al., 1987; Kapuret al., 1992; Lighten et al., 1998; of blastocysts to Ishikawa cells, as well as blastocyst invasiveness. .o
u
p
SlaterandMurphy,1999).AtthetimeofimplantationintheratIGF1is TheseactionsofIGF1wereallpreventedbyinhibitionofthePI3K/Akt .c
o
stronglyexpressedinthebasallaminaandtheapicalsurfaceofuterine pathway.ThesedatasuggestanimportantroleforIGF1intheacquisition m
/h
epithelialcells,whicharethesitesoftrophoblastinvasionandattach- ofblastocystadhesioncompetenceduetoregulationoffibronectinex- u
m
ment, respectively (Slater and Murphy, 1999). Additionally IGF1 is pression.Furthermore,ourresultsindicatethatadditionofIGF1tothe re
p
secretedbymouseembryos(Inzunzaetal.,2010).TheIGF1receptor culturesystemmayimprovethesuccessofhumanassistedreproduction /a
(IGF1R)isexpressedthroughoutmousepreimplantationstagesofdevel- byimprovingtheadhesioncompetenceofblastocysts. rtic
le
opment(Inzunzaetal.,2010)andisexpressedapicallyontrophoblast -a
b
cells(Bedzhovetal.,2012),whicharethecellsthatmakefirstcontact Materials and Methods stra
with the uterine epithelium. High levels of IGF1, which cause IGF1R c
t/3
down-regulation,decreasenormalblastocystimplantationsitesandin- 0
Useofanimalsandethicalapproval,ovulation /2
creaseresorptionsites(Pintoetal.,2002).Additionally,beadssoaked /2
inductionandembryocollectionandculture 8
inIGF1elicitadecidualresponseintheuterusthatmimicsthatofablasto- 4
/7
cyst(Pariaetal.,2001).DespitethisknowledgeofIGF1/IGF1Rexpres- Proceduresinvolvingtheuseofanimalswereconductedinaccordancewith 27
0
sion in the embryo and uterine tissue, it is not knownwhether IGF1 theAustralianCodeofPracticeforUseofAnimalsinResearchandwere 5
9
affectsblastocyst adhesion competencyor the mechanisms by which approvedbytheUniversityofSydneyAnimalCareandEthicsCommittee. b
y
IGF1influencesearlyimplantation. TheQuackenbushSwiss(QS)strainofmicewasused(AnimalResource gu
Centre,Perth,Australia).Micewerehousedundera12-hlight:12-hdark e
Acquisition of adhesion competence by the blastocyst involves an cycle (light; 06:00–18:00), and housed in a conventional holding facility st o
accumulationofintegrinsontheapicalsurfaceoftrophoblastcells.Im- n
where the temperature was maintained at (cid:2)20–228C and the humidity 1
plantationdepends,inpart,onthebridgingbetweenintegrinsonthe 2
kept at 55%. Mice had free access to water and commercially prepared A
endometriumandtheblastocystbyextracellularmatrix(ECM)proteins p
suchasfibronectin(Kanekoetal.,2013).Integrina5b1subunitsincrease pcaeglleedts.siFnegmly.alOevmuilcaetiowneirneduccatgieodnionfgfreomuaplseomfic1e0(w4h–il1s0t mwaeleekms oicled)wweraes ril 20
1
ontheapicalsurfaceofadhesioncompetentmouseblastocysts,andthis achieved by intraperitoneal injection of 10I.U. of pregnant mare serum 9
correlateswithanincreasedabilitytobindfibronectin(SchultzandArmant, gonadotrophin(PMSG;Intervet,Sydney,Australia)followed48hlaterby
1995;Schultzetal.,1997;Wangetal.,2002).MostECMproteinsbindto intraperitonealinjectionof10I.U.ofhCG(Intervet)aspreviouslydescribed
multipleintegrins,forexamplefibronectin,whichbindsthea5b1,avb3 (Nasr-Esfahanietal.,1990).Superovulatedfemalemicewerethenpaired
andaIIb3integrinsinmouseblastocysts(Sutherlandetal.,1993;Schultz withastudmaleQSmouse(10–30weeksold)overnight.Thepresenceof
avaginalplugthefollowingdayindicatedsuccessfulmatingandthiswascon-
andArmant1995;Yelianetal.,1995;Schultzetal.,1997).
sideredtobeDay1ofpregnancy.
