ProductionofPigletsafterCryopreservationofEmbryosUsingaCentrifugation-BasedMethodforDelipationWithoutMicromanipulation1RongfengLi,3,4CliftonN.Murphy,3LeeSpate,3DavidWax,3ClayIsom,3AugustRieke,3EricM.Walters,4MelissaSamuel,3,4andRandallS.Prather2,3,4DivisionofAnimalScience,3NationalSwineResourceandResearchCenter,4UniversityofMissouri-Columbia,Columbia,MissouriABSTRACT
Itisstilldifficulttosuccessfullycryopreserveinvitro-produced(IVP)swineembryos,astheyaresensitivetochillingduetotheabundanceofintracellularlipids.Mechanicaldelipationthroughmicromanipulationissuccessful,butthismethodincreasesthepotentialofpathogentransmissionbecauseofthedamageinflicteduponthezonapellucidaduringmicromanipulation,anditislaborintensive.ReportedhereisamethodtoremovethelipidofIVPporcineembryos,withoutsignificantlycompromisingthezonapellucida,bytrypsintreatingtheembryosorexposingtheembryotoahigh-osmolalitysolutiontoenlargetheperivitellinespacesothatthelipidcouldbepolarizedandseparatedcompletelyaftersubsequentcentrifugationwithoutmicromanipulation.Theproceduresworkbothfornucleartransfer-derivedembryosandinvitro-fertilizedembryos.Bothmethodsprovideahigh-throughputprocessthatleavesthezonapellucidaintact(orrelativelyintactforthetrypsintreatment)toaidinpreventingdiseasetransmission.Itisalsodemonstratedthatthisprocedureresultsinviablepiglets,aclaimthatcouldnotbemadeinmanypreviousreports.Althoughtheefficienciesofcryopreservationhavenotbeendramaticallyimproved,theseproceduresallowasinglepersontoprocessverylargenumbersofembryoswithoutthenecessityofmanipulatingeachindividualembryoonamicromanipulator.Suchhigh-throughputprocessingovercomesthelackofhighefficiency(i.e.,thesystemcanbeoverloadedwithembryosfortransfertosurrogates).
cryopreservation,embryo,invitrofertilization,osmoticstress
INTRODUCTION
Successfulcryopreservationofearlymammalianembryosprovidesopportunitiesforthepreservationofgermplasm,andthemovementofgeneticsdomesticallyandinternationally.Unfortunately,thepigembryohasbeenmoredifficulttocryopreservethanmanymammalianembryos.Significantadvanceshavebeenmadetowardthesuccessfulcryopreserva-tionofpigembryosbasedontheobservationthatpigembryosareverysensitivetohypothermicconditions,andthatremovalofintracellularlipids(delipation)appearstoalleviatethissensitivity[1–4].Moststudieshavefocusedoninvivo-producedembryos,astheyareconsideredtobemore
SupportedinpartbyNationalInstitutesofHealth,NationalCenterforResearchResourcesgrantsR01RR13438andU42RR018877,andbyFoodforthe21stCentury.2Correspondence:FAX:5738847827;e-mail:pratherr@missouri.eduReceived:9September2008.Firstdecision:2October2008.Accepted:20November2008.
Ó2009bytheSocietyfortheStudyofReproduction,Inc.eISSN:1259-7268http://www.biolreprod.orgISSN:0006-3363
1developmentallycompetentthaninvitro-produced(IVP)embryos.Alternativestomechanicaldelipationincludedesta-bilizingthecytoskeleton[5]andalteringthevitrificationconditions[6–9].Mostrecently,asolid-surfacecryopreserva-tionprocedurewasdevelopedforthepigthatshowsgreatpromiseforearlyembryos[9],andhasbeenshowntoworkonIVPembryos.
IVPembryosareevenmoredifficulttocryopreserve[10].TherearethreereportsofthecryopreservationofIVPembryosthatresultedinpiglets:twousedmechanicaldelipationthroughcentrifugationandmicromanipulation[10,11],andtheotherusedsolid-surfacevitrification[9].However,mechanicaldelipationincreasesthepotentialofpathogentransmission,becauseofthedamageinflicteduponthezonapellucidaduringmicromanipulation.Itisalsolaborintensiveandtimeconsuming.Developmentofapractical,noninvasivemeansoflipidremovalforhigh-throughputcryopreservationofIVPporcineembryosisstillnecessaryforresearchandcommercialpurposes.
Aftercentrifugationofthepigoocyteorembryowithanintactzonapellucida,thepolarizedlipiddropletstendtoremainconnectedwiththecytoplasmoftheoocyteorblastomereoftheembryoviaabridge-likestructure[12].Thepolarizedlipiddropletscanredistributeintotheoocyteorblastomereduringsubsequentcultureorcryopreservationprocedures.Thus,invivo-derivedembryosneedtobecryopreservedimmediatelyaftercentrifugationinordertopreventlipidredistributionpriortocryopreservation[13].Iftheperivitellinespaceisenlarged,thebridge-likestructurewillbreakaftercentrifugation,andthelipiddropletswillnotredistributeintothecytoplasmoftheoocyteortheblastomereoftheembryo,butwillstaywithintheintactzonapellucida.Atleasttwomethodsmightmaketheperivitellinespacelarger.Oneistoswellthezonapellucidathroughpartialenzymaticdigestion(suchastrypsin,pronase,etc.).Anotheroptionistocondensethevolumeoftheoocyteorembryobyhigh-osmolalitytreatment.Twogroups[14,15]demonstratedthatpartialenzymaticdigestionandsubsequentcentrifugationimprovedthecryopreservationsurvivalofpigparthenogeneticembryosandhand-madeclonedembryos.Butneitherreportedanypigletsproducedbyusingacombinationofenzymaticdigestionandcentrifugation.
Thelipiddropletsareabundantandlargeintheearly-stageporcineembryo,andgraduallydeclineinsizeandabundanceastheembryoadvancestoandbeyondtheblastocyststage[16,17].Coincidentwithreducedlipidcontentisanincreasedfreezabilityoflate-stageembryos.Late-stageembryos(blasto-cyst)withlowlipidcontentandsmaller-sizedlipiddropletssurvivecryopreservationbetterthanearly-stageembryos(two,four,andeightcell)[18–20].Interestingly,thelargelipiddropletsintheearly-stageembryosareeasiertoremovebycentrifugationthanthesmallerdropletsinthelater-stageembryos.563
564LIETAL.
matured,Basedontheaboveobservations,wetreatedIVP(invitro-theembryo,zonainvitro-fertilized)pigembryoswithtrypsintoswellembryosfollowedpellucida,cryopreservation.wereculturedbycentrifugationorwithhighosmolalitytoshrinkthetothetoseparatethelipids.TheseforThehigh-osmolalityblastocysttreatmentstageandwassubjectedtopigletsnuclearIVPresultedtransfer-derived(NT)transgenicembryos.alsousedLivedoesn’tandnaturecompromiseNTembryos.fromboththeThetrypsin-andhighosmolality-treatedzonahighpellucida,osmolalityandtreatment,high-throughputwhichcommercialoftheproceduremakeitattractiveforpossiblerefinements,limitationforswineapplication.commercializationembryoItcryopreservationisanticipatedthat,withfurtherofembryotransfer.willnolongerbeaMATERIALSANDMETHODSGeneralExperimentalDesign
Thestudiesreportedhereincludethreesetsofexperiments.ThefirstsetofexperimentsevaluatedcryopreservedIVPblastocyststhatwereearlierdelipatedatthetwo-tofour-cellstageusingacombinationoftrypsinandcentrifugationtreatments.Inordertoobtainahighpercentageoflipidseparation,invitro-maturedoocytesweretreatedwithtrypsin,sothatoocytesratherthanembryoscouldbecryopreserved.Unfortunately,theseoocytesparthenogeneticallyactivatedaftertrypsintreatment,and12.5%(8/64)developedtotheblastocyststageinvitro.Next,wetreatedcleavedembryosat28–30hafterinseminationtoavoidanyparthenogeneticactivation.IVPembryosweretreatedwithtrypsinandtrypsinpluscentrifugation.Theuntreatedembryosservedascontrol.Thefirstexperimentinvestigatedthesensitivityofdifferentstagesofembryos(two-,three-tofour-,and!four-cell-stageembryos)tothetrypsintreatment.Thenumberofnucleiandthepercentagethathatchedwerealsodetermined.Althoughthetrypsin-treatedzonapellucidawasthinner,theseembryosdidnothatchatanincreasedpercentagecomparedtotheuntreatedembryos.Wethereforeattemptedtoprovideadditionalassistance:thezonapellucidawaseitherpuncturedwithamicropipette,orcompletelyremovedbyusingpronaseaftervitrificationandwarming.Basedontheresultsoftheaboveexperiments,thecombinationoftrypsintreatmentandpronasetreatmentwasusedtocryopreserveIVPembryospriortoembryotransfer.
Thesecondsetofexperimentsevaluatedcryopreservationoflipid-separatedIVPblastocystsderivedfromhigh-osmolalitytreatmentandcentrifugationofzygotes.Lipidseparation,embryodevelopment,andhatchingweremeasuredafterexposureofzygotestodifferentosmolalities(300,350,400,450,and500mOsmbyaddingNaCl)anddifferentcentrifugationdurations(6,12,and20min).Next,sucrosewasusedtomakemediawithevenhigherosmolalities(600and800mOsm),andthenlipidseparation,embryodevelopment,andhatchingweredetermined.Thehatchingabilityoffreshembryoswasnotadverselyaffected,aswithtrypsintreatment.However,thehatchingabilityofcryopreservedembryoswasobviouslycompromisedbythecryopreservationprocess.Thecombinationtreatmentsof350mOsmplus6-mincentrifugation(350þ6),400þ6,450þ12,and450þ20werechosenforcryopreservationpriortoembryotransfer.Thezonapellucidawasremovedwithpronasebeforetransfer.
Thethirdsetofexperimentsevaluatedcryopreservationoflipid-separatedNTblastocystsderivedfromhigh-osmolalitytreatmentandcentrifugationattheone-cellstage.Basedontheresultsofthesecondsetofexperiments(describedabove),andtoavoidpotentiallynegativeeffectsofexcessivelyhighosmolality(e.g.,450;500mOsm)ondevelopmentoftheNTembryo,350and400mOsmwerechosenfortheNTembryotreatment.High-osmolalitymediaat350or400mOsmweremadebyaddingNaClorsucrose.TheNTembryosweretreatedwiththesemediaandcentrifuged.Insomecases,thelipidseparationwasnotascompleteaswiththeIVPembryos,sothelipid-unseparatedembryosweretreatedandcentrifugedasecondtime.Theblastocystsderivedfromlipidseparationwerecryopreservedandtransferredintosurrogates.Beforetransfer,thezonapellucidawassoftenedorremovedwithpronase.