Theprocessofintegrinactivation,whichoccursviainside-outsignal-
FemalemiceweresacrificedonDay4ofpregnancy,90–94hafterhCG
ling,regulatesintegrinaffinityforECMproteins.Thisinside-outsignalling
administration, in order to recover early blastocysts. The oviducts and
ismediatedbyanumberofsignallingpathways,suchasthePI3kinase
uterinehornswereflushedwithHepes-bufferedmodifiedsynthetichuman
(PI3K)/Aktpathway(Somanathetal.,2007).AktisactivatedbyIGF1 tubalfluidmedium(Hepesmod-HTF; (cid:2)300mosM/l,pH7.4)containing
inmouseblastocysts(GreenandDay,2013)andtreatmentoftropho- 0.3mg/ml bovine serum albumin (BSA; Sigma-Aldrich; St Louis, MO,
blast cells with IGF1 increases fibronectin binding mediated by a5b1 USA).Blastocystswerethencollectedandculturedfor24–28h,toenable
andavb3(Kabir-Salmanietal.,2002,2003,2004). hatchingfromthezonapellucida(Day5Blastocysts),inPotassiumsimplex
286 Greenetal.
optimizedmedium(KSOM;(cid:2)260mosM/l,pH7.4)containing0.3mg/ml 500mMtoinduceapoptosisinblastocystsandalthough250mMdidnotin-
BSA. All media was made up from stock solutions prepared from tissue creaseapoptosisinblastocysts(Rileyetal.,2006),thisconcentrationwas
culturegradereagents(Sigma-Aldrich)aspreviouslydescribed(Greenand showntodecreaseblastocysthatching(Rileyetal.,2005).Inthepresent
Day,2013).AllblastocystsusedinthisstudywerecollectedonDay4and studywechosetousea5mMLY294002,aconcentrationmuchcloserto
allowed to hatch overnight before treatment (all blastocysts used were theIC50forthedrug.
hatchedornearcompletionofhatching).
RecombinantmouseIGF1(R&DSystems;Minneapolis,MNUSA)was Ishikawacellculture
reconstituted at 100mg/ml in sterile phosphate-buffered saline (PBS;
TheIshikawacelllinewasagiftfromProfessorChrisMurphy,Universityof
AMRESCO;Solon,OH,USA).MaturemouseIGF1shares94%aminoacid
Sydney.Cellswerethawedandplatedon10mmglasscoverslips(Menzel
sequencehomologywithhumanIGF1andexhibitsspeciescrossreactivity
Glaser;Braunschweig,Germany)in24-wellplates(Corning,NY,USA)for
(Belletal.,1986).Specifically,R&DSystemstesttheirmouseIGF1activity
co-cultureexperimentsordirectlyonto6wellpates(Corning,NY,USA)
onhuman MCF-7breastcancercellproliferation.Thusmouse IGF1was
for western blot analysis with Dulbecco’s modified Eagle’s medium
usedinalloftheexperiments.IGF1at10ng/mlhasbeenusedtoseepositive
(DMEM; GIBCO, Grand Island, NY, USA) containing 10% (v/v) fetal D
effectsonembryodevelopment(GreenandDay,2013).Higherconcentra- o
bovineserum(FBS;BovogenBiologicalsPtyLtd,Essendon,VIC,Australia) w
tions,suchas30,100and1000ng/mlIGF1,areknowntohavedetrimental n
and5000I.U.ofpenicillinand5000mgofstreptomycinperml(Invitrogen, lo
effectsonblastocystsandcauseIGF1Rdown-regulation(Chietal.,2000; a
Camarillo,CA,USA). d
Green and Day, 2013), and this range of IGF1 concentrations has been ed
usedinthisstudytoinvestigatethemechanismsofIGF1inattachmentof Co-cultureofmouseblastocystswithIshikawa fro
m
theembryotouterineepithelialcells. cellsinthepresenceandabsenceofIGF1 h
TodetermineadirecteffectofIGF1throughtheIGF1R,anIGF1Rneutral- ttp
s
izingantibody(IGF1RnAb;AF-305-NA;R&DSystems)wasreconstitutedat Attachmentexperiment1(Fig.1)wasperformedwithco-cultureofIshikawa ://a
0.2mg/mlinPBSandusedatafinalconcentrationof2mg/ml. cellsandblastocystsinthepresenceandabsenceofIGF1.Ishikawacellsthat c
a
LY294002isaspecificinhibitorofPI3KwithanIC50of1.40mM(Vlahos werenearconfluencewereculturedineitherserummedium(containing10% de
m
et al., 1994). Previously LY294002 has been used at concentrations of FBS)orinserumstarved(SS)medium(0.01%FBS)for2hpriortoreceiving ic
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Figure1 TimelineofexperimentsusedtostudyattachmentofmouseblastocyststohumanIshikawacellsinvitro.BL:blastocyst.
IGF1Increasesmouseblastocystattachmentinvitro 287
blastocysts.After2h,SSIshikawacellswerethenculturedinthepresenceor or the blastocysts, at 2.5mm intervals. This provides us with sections
absenceof10ng/mlIGF1.Blastocystswereisolatedfromsixmiceineach throughout the whole blastocyst so that we can visualize the inner cell
experiment.Between4and12Day5blastocystsweretransferredperwell mass (ICM) in relation to the rest of the embryo.Images were analysed
ontotheIshikawacellsineachofthetreatmentgroups.Blastocystswereran- usingLSMImageBrowsersoftware(CarlZeiss)andthelocalizationofpro-
domlyallocatedtotreatmentgroupsandtreatmentgroupswereperformed teinsisdescribedinrelationtotheICM.Anunbiasedthirdpartyperformed
induplicate.Initially,co-cultureswereincubatedundisturbedfor24hand ablindedanalysisoftheimmunofluorescenceimages.