CollectionofPorcineOocytes,InVitroMaturation,andFertilization
AllchemicalswerepurchasedfromSigma(St.Louis,MO),unlessotherwiseindicated.Ovarieswerecollectedfromprepubertalgiltsatalocalabattoirandtransportedtothelaboratoryin0.9%NaClat30–358C.Cumulusoocytecomplexeswereaspiratedfromantralfollicles(3–6mmindiameter)withasyringeandan18-gaugeneedle.Approximately50oocyteswere
culturedin500llmaturationmedium,TCM199(no.31100035;Gibco,GrandIsland,NY)with0.1%polyvinylalcohol(PVA),3.05mmol/Lglucose,0.91mmol/Lsodiumpyruvate,0.57mmol/Lcysteine,0.5lg/mlluteinizinghormone(LH),0.5lg/mlfollicle-stimulatinghormone(FSH),10ng/mlepidermalgrowthfactor(EGF),75lg/mlpenicillin,and50lg/mlstreptomycinfor40–44hat38.58C,5%CO2,inhumidifiedair.Aftermaturationthecumuluscellswereremovedfromtheoocytesbyvortexingfor4mininTyrodelactate(TL)-Hepes[21]supplementedwith0.1%PVAand0.1%hyaluronidase.Denudedoocyteswithavisiblepolarbodywereselectedinoocytemanipulationmedium(stocksolution:TCM199,with0.6mmol/LNaHCO2.9mmol/LHepes,50lg/mlpenicillin,60lg/mlstreptomycin,30mmol/L3,NaCl,and3mg/mlbovineserumalbumin[BSA]).ForIVF,30–40oocyteswithapolarbodyweretransferredto50-llmicrodropsofIVFmedium(amodifiedTris-bufferedmedium,with113.1mmol/LNaCl,3mmol/LKCl,7.5mmol/LCaCl2,5mmol/Lsodiumpyruvate,11mmol/Lglucose,20mmol/LTris,2mmol/Lcaffeine,and2mg/mlBSA).One0.1-mlfrozensemenpelletwasthawedinto3-mlspermwash(PBS[Gibco],with1mg/mlBSA,75lg/mlpenicillin,and50lg/mlstreptomycin)at398C,andthenlayeredonto60%percollinPBSforcentrifugation(6003g,10min).Aftercentrifugation,thepelletwastransferredinto10-mlspermwashandcentrifuged(6003g,5min)forasecondwash.Thesupernatantwasremovedandthespermpelletwassuspendedat0.53106sperm/mlinIVFmedium.Thespermsuspension(50ll)wasaddedtoa50-llIVFdropwithoocytesandculturedat38.58C,5%CO2,inhumidifiedairfor4h.
NuclearTransfer
Sow-derivedoocyteswerepurchasedfromBoMed,Inc.(Madison,WI),andshippedovernightinmaturationmedium(TCM199,with2.9mmol/LHepes,10%porcinefollicularfluid,0.5lg/mlp-FSH,5lg/mlinsulin,10ng/mlEGF,25mg/Lgentamycin,0.91mmol/Lpyruvate,0.57mmol/Lcysteine),andmovedtofreshmediumafter24h.Afteratotal42;44hofmaturation,thecumuluscellswereremovedfromtheoocytesbyvortexingfor4mininTL-Hepessupplementedwith0.1%PVAand0.1%hyaluronidase.Thefirstpolarbodyandtheadjacentcytoplasmfromtheseoocyteswereaspiratedwhileinmanipulationmediumwith7.0lg/mlcytochalasinB.Adonorcellwasthentransferredintotheperivitellinespace.Fusionandactivationwereaccomplishedsimultaneouslywithtwo30-lsecpulsesof1.2kV/cminfusion/activationmedium(0.3Mmannitol,1.0mMCaCl2,0.1mMMgCl2,0.5mMHepes)or,alternatively,inafusion-onlymediumwithalowerconcentrationofcalcium(0.3Mmannitol,0.1mMCaCl2,0.1mMMgCl2,and0.5mMHepeswasusedforfusion,thenthefusedoocyteswereexposedto200lMthimerosalfor10mininthedark,andthen8mMdithiothreitolfor30mintoaccomplishactivation[22]).
CultureofIVPEmbryos
TheIVPandNTembryoswereculturedinPZM3(108.0mmol/LNaCl,10.0mmol/LKCl,0.35mmol/LKH2PO4,0.4mmol/LMgSOCa(lactate)4Á7H2O,25.07mmol/LNaHCO1.0mmol/Lglutamine,3,0.2mmol/LNa-pyruvate,2.0mmol/L5.0mmol/Lhypotaurine,20ml/LEaglebasal2Á5Hmedium2O,aminoacidsolution,10ml/LmodifiedEaglemediumaminoacidsolution,0.05mg/mlgentamicin,3mg/mlBSA,28862mOsm,pH7.360.2)at38.58C,5%CO2inhumidifiedair.