checkedforattachment.Howeverasnoblastocystshadadheredat24h
(uptoDay6),co-cultureswereincubatedundisturbedfor48h(uptoDay7). Westernblotanalysis
BlastocystattachmenttoIshikawacellswasdeterminedat48hbyblowing
AllwesternblotdataonIshikawacellsarefromIshikawacellsculturedinthe
mediawithaglassmouthpipetteattheblastocystswhilstexaminingtheir
absenceofblastocysts.NearconfluentIshikawacellswereallocatedtoserum
positionunderadissectingmicroscope(WildM3microscope;LeicaMicro-
mediumorSSmediuminthepresenceorabsenceof10ng/mlIGF1for48h.
systems,Wetlar,Germany).Embryosthatdidnotfloatawaywereconsid-
Similarlyallwesternblotsonblastocystswereperformedintheabsenceof
eredto have attached(Singh et al., 2010).Co-culture experiments were
Ishikawacells.Blastocystswereisolatedfromatleasteightmiceineachex- D
performed in duplicate and repeated three separate times. Data were o
periment.Day5blastocystswereallocatedrandomlytoserummediumor w
pooledfromthethreeseparateexperimentsandcountedastheproportion n
SSmediuminthepresenceorabsenceof10ng/mlIGF1andculturedfor lo
ofblastocyststhathadattachedtotheIshikawacellsoutofthetotalnumber a
48honnon-adherentculturedishes(Corning).After48h,cellsorblasto- d
ofofrbelaascthoceyxsptsearidmdeendttaolgtrhoeuIsph.ikawacells.Between14and19micewereused cByesvtesrwly,erMeAw,asUhSeAd)in+c1olmdMPBSPManSdFly(Ssiegdmwa)i.thCloyslliescbtiuofnfesrw(CeerellSriegpnealalitnegd, ed from
until there were 120 embryos to run per lane, per experiment. Protein h
Pretreatmentofblastocystsbeforeco-culture assayswerecarriedoutonIshikawacellsaccordingtothemanufacturer’s ttp
s
withIshikawacells instructions(DCproteinassaykit,Bio-Rad).Proteinsampleswerediluted ://a
with6×Laemmlibuffer(35mMTris–HCl,pH6.8,10.28%(w/v)sodium ca
Attachmentexperiment2(Fig.1)wasperformedbypre-treatingblastocysts d
beforetheirco-culturewithIshikawacells.Blastocystswereisolatedfrom dodecyl sulphate (SDS), 36% (v/v) glycerol, 0.05% (w/v) bromophenol em
blue;Sigma)andheatedfor5minat1008C.Proteinswereelectrophoresed ic
between5and10miceineachexperiment.Day5blastocystswerecultured .o
onan8%SDS–polyacrylamidegel,transferredtoanitrocellulosetransfer u
in the presence of serum, with or without 2mg/ml IGF1R nAb or in SS p
membrane (Hy-BLOT, Australia) and then incubated in blocking buffer .c
medium in the presence or absence of 10, 100 or 1000ng/ml IGF1 or o
(Odyssey; Li-cor Biosciences, Lincoln, NE, USA) overnight at 48C with m
with 10ng/ml IGF1 in the presence of 2mg/ml IGF1R nAb or 5mM /h
gentle shaking. Membranes were probedwith primaryantibodies (rabbit u
LY294002(PI3kinaseinhibitor;Selleck,Houston,TX,USA)for24h.Blasto- m
cysts(nowday6blastocysts)werethenwashedandculturedonIshikawa anti-E-cadherin;NovusBiologicals;#NB110-56937,rabbitanti-pFAK;Invi- re
p
cellsfor24hinSSmediumandassessedforattachmentasabove.Experi- trogen; #44-624G, or rabbit anti-integrin b3; Santa Cruz Biotechnology; /a
mentswererepeatedthreetoninetimesandthedatapooledtocalculate #sc-14009ormouseanti-a-tubulin;Sigma;#T9026)overnightat48Cin rtic
theproportionofattachedembryosforeachtreatment. blocking buffer+0.1% Tween-20. Membranes were washed in Tris- le-a
buffered saline+Tween 20 (TBST; 10mM Tris–HCl, pH 7.6, 150mM bs
Immunofluorescence NaCland0.1%Tween20)andsubsequentlyincubatedfor2hwitheither trac
1:4000Donkeyanti-RabbitIRDye800CW(Odyssey)or1:4000Donkey t/3
Co-cultures,fromattachmentexperiment1wereexaminedfortotalfocal anti-Mouse IRDye 680LT (Odyssey). Proteins were visualized using the 0/2
adhesionkinase(FAK)proteinexpression.Blastocystswereisolatedfrom Odyssey infrared imager and Odyssey application software, version 3 /28
betweenthreeandfivemiceineachexperiment.Day5blastocystswerecul- (Odyssey).DensitometrywasperformedusingImage-Jsoftware(National 4/7
turedintheabsenceofIshikawacellsatlowembryodensityinSSmediumin InstitutesofHealth,Bethesda,MD,USA)orOdysseyprogram. 27
0
thepresenceorabsenceof10ng/mlIGF1or5mMLY294002andwere 5
9
examinedforfibronectinexpressionat24,48or72h.Co-culturesorsepar- Flowcytometry b
y
ately cultured blastocysts were fixed in 4% paraformaldehyde (PFA) for g
15min,washedinPBS+1mg/mlPolyvinylalcohol(PBS+PVA)andthen Surfaceexpressionofintegrinavb3,b3andb1onIshikawacells,culturedin ue
s
permeabilisedwithPBS+PVA+0.3%TritonX100for30minandthen theabsenceofblastocysts,wasanalysedbyflowcytometryafter48hculture t o
washedwithPBS+PVA.BlockingwascarriedoutinPBS+PVA+0.1% inserummediumorSSmediuminthepresenceorabsenceof10ng/mlIGF1. n 1
Tween-20+0.7% BSA (PPTB) for 30min. Primary and secondary anti- Cellswereremovedfromthecultureplatewith0.5%trypsin(LifeTechnol- 2 A
ogies).Cellswereresuspendedin600mlofPBS+0.3%BSAandincubated p
bbooddiieessw[rearbebidtiluatnetdi-inintePgPriTnBb.S3a;mSpalnetsawCerruezinBciuobteactehdnowloitghy;pr#imsca-r1y4a0n0t9i-, withprimaryantibodiespre-conjugated(1:200anti-humanintegrinavb3; ril 20
eBioscience;SanDiego,CA,USA;#11-0519-41,anti-humanintegrinb3; 1
rabbitanti-FAK;Sigma;#F-2918orrabbitanti-fibronectin(unattachedblas- 9
eBioscience; #11-0619-41 or anti-human integrin b1; eBioscience;
tocysts);NovusBiologicals,Littleton,CO,USA;#NBP1-91258,orrabbit
#11-0299-41)orisotype controlantibodies(ArmenianhamsterIgG/Rat
anti-fibronectin antibody (for blastocysts attached to coverslips); Calbio-
IgG2a/2b; Biolegend; SanDiego, CA,USA;#78023) for30minonice.
chem,EMDChemicals,Gibbstown,NJ,USA]overnightat48Candthen
Sampleswerethenwashedandresuspendedin200mlPBS+0.1%(v/v)
washedinPPTBbeforeincubationwiththesecondaryantibody(anti-rabbit
propidium iodide (Sigma). Analyses were performed using the FACS
Alexa488;LifeTechnologies,Carlsbad,CA,USA)for2hatroomtempera-
Calibur (Becton Dickinson, San Jose, CA, USA) and Cell Quest (Becton
ture.SampleswerethenwashedthreetimesinPPTB,withthefinalwash
Dickinson)andFlowJo(Treestar,SanCarlos,CA,USA)softwarepackages.
lasting10minandthenmountedin5mlVectashieldcontaining1.5mg/ml
4′,6′-diamidino-2-phenylindole (DAPI; Vector Laboratories, Burlingame, Aminimumof10000cellswereanalysedpersample.
CA,USA).Isotypecontrolswereperformed,inwhichcellswereincubated
Blastocystoutgrowth
withpurifiedrabbitIgG(Sigma)inplaceoftheprimaryantibody.Fluores-
cencewasvisualizedusingtheLSM510Metaconfocalmicroscope(Carl Blastocystswereisolatedfromatleastfivemiceineachexperiment.Day5
Zeiss,Germany)usinga405nmlaserandArgonlaser(458,477,488and blastocystswereculturedon24-wellplates(Corning)thatwereeitherun-
514nmlines)at40×objective.AZ-stackwastakenthroughtheco-cultures coated,orcoatedwithfibronectin.Tocoatwells,fibronectinwasmadeto
288 Greenetal.
aconcentrationof25mg/mlinPBS,andenoughsolutionwasaddedtocover
eachwellandleftat48Covernight.Blastocystswereallowedtooutgrow,un-
disturbedfor72hinserummediumorinSSmediuminthepresenceor
absenceof10ng/mlIGF1withorwithout5mMLY294002(Selleck).At
72hblastocystoutgrowthsweremicrographedontheAxiovert35micro-
scope (Carl Zeiss) using a Axiocam ICc5 camera (Carl Zeiss) and Zen
imagesoftware(CarlZeiss).Forthoseblastocyststhatwereattachedto
thewell,theareaofoutgrowth(arbitraryunits),alongwithblastocystarea
was measured using Image-J software (National Institute of Health) by
tracing around the outgrown cells using the freehand area selection tool
andthenthewandtooltoobtainanareavalueinarbitraryunits.Experiments
wererepeatedthreetoeighttimesandthedatapooledtocalculatethe
averageareaofoutgrowth.