TrypsinandHigh-OsmolalityTreatmentofIVPandNTEmbryos
Tomakehigh-osmolalitymedium,theoocytemanipulationmediumstockwasusedasthebasicmedium.Theosmolalityofthisstockwas300–310mOsm.Adding0.016gNaClto10mlofthisstockincreasedtheosmolality50mOsm.Whensucrosewasusedtoincreaseosmolality,1Msucrose(madewiththeoocytemanipulationstockassolvent)wasdilutedtomake350-,400-,500-,600-,and800-to850-mOsmsolutions.Osmolalitywasverifiedbyusingavaporpressureosmometer(Model5520;Wescor,Logan,UT).
TheIVPembryosweretreatedwithtrypsinorhighosmolality.Forthetrypsintreatment,cleavedembryoswereselectedat28–30hafterinseminationandtreatedwith2%–4%trypsin(T1426;Sigma)inPBSfor2–15min.Aftertrypsintreatment,theembryoswerecentrifugedat134003gfor5mininoocytemanipulationmediumwith7.0lg/mlcytochalasinBandthenculturedinPZM3.TodeterminethenumberofnucleiinDay6(D6)blastocysts,4lg/mlHoechst33342wasusedtostainthenuclei,whichwereexaminedbyepifluorescentmicroscopy.Asanadditionalindicatorofembryoviability,D5andD6blastocystsweremovedtofreshPZM3orBuffaloratliver(BRL)cell-conditionedmedium(TCM199,with26.2mmol/LNaHCO3,0.2mmol/LNa-Pyruvate,and10%fetalbovineserum[FBS])toinvestigatetheabilityofembryostohatchandattachtothebottomofthedish.Forthehighosmolality
HIGHTHROUGHPUTCRYOPRESERVATIONFORSWINEEMBRYOS
treatment,at18–20hafterinsemination,theembryoswereequilibratedin2mlmanipulationmediumwithdifferentosmolalities(300,350,400,450,and500mOsm),7.0lg/mlcytochalasinB,and0.1mg/mlBSAfor6min,andthencentrifugedin0.35mlofthesamemediumin1.5mleppendorftubes(30–50embryoswereputineachtube)for6,12,or20minat134003g.A6-minequilibrationperiodwaschosenbecauseithasbeenshownpreviouslytobesufficientforsperm[23].Aftercentrifugation,theembryosweretransferredinto2mloocytemanipulationmedium,washedinPZM3threetimes,andthenculturedinPZM3.After12h,lipidseparationwaschecked,andembryosinwhichthelipidshadnotseparatedwereremoved,whilethelipid-separatedembryoswereculturedinPZM3tochecktheblastocystandhatchingpercentages.
TheNTembryosweretreatedwith350or400mOsmbyaddingNaClorsucrose.PVAat0.1mg/mlconcentrationwasaddedinsteadofBSAwhensucrosewasused.TheNTembryosweretreatedthesameastheIVPembryos14–18hafterfusion/activation,andthenculturedinPZM3medium.After12-hculture,thelipidseparationwaschecked,andtheembryosinwhichthelipidshadnotseparatedwereremovedandtreatedagainwithmediumatthesameosmolalityandcheckedagainforlipidseparation12haftertreatment.Thelipid-separatedembryosfromboththefirstandsecondhigh-osmolalitytreatmentswereculturedinPZM3.Interestingly,thereappearedtobenoleakagethroughthesmallholemadeinthezonapellucidabythetransferpipette.
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FIG.1.DevelopmentofIVPembryosaftertrypsintreatmentortrypsintreatmentpluscentrifugation.Differentsuperscriptswithinatreatmentaredifferent:a,bandc,d(P,0.01).Summaryoffourreplications(n¼701).Valuesshownaremeans6SEM.Therewasnodifferenceinthetrypsin-treatedembryos.
VitrificationandEmbryoTransfer
Vitrificationofembryoswascarriedoutbyusingtheopenpulledstraw(OPS)method.TheOPSstrawswerepurchasedfromLECInstrumentsP/L.Allsolutionsusedduringvitrificationwerepreparedwithholdingmedium(oocytemanipulationmediumwith20%FCSinsteadof3mg/mlBSA).D5andD6IVPandNTblastocysts(dayofNTorinseminationwasconsideredD0)wereplacedinequilibrationsolution(10%ethyleneglycol,10%dimethylsulfoxide[DMSO])for2min,followedbyexposuretovitrificationsolution(20%ethyleneglycol,20%DMSO).EmbryoswereloadedintoanOPSandimmediatelyplungedintoliquidnitrogen.Allprocessesbeforeplungingintonitrogenwereconductedonawarmstageat38.588C.Thetimefromexposuretothevitrificationsolutiontoplungingwas25–30sec.
Forembryowarming,bothcryopreservedIVPandNTembryoswerewarmedbyimmersingtheendoftheOPSinto0.3Msucrosefor5minat38.58C,thentransferringthethawedembryosto0.2Msucrosefor5min,andthentoholdingmediumfor5min.ThewarmedembryoswereculturedinBRLcell-conditionedmediumtocheckreexpansionorusedfortheassistedhatchingexperimentorembryotransferaftersofteningorremovingzonapellucidawith0.5%pronase.