D
o
w
n
Statisticalanalysis lo
a
d
Embryoscollectedfrommultiplemicewererandomlyallocatedbetween Figure2 Insulin-likegrowthfactor1(IGF1)improvesattachmentof ed
treatmentsforallexperiments.Chi-squaredanalysisoftheoverallpropor- mouseblastocyststoIshikawacellsinvitro.Attachmentofblastocyststo fro
m
tionofblastocystsattachedtoIshikawacellco-cultureswasperformedon Ishikawacellsat48haftercultureinserummediumorserumstarved h
pooledproportiondatafromthreeseparateexperiments(MicrosoftOffice (SS) medium in the presence or absence of 10ng/ml IGF1. Results ttp
s
Excel,Berkshire,UK).Westernblotanalysiswasperformedthreetimes.The aredisplayedasthepercentageattachmentofblastocyststotheIshi- ://a
opticaldensityofproteinbandswasnormalizedtoa-tubulinandexpressed kawacells.Nvaluesinparenthesesrepresentthetotalnumberofblas- c
a
relativetothecontrol,followedbystatisticalanalysisusingunpairedt-tests tocysts added to the Ishikawa cells, pooled from at least three de
m
(Microsoft Office Excel). Flow cytometry analysis was performed three experiments.Chi-squareanalysiswasusedtocomparethetreatment ic
times,followedbystatisticalanalysisoftheaveragefluorescenceintensity, groups.**indicatesP,0.01. .o
u
usingunpairedt-tests(MicrosoftOfficeExcel).Embryooutgrowthexperi- p.c
o
mentswereperformedatleastthreetimes,followedbystatisticalanalysis m
usingKruskal–WallistestfollowedbyDunnmultiplecomparisontestfor /h
non-parametricdata(GraphPadPrismversion6.00forWindows,GraphPad AlthoughIGF1hadnoeffectontotalexpressionofintegrinb3inIshi- um
kawa cells, it is the integrin expression on the apical surface that is re
Software, La Jolla, CA, USA, www.graphpad.com). A P,0.05 was p
consideredstatisticallysignificant. rseioqnuiorefdafvobr3in,tber3acatniodnbw1ithinEteCgMrinpsrowtaesinisn.vTehsteigraetfoedre,insuIsrfhaickeawexapcreelsls- /article
using flow cytometry. Integrins avb3, b3 and b1 were all expressed -ab
s
Results onthecellsurfaceinIshikawacells,asindicatedbythegreaterfluores- tra
c
cence seen with the presence of each antibody compared with the t/3
EffectofIGF1onattachmentofmouse isotypecontrol(Fig.3C).However,therewasnodifferenceinsurface 0/2
blastocyststoIshikawacellsinvitro expressionofintegrinsavb3,b3orb1betweenanyofthetreatment /28
4
groups(Fig.3C). /7
Blastocystattachmentwasassessedinthepresenceorabsenceof10ng/ml 2
7
0
IGF1(attachmentexperiment1)after24and48h,beforefixationwith 5
9
PFA(Singhetal.,2010).At24hnoblastocystshadattachedtotheIshi- b
y
kawacellsinanyofthetreatmentgroups(datanotshown).Therefore EffectofIGF1onphosphorylationofFAKand gu
e
attachment was assessed at 48h. The proportion of blastocysts that s
totalFAKexpressionlevelsinIshikawacells t o
attached to Ishikawa cells under SS conditions (27 attached/50) was n
andmouseblastocysts 1
reduced compared with attachment in the presence of serum (39 2
A
attached/47; Fig.2). The presence of IGF1increasedthe proportion FAK signalling is linked to integrin activation (Kornberg et al., 1992). p
ofblastocystattachment(IGF1:39attached/46)comparedwithattach- Therefore,activationofFAKsignallingpathways,indicatedbyphosphor- ril 2
0
mentinSSconditions(Fig.2),tothelevelseeninthepresenceofserum. ylation of FAK (p-FAK) was investigated by western blot analysis in 19
Ishikawacells and blastocysts.Phosphorylationof FAK wasincreased
in Ishikawa cells after 48h treatment with 10ng/ml IGF1 (Fig. 4A).
EffectofIGF1onE-cadherinandintegrinb3
ImmunofluorescencewasusedtoshowthattotalFAKexpressioninIshi-
proteinlevelsandintegrinavb3,b3andb1
kawacellswasincreasedontheapicalsurfaceinco-culturesaftertreat-
surfaceexpressioninIshikawacells mentwithIGF1for48h.Similarly,totalFAKexpressionwasincreasedin
AsIGF1increasedattachmentofblastocyststoIshikawacells,theeffect blastocysts that were attached to Ishikawa cells in co-cultures in the
ofIGF1onseveraladhesionproteinswasinvestigatedinIshikawacells presenceofIGF1incomparisontoco-culturesintheabsenceofIGF1
bywesternblot.TherewasnodifferenceinE-cadherinexpressionin (Fig.4B).TherabbitIgGisotypecontrolhadnodetectableimmunofluor-
Ishikawacellsbetweentheserum,SSorSSplusIGF1treatmentgroups escence staining in either the blastocyst or Ishikawa cells (data not
(Fig.3A).Furthermore,IGF1hadnoeffectonintegrinb3expression shown). Western blots were performed on unattached blastocysts
inIshikawacells;however,expressionofintegrinb3wasincreasedin andshowednoeffectonphosphorylationofFAKortotalFAKlevelsin
thepresenceofserum(Fig.3B). blastocystsafterIGF1treatmentfor48h(Fig.4CandD).
IGF1Increasesmouseblastocystattachmentinvitro 289
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Figure3 IGF1hasnoeffectonE-cadherinorintegrinb3proteinlevelsorintegrinavb3,b3andb1surfaceexpressioninIshikawacells.Ishikawacells
wereculturedfor48hinserummediumorSSmediuminthepresenceorabsenceof10ng/mlIGF1.(A)RepresentativewesternblotshowingE-cadherin
expression.Westernblotswereprobedforanti-E-cadherin(green)andanti-a-tubulin(red).GraphshowingaverageE-cadherinbandintensities(+SEM,
n¼3)normalizedtoa-tubulinandexpressedrelativetotheSScontrol.(B)Representativewesternblotshowingintegrinb3expression(green)and
a-tubulin(red).Graphshowsaverageintegrinb3bandintensities(+SEM,n¼3)normalizedtoa-tubulinandexpressedrelativetotheSScontrol.Analysis
ofvariancewithTukey’sposthocanalysiswasusedtocomparethetreatmentgroups.*indicatesP,0.05(C)Representativeflowcytometryhistogram
showingsurfaceexpressionofintegrinavb3,b3andb1inIshikawacells.MW:molecularweight.
290 Greenetal.
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Figure4 IGF1increasesfocaladhesionkinase(FAK)phosphorylationandtotalFAKexpressionlevelsinIshikawacells.Ishikawacellsorblastocystswere A
p
pcureltsusrieodnfionrI4sh8ikhaiwnaSScemlles.diWumesintetrhnebplroetssewnceereorparobbseendcfeoorfa1n0ti-npg-/FmAlKIG(gFr1e.e(nA))aRnedparenstie-nat-attuivbeulwine(srteedrn).bGlortasphhoswhionwgpshaovseprahgoerypla-yFeAdKFbAaKnd(pi-nFtAenKs)iteiexs- ril 20
1
(+SEM,n¼3)inIshikawacells,normalizedtoa-tubulinandexpressedrelativetotheSScontrol.(B)Representativeconfocalimageshowingtotal 9
FAKexpressionandlocalization(green)in blastocysts attachedtoIshikawacells.Plain arrowsindicateFAKstainingonIshikawaapical cellsurface.
DottedarrowindicatesincreasedFAKstainingonIGF1treatedblastocyst.Scalebarsrepresent20mm.Nineattachmentsiteswerestainedacross3experi-
mentsintheSSgroupand10attachmentsiteswerestainedacross3experimentsintheSSplusIGF1group.(C)Representativewesternblotshowingp-FAK
expressioninblastocysts.Westernblotswereprobedforanti-p-FAK(green)andanti-a-tubulin(lowerpanel).Onehundredandtwentyblastocystswere
runineachlane.Graphshowsaveragep-FAKbandintensities(+SEM,n¼3)inblastocysts,normalizedtoa-tubulinandexpressedrelativetotheSS
control. (D) Representativewesternblot showing total FAK expression in blastocysts. Western blots were probed for anti-total-FAK (green) and
anti-a-tubulin(lowerpanel).Onehundredandtwentyblastocystswererunineachlane.GraphshowsaveragetotalFAKbandintensities(+SEM,
n¼3)inblastocysts,normalizedtoa-tubulinandexpressedrelativetotheSScontrol.Student’sT-testwasusedtocomparethecontroltothetreatment
groups.
IGF1Increasesmouseblastocystattachmentinvitro 291
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Fibronectinlocalizationinearlyandlate 70
fibronectin expression. The localization at 48h was comparable 5
stageblastocysts between the SS control and IGF1 group. At 72h (Day 8 blastocyst), 9 b
y
FibronectinisanimportantECMproteinthatissecretedbyblastocysts there was a distinct polarization of fibronectin in the trophectoderm gu
e
butnotbyIshikawacells(Kanekoetal.,2013).Theexpressionandlocal- ononesideoftheblastocystandthelocalizationwascytoplasmicand st o
izationoffibronectininblastocystswasinvestigatedbyimmunofluores- punctate.TherewasnostainingintheICM(Fig.5EandF).At72hin n
1
cenceover72hofculture,inthepresenceorabsenceofIGF1.Here theIGF1treatedgroup,fibronectinstainingwasofamuchhigherinten- 2 A
wedescribethechangeinfibronectinexpressionovertimeaswellas sity,comparedwiththeSSgroup(Fig.5EversusF).Additionally,IGF1 p
anIGF-inducedincreaseinfibronectinexpressionovertime.Day5blas- treatment resulted in a distinct apical localization of fibronectin. The ril 2
0
tocystswereculturedinSSmediuminthepresenceorabsenceof10ng/ polarizedfibronectinstainingseenat48and72hinblastocystspartially 19
mlIGF1andcollectedat24,48and72handthenimmunostainedfor coversthetrophoblastcellsattheembryonicpoleandextendsfromthe
fibronectin.At24h(nowday6blastocyst),fibronectinwaslocatedin embryonicpoletotheabembryonicpoleononesideoftheblastocyst.