Forassistedhatching,embryoswereeitherpuncturedbymanipulationbyusinga20-lmouterdiameterpipette,orstrippedoftheirzonapellucidabyexposureto0.5%pronasefor20–30secandthenculturedinBRLcell-conditionedTCM199.Tosoftenorremovethezonapellucidabeforetransfer,theembryosweretreatedin0.5%pronase(inPBS)for10–15sec(tosoften)or20–30sec(toremove),andthentransferredintoholdingmediumimmediatelybeforethezonapellucidacompletelydisappeared.Theblastocystswiththezonapellucidasoftenedorremovedweresurgicallytransferredintotheampullary-isthmicjunctionoftheoviductofasurrogate4or5daysafterobservedestrus.AllexperimentswithanimalswereapprovedbytheInstitutionalAnimalCareandUseCommittee.
onpercentlipidseparation,percentblastocyst,percenthatching,thepercentageofembryoattachmentonthebottomofdishafterhatching,andthenumberofnucleiintheblastocystsweretestedasappropriateineachexperiment.Percentagedatawerearcsinetransformedpriortoanalysis.Differencesbetweentreatmentgroupswereassessedviaposthoc,pairwisecomparisonsofleastsquaresmeanvalues.SignificancewasassignedtoPvalueslessthan0.05.
RESULTS
CryopreservationofLipid-SeparatedIVPBlastocystsDerivedfromTrypsin-TreatedandCentrifugedCleavage-StageEmbryos
Overall,whileneithertrypsintreatmentnortrypsintreatmentpluscentrifugationaffectedthepercentageofembryosthatdevelopedtotheblastocyststageorthenumberofnucleiintheblastocysts(34.262.7vs.37.762.7,trypsinþcentrifugationandcontrol,respectively)therewasasignificantdifferenceindevelopmenttotheblastocyststagefromtwo-cellversusmorethanfour-cellstageembryosfortwoofthetreatments(Fig.1;SupplementalTableS1availableatwww.biolreprod.org).Thismaybebecausethoseembryosthatarealreadybeyondthefour-cellstageat28–30hpostinseminationmaybearesultoffragmentation,andarethusoflowerquality.Inaddition,thepercentblastocystfromthetrypsinpluscentrifugationtreatmentforthethree-tofour-cell-stageembryoswashigherthanthecontrolsortrypsin-onlytreatments.Whilethetrypsin-treatedzonapellucidawasthinner,theseembryosdidnothatchwhenculturedinPZM3.BRLcell-conditionedTCM199with10%FBSimprovedthehatchingpercentageoftrypsin-treatedandcentrifugedembryosto15.4%,whichwasstilllowerthanthecontrols(61.3%),buthigherthanPZM3alone(0%).Theonly
StatisticAnalysis
AlldataweresubjectedtoANOVAbyusingthePROCGLMcommandsinSAS(SasInstitute,Cary,NC).Themaineffectsoftrypsintreatment,centrifugationtime,zonapellucidaremovalorpuncture,osmolality,andsolute
TABLE1.Hatchingabilityoftrypsin-andcentrifugation-treatedblastocystsaftervitrification-warmingandcultureinBRLcellconditionedTCM199.*
No.re-expandedafterovernightculture(%,mean6SEM)27(100.060)31(96.963.3)33(97.164.2)
No.ofblastocyststhathatched(%,mean6SEM)0(0)a1(3.262.6)a33(97.164.2)b No.ofembryosattachedtothebottomofthedish
afterhatching(%,mean6SEM)
00
5(14.768.3)
TreatmentControlPunctured
Pronasetreated
No.ofblastocystsfrozen-thawed
273234
*Summaryofthreereplications. Thezonapellucidawasremovedwiththepronasetreatment.a,bDifferentsuperscriptswithinacolumnaresignificantlydifferent,P,0.01.
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FIG.2.Trypsintreatedembryos.a)Beforetrypsintreatment.b)Aftertrypsintreatmentandcentrifugation.candd)Freshblasto-cysts.e)Collapsedblastocystaftervitrifica-tionandwarming.f)Collapsedblastocystsafterzonaremoval.Bar¼50lm.
LIETAL.
outgrowthandattachmentthatwasobservedwaswithconditionedTCM199inthecontrols(8%ofthosethathatched)(SupplementalTableS2).
Aftervitrificationandwarming,good-qualityembryos(embryosthatappearedtobemorphologicallynormal)wereselectedforanassistedhatchingexperiment.Theembryoshadtheirzonapellucidapunctured,weretreatedwithpronase,orwereusedascontrols.Thepercentagethatreexpandedwasnotdifferentforthedifferenttreatments(96%–100%).Onlythoseembryosthathadtheirzonapellucidaremovedwithpronaseattachedtothedish,andonlyoneembryohatchedofthe32thathadtheirzonapellucidapunctured(Table1).Afterzonaremoval,14.7%ofthevitrified-warmedembryosattachedonthebottomofthedishandcontinuedtogrowinBRLcell-conditionedmedium.Developmentalcompetencewasdeterminedbytransfertofoursurrogates;twosurrogatesreceived50vitrified-warmedandzona-removedembryos,andanothertworeceived25or24vitrified-warmedandzona-removedembryos.Onesurro-gatefromeachgroupmaintainedthepregnancyanddeliveredninepiglets(eightmalesandonefemale)andfivepiglets(allofthemweremale),respectively(SupplementalTableS3).PhotosofembryosbeforeandaftertrypsintreatmentorvitrificationareshowninFigure2.