thecytoplasm,nucleus,cell–celljunctionsandonthebasalandapical ThisisthesidethatattachedtoIshikawacellsinvitro.
surfaceofthecellsinthetrophectoderm(Fig.5AandB).TheICMalso The addition of the PI3 kinase inhibitor, 5mM LY294002, to IGF1
expressed fibronectin but this was restricted to the cells facing the treatedblastocystsaffectedtheoverallpolarizationoffibronectinand
blastocoel.IGF1treatmenthadnoeffectonfibronectinlocalizationor comparedwiththeintenseapicalstainingseeninIGF1treatedblasto-
stainingintensityat24h.IGF1treatmentincreasedfibronectinstaining cyststhelocalizationoffibronectininLY294002treatedblastocystsin
intensityat48hcomparedwiththeSScontrol(Fig.5CversusD).At thepresenceofIGF1,wasmorecytoplasmicandstrongstaininginthe
48h (Day 7 blastocyst), fibronectin was located in the cytoplasm, cell–celljunctionswaspresent(Fig.5FversusG).
nucleus,cell–celljunctionsandonthebasalandapicalsurfaceofthe Toensurethepolarizedfibronectinstainingwasfunctionallyrelatedto
trophectoderm(Fig.5CandD)andthisstainingwaspolarizedtoone attachment,blastocyststreatedwithIGF1thathadadheredtoglasscov-
side of the blastocyst. At 48h the majority of ICM cells had lost erslips(72h)wereimagedforfibronectinstaining.Theattachedsurface
292 Greenetal.
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IGF1Increasesmouseblastocystattachmentinvitro 293
of theblastocystexpressedfibronectin. Thesurfaceof theblastocyst withthePI3KinhibitorLY294002inthepresenceorabsenceofIGF1
facingawayfromthecoverslipdidnotexpressfibronectin(Fig.5H). reducedblastocystattachmenttoIshikawacellscomparedwithSSor
IGF1 pretreatment conditions, respectively (Fig. 7E). Pretreatment of
EffectofIGF1onblastocystoutgrowth blastocystswithhigherconcentrationsofIGF1(100and1000ng/ml)
didnotincreaseblastocystattachmentcomparedwiththeSScontrol.
Fibronectin is known to increase blastocyst outgrowth in serum free
medium to the level seen in the presence of serum (Armant et al.,
1986).Thereforeblastocystoutgrowthwasmeasuredafter72h(now Discussion
day8blastocysts)ofcultureoneitheruncoatedwellsorwellscoated
with fibronectin, in serum medium or SS medium in the presence or Anumberofgrowthfactorsandtheirreceptorsareexpressedbyboth
absenceof10ng/mlIGF1or5mMLY294002.Cultureofblastocysts thepreimplantationembryoandthereproductivetractandtheseinflu-
inserummediumsignificantlyincreasedtheaverageareaofblastocyst ence preimplantation embryo development and implantation (Hardy
outgrowth compared with SS blastocysts and blastocysts cultured in andSpanos,2002;Armant,2005).Inparticular,growthfactorssuchas D
o
thepresenceofIGF1(Fig.6H)andthiswasseenasanincreaseintroph- IGF1,whichareproducedbytheembryoandattheimplantationsite, wn
ectodermspreadingacrosstheculturedish(Fig.6A–C).UnderSScon- arethoughttoplayaroleintheco-ordinationofblastocystimplantation loa
d
ditions blastocyst outgrowth was increased by the presence of IGF1 abilitywithuterinereceptivity(Wangetal.,2000;Armant,2005).Inthe ed
whenblastocystswereculturedonuncoatedwells(Fig.6B,CandH). presentstudyweusedaninvitromodelforembryoattachmenttoinves- fro
m
Blastocyst outgrowth in SS media was increased when blastocysts tigate the effect of IGF1 on mouse blastocyst adhesion competency. h
wereculturedonfibronectincoatedwells(Fig.6B,DandH).Theout- Co-cultureofblastocystswithIshikawacellsfor48h,underSScondi- ttp
s
growthofblastocystsinthepresenceofIGF1wasnotimprovedbyfibro- tions, decreased blastocyst attachmentcomparedwith attachment in ://a
c
nectin(Fig.6CversusEandH).OutgrowthinthepresenceofIGF1and thepresenceofserum,whichiswellknowntocontainalargenumber a
d
LY294002oneitheruncoatedorfibronectincoatedwellswasdecreased ofgrowthfactors.AdditionofIGF1totheco-cultures,intheabsence em