CryopreservationofLipid-SeparatedIVPBlastocysts
DerivedfromZygotesTreatedwithHigh-OsmolalityMediaandCentrifugation
Bothincreasingosmolalityandextendingthecentrifugationtimeenhancedthepercentageoflipidseparation(Fig.3;SupplementalTableS4).For6-,12-,and20-mincentrifugation,thepercentageofdelipatedembryoswasthehighest,at450,500,and400mOsm,respectively.Ingeneral,thelipidseparationwasmorecompleteifthecentrifugationtimewaslonger(e.g.,20minwasmoreeffectivethan6minwhentheosmolalitywas400orlower).Theseembryoshatchedfrom0%to4.2%pertreatment.Evenhigherosmolality(600mOsm),achievedbyusingsucroseinplaceofNaCl,couldnotimprovethelipidseparation.Infact,theoppositewastrue,asthepercentageofembryosinwhichlipidseparationoccurreddecreasedastheosmolalitywasincreased,andtherewasalmostnoseparationwhentheosmolalityreached800mOsm(Fig.4;SupplementalTableS5).Theabilityoflipid-separatedembryostodeveloptotheblastocyststagewasadverselyaffectedby600mOsmorhigherosmolality.Thegeneralefficiencies(howmanylipid-separatedblastocystscouldbeproducedfromtheinitial
FIG.3.LipidseparationofIVPembryosaftertreatmentwithdifferentosmolalitiesanddifferentcentrifugationtimesat18–20hafterinsemina-tion.ErrorbarsrepresentSEM.DetailsofsignificantdifferencescanbefoundinSupplementalTableS4.
FIG.4.LipidseparationanddevelopmentofIVPembryosaftertreatmentwithdifferentosmolalitiesandcentrifugationfor6min.Inthisexperiment,400mOsmwasusedfortheNaCllipidseparationanddevelopmenttotheblastocyststage(n¼721,threereplications).DetailsofsignificantdifferencescanbefoundinSupplementalTableS5.Differentfootnotesymbols(a,b,c)withinatreatmentaredifferentP,0.05.
HIGHTHROUGHPUTCRYOPRESERVATIONFORSWINEEMBRYOS567
TABLE2.Re-expansionandhatchingofhighosmolality-treatedembryosaftervitrification-warmingandcultureinBRLcellconditionedmedium.*
Precentrifugationandcentrifugationtime(min)
6666þþþþ661220
No.ofblastocystsvitrified-warmed
888944535950
No.ofre-expandedblastocystsafter12hculture(%6SEM)72693537(81.8(77.5(79.5(71.2
66668.0)a0.2)a4.2)a2.3)aNo.ofhatchingblastocysts(%6SEM)
2(2.361.0)b0b0b0b0b20(40.0611.4)aNo.ofembryosattachedtothebottomofthedishafterhatching(%6SEM)
0b0b0b0b0b4(8.067.1)aOsmolality(mOsm)350400450450
Control(vitrified-warmednormalblastocyst)Control(freshnormalblastocyst)
8(13.662.7)b-
*Summaryofthreereplications.a,bDifferentsuperscriptswithinacolumnaresignificantlydifferent,P,0.05.
oocytes)werelowerwhen300mOsmand6mincentrifugation,500mOsmand20mincentrifugation(SupplementalTableS4)andhigherthan500mOsmtreatments(SupplementalTableS5)wereapplied.Theothertreatmentshadsimilarefficienciesofblastocystproductioncomparedwiththecontrols.Althoughtheblastocystsderivedfromdifferentosmolalityandcentrifugationtreatmentsurvivedcryopreservation,thehatchingabilitywascompromisedbythecryopreservationprocess.Only2.3%oftheblastocystsderivedfromthe350þ6treatmenthatchedaftervitrificationandwarming,andnoneoftheblastocystsderivedfrom400–450mOsmtreatmenthatched,while40%ofthecontrolshatched,and8%attachedtothedish(Table2).
Onehundredvitrified-warmedzona-intactembryosweretransferredtotwosurrogates(50embryos/surrogate),inwhichnoneestablishedapregnancy.Subsequently,175vitrified-warmedembryoswithzonaremovedafterwarmingweretransferredtosevensurrogates(25embryos/surrogate).Threesurrogatesestablishedpregnanciesandfarrowed,producingnormaloffspring(Table3).Theconclusionsfromthissectionarethatzonaremovalisprerequisiteforestablishingpregnancywithcryopreserved-warmedporcineIVPembryos.Inaddition,centrifugationfrom6to20minandexposuretoosmolalitiesrangingfrom350to450mOsm(Fig.5)iscompatiblewithtermdevelopment.Thus,acombinationofosmolarityandcentrifugationinthisrangewillworkforproducingpigembryosforcryopreservation,asshowninFigure6.CryopreservationofLipid-SeparatedNTBlastocystsDerivedfromHigh-OsmolalityTreatmentandCentrifugation
ThepercentageofNTembryoswithcompletelipidseparationwasnumericallylowerthanthatoftheIVPembryos.