comparedwithoutgrowthinIGF1aloneoneitheruncoatedorfibronec- of serum, increased attachment to a level similar to that observed in ic.o
u
tincoatedwells,respectively(Fig.6F–H). thepresenceofserum,suggestingthatIGF1alonecansupportattach- p
.c
mentofblastocyststoIshikawacellsinvitro.Furthermore,pretreatment o
m
EffectofpretreatmentwithIGF1,anIGF1R ofDay5blastocystswithIGF1for24hwassufficienttoimproveblasto- /hu
cyst attachment to Ishikawa cells compared with pretreatment in SS m
nAborLY294002onattachmentofmouse re
medium.Thissuggeststhattheinvitroculturedembryocanbemanipu- p
blastocyststoIshikawacells /a
latedbygrowthfactors,normallyfoundinvivo,toincreasetheblasto- rtic
Inattachmentexperiment2,Day5blastocystswereculturedinserum cyst’sadhesiveability. le
-a
mediumcontaining2mg/mlIGF1RnAborinSSmediuminthepresence TheeffectsofIGF1onblastocystattachmentweremediatedbythe b
s
orabsenceof10,100or1000ng/mlIGF1orwith10ng/mlIGF1inthe IGF1RasanIGF1RnAbnegatedthepositiveeffectsofIGF1pretreat- tra
c
presenceof2mg/mlIGF1RnAbor5mMLY294002for24h.Attheend mentonblastocystattachment.IntheblastocystIGF1isknowntophos- t/3
0
ofthispretreatmentperiodthehealthofblastocystsculturedinLY294002 phorylateAkt(GreenandDay,2013),asignallingmoleculedownstream /2
/2
wasassessedandblastocysts(Day6blastocysts)wereofhealthyappear- ofthePI3Kpathway.InthepresentstudythePI3KinhibitorLY294002 8
4
ance(Fig.7A–D).BlastocystswerewashedinSSmediumandthencul- prevented the increase in attachment mediated by IGF1. LY294002 /7
2
7
tured onIshikawacellsfor24h,underSS conditionsandassessedfor alsodecreasedattachmentinSSblastocysts,suggestingthateveninSS 0
5
9
attachment(onDay7).Allblastocystsinalltreatmentgroupsremained conditionstheremaybeautocrinegrowthfactorstimulationorconstitu- b
y
ofnormalappearanceaftertheco-cultureperiod(datanotshown). tive activation of the PI3K pathway to influence attachment. In the g
u
Pretreatmentofblastocystswithserumfor24hdidnotimproveat- presentstudy,pretreatmentwithserumdidnotimproveblastocystat- e
s
tachmentformtheserumstavedgroup(Fig.7E).Pretreatmentofblasto- tachment,infactattachmentintheseconditionswasreducedcompared t o
n
cysts with an IGF1R nAb in the presence of serum had no effect on withattachmentafterIGF1pretreatment.Highlevelsofinsulinandother 1
2
blastocyst attachment compared with pretreatment with serum growthfactorsthatarepresentinserummayhavenegativeeffectson A
p
(Fig.7E).Pretreatmentwith10ng/mlIGF1for24hincreasedblastocyst blastocystattachment,althoughserumappearstopositivelyinfluence ril 2
0
attachmenttoIshikawacellscomparedwithpretreatmentinSSmedium Ishikawacellstomediateattachment. 1
9
ormediumcontainingserum(Fig.7E).ThepositiveeffectofIGF1pre- High concentrations of IGF1 are known to be detrimental to the
treatmentonattachmentwasreducedbytheIGF1RnAb,tothelevel blastocyst by decreasing blastocyst hatching (Green and Day, 2013)
seeninSSconditions(Fig.7E).Additionally,pretreatmentofblastocysts and increasing apoptosis (Chi et al., 2000). High concentrations of
Figure5 IGF1increasesapicalfibronectininlatestageblastocysts.Representativeconfocalimagesofblastocystsstainedforfibronectin(Green)and
DAPI(Blue).Day5blastocystswereculturedinSSmediumfor24(AandB),48(CandD)or72h(E–H)intheabsence(A,CandE)orpresence
(B,DandF)of10ng/mlIGF1orinthepresenceofaPI3Kinaseinhibitor(5mMLY294002+10ng/mlIGF1)(G)orinthepresenceof10ng/ml
IGF1,attachedtoacoverslip(H).Scalebarsrepresent20mm.Redlineindicateslocationofcoverslip.Arrowindicateslocationoftheinnercellmass
(ICM).Thefollowingtotalnumberofblastocystswereanalysed,fromatleast3experimentalrepeatsineachtreatment;24intheSSgroup(24h),21in
SSplusIGF1(24h),26inSS(48h),30inSSplusIGF1(48h),40inSS(72h),59inSSplusIGF1(72h),13inSSplusIGF1+LY294002(48h)and15blas-
tocystswereanalysedthatwereattachedtoacoverslip(72h).