Extendingthecentrifugationtimeenhancedthelipidsepara-tion,butincreasingtheosmolalitydidnot.Bytreatingthelipid-unseparatedembryoswithhighosmolalityforasecondtime,thetotallipidseparationfrombothfirstandsecondtreatmentstendedtobesimilarbetweenthedifferentosmolalityandcentrifugationtreatments(Table4).Thehighestpercentageoflipidseparationwasobtainedfrom350mOsmand20-mincentrifugation.SucroseandNaClwerenotdifferentinregardtolipidseparationwhentheywereusedtomakehigh-osmolalitymedium(Table4).Threeembryotransferswereperformed;foreachtransfer,80;90embryoswiththezonapellucidasoftenedorremovedbypronasetreatmentweretransferredintothesurrogate.Oneofthethreeestablishedapregnancy,andonedeliveredasinglemalepiglet(Table5).Thephotosofembryosafterhigh-osmolalitytreatment,andbeforeandaftervitrification,areshowninFigure7.DISCUSSION
Pigsareanincreasinglyimportantmodelforstudyingandunderstandinghumanmedicine,inadditiontotheirextensiveroleinagriculture.Invitroproductionofpigembryosorbynucleartransferhasbeenusedtocreatediseasemodelsorpotentialorgandonorsforxenotransplantation[24–26].Asaresult,thedemandforeffectivecryopreservationofIVPembryoshasdramaticallyincreased.However,apracticalandefficientprotocolforthispurposeiscurrentlyunavailable.Here,wereporttwopracticalprotocolstoproducepigembryosinvitro,whichcanbesuccessfullycryopreserved.Theyaretrypsinorhigh-osmolalitytreatmentofIVPembryosandsubsequenthigh-speedcentrifugation.Bothmethodscanenlargetheperivitelinespacebypartialzonapellucidadigestion(trypsin)oroocytecytoplasmiccondensation(high
TABLE3.TransferofIVPembryosderivedfromhighosmolalitytreatmentandcentrifugationaftervitrificationandwarming.*Osmolality(mOsm)350350350400400450450450450
ChemicalNaClNaClNaClNaClNaClNaClNaClNaClNaCl
Centrifugationtime(min)
6666620201212
No.ofembryostransferred
255050252525252525
Zonaremovalaftervitrificationandwarming
YesNoNoYesYesYesYesYesYes
Outcome
3Males2FemalesReturnedtoReturnedtoReturnedto2Females2MalesReturnedto2Females1MaleReturnedtoReturnedto
estrusonDay19estrusonDay27estrusonDay21estrusonDay25estrusonDay24estrusonDay20
*Twosurrogateswerenotincludedintheabovedataduetoreproductivetractinfections.
568LIETAL.
FIG.5.Zygotestreatedwithdifferentosmolarities.Row1:immediatelyafterhigh-osmolalitytreatment.Row2:immediatelyaftercentrifugation.Thebridge-likestructurecouldbeseeninthe300-mOsmgroup(andinsert).Completelipidseparationwasobservedinoocytestreatedwith400mOsm.Largebridge-likestructureswerepresentinoocytestreatedwith600or800mOsmandcentrifuged(600and800mOsmachievedwithsucrose).Bar¼50lm.
osmolality).Withcentrifugation,thelipidscouldbeseparatedcompletelyfromthecytoplasm.Bothmethodsprovideahigh-throughputprocessthatleavesthezonapellucidaintact(orrelativelyintact,asisthecaseforthetrypsintreatment)toaidinpreventingdiseasetransmission.Itisalsodemonstratedthatthisprocedureresultsinviablepiglets,aclaimthatcouldnotbemadeinmanypreviousreports.Althoughtheefficienciesofcryopreservationhavenotbeendramaticallyimproved,theseproceduresallowasinglepersontoprocessverylargenumbersofembryoswithoutthenecessityofmanipulatingeachindividualembryoonamicromanipulator.Asummaryofthedatafromthefirstandsecondsetsofexperiments(exceptforthoseembryosinwhichthezonapellucidawasnotremovedpriortoembryotransfer)shows324embryostransferredto11surrogates,resultingin5pregnanciesand26offspring.The
FIG.6.IVPembryosafterhigh-osmolalitytreatmentandcentrifugation.a)Afterhigh-osmolalitytreatmentandseveral-hourcul-ture.bandc)Afterhigh-osmolalitytreat-mentandculturetoblastocyst.d)Afterwarming.e)Afterremovalofthezonapellucida.f)ReexpansionafterinvitrocultureinBRLcell-conditionedmedium.Bar¼50lm(a,b,d,ande)and150lm(candf).
timerequiredtoprocess324embryosis30–40min.Ifthepreviousproceduresthatrequiremicromanipulationwereused,itwouldrequirenotonlytheexpensiveequipmentandthespecializedtraining,butalsoabout5additionalhoursoflabor.Suchhigh-throughputprocessingovercomesthelackofhighefficiency(i.e.,thesystemcanbeoverloadedwithembryosfortransfertosurrogates).
Lipidscouldbeseparatedwithoutcompromisingdevelop-menttotheblastocyststagewithanosmolalityof450mOsmand6-mincentrifugation.Byincreasingthecentrifugationtimeto20min,350mOsmresultedinmorethan90%oftheembryoshavinglipidseparation.Anumberofthedifferentcombinationsofosmolalityanddurationofcentrifugationresultedinnotonlyblastocyst-stageembryos,butalsonormaloffspring.Forpracticalapplications,wesuggestusingthehigh-
HIGHTHROUGHPUTCRYOPRESERVATIONFORSWINEEMBRYOS
TABLE4.LipidseparationofNTembryosaftertreatmentwithdifferentosmolalitiesandcentrifugationtimesat14–18hafterfusion.*
Lipidseparatedembryos
Chemicalusedtomakehigh
OsmolalityosmolalityCentrifugation(mOsm)mediumtime(min)400400400400400350Control
SucroseSucroseNaClNaClNaClNaCl
666122020
1sthighosmolalitytreatmentandcentrifugation
1stþ2ndosmolalitytreatmentandcentrifugationNo.44467189100491653-Percentage(mean6SEM)83.660.9c82.081.784.789.391.7
66666-1.4c0.2c0.7bc1.7ab0.7a569
Lipidseparatedblastocysts
Percentage(mean6SEM)
/LipidseparatedNo.embryos731152220621168
16.462.1ab17.124.720.012.617.813.3
6666660.8ab2.5a6.8ab2.3b2.5ab3.3b/Totalembryos13.761.614.120.217.011.316.313.3
6666660.82.05.72.12.43.3
ActivationandfusionofNTembryosLowcalciumþThiþDTT ElectricalElectricalElectricalElectricalElectrical
Total
Percentageno.of
embryosNo.(mean6SEM)53181810911855071260
3365466788415570-63.362.5d66.761.574.675.580.1
61.7bcd60.4cd65.1abc62.3ab62.9a-
*Summaryoftwotosixreplicates. Thi,thimerosal;DTT,dithiothreitol.a,b,c,dDifferentsuperscriptswithinacolumnaresignificantlydifferent,P,0.05.
osmolalitytreatmentinplaceoftrypsinbecauseofthevariabilityofthetrypsin,asdescribedbelow.
Interestingly,fortheIVPembryos,400mOsmappearedtobeanoptimalosmolalityforlipidseparation,butitdidnotworkaswellonNTembryos.ThereweremanydifferencesbetweenIVPandNTembryos.ThefirstwasthattheNTembryoshadasmallervolumeofcytoplasm,sincesomecytoplasmwasremovedduringenucleation.Theosmolality,therefore,didnotneedtogotoashighas400mOsmtoprovideextraspaceinthezonapellucida.However,the350mOsmand20-mincentrifugationresultedinanumericallylowerpercentageoflipidseparationintheNTgroupascomparedwiththeIVPgroup.Interestinglywhenwetried300mOsm(unpublisheddata),thelipidseparationwassimilartothatfor350mOsm.Asecondroundoftreatmentsforthoseembryosthatdidnotachievecompletelipidseparation12hafterthefirsttreatmentimprovedtheoverallpercentageofNTembryosthathadcompleteseparation.AseconddifferencebetweentheIVPandNTembryoswasthattheywerederivedfromgiltandsowoocytes,respectively,andculturedindifferentmaturationmedia.Thedifferentovarysourcesanddifferentmaturationcultureconditionscouldresultindifferentcytoplasmicproperties,andthusaffecttheresults.AthirddifferenceisthatthephysicalcharacteristicsofthecytoplasmorplasmamembranemaybedifferentintheNTembryo,becausetheparthenogeneticactivationmaynotexactlyreplicatetheeventsthatnormallyoccuratfertilization.
Itcannotbecategoricallystatedthatalargerperivitellinespacewillnecessarilyresultinahigherpercentageoflipidseparation.Thelowercytoplasmicvolumeandlargerperivitel-linespaceafter600mOsmtreatmenthadonly24.5%lipidseparation.Whentheosmolalityreached800mOsm,almostnoneofthelipidscouldbeseparated.Duringthecentrifuga-tion,thelipidfractionwillrisetothetop,duetoitslowerdensity,whilethenonlipidfractionofthecytoplasmwillstay
TABLE5.TransferofNTembryosaftervitrificationandwarming.*Osmolality(mOsm)400400350
ChemicalSucroseNaClNaCl
Centrifugation(min)
66–2020
atthebottom,dueitshigherdensity.Thehighosmolality(600or800mOsm)causedanextremechangeinthepropertiesoftheoocytecytoplasm,sothatthecytoplasmdidnotseparate.Theremaybeapositiveeffectofhigherosmolalityonearlyembryodevelopment[27],astheembryosappeartohaveareducedlevelofapoptosisthroughregulationofBax-a/Bcl-xlgeneexpression[28].Inourstudy,theembryoswereexposednotonlytohighosmolality,butalsotohigh-speedcentrifu-gation.WhiledevelopmentwasnotimprovedabovethatofcontrolswhentheIVPembryosweretreatedwithhigh-osmolalitymediumfollowedbycentrifugation,therewasanimprovementfortheNTembryos(400mOsm[byaddingNaCl]significantlyimprovedtheNTembryodevelopment).However,developmenttoterminhigh-osmolalitytreatmentwasnotashighasanticipated.
Twogroups[29,30]foundthatthepercenthatchingofinvivo-derivedpigmorula/earlyblastocyststhathadbeencentrifuged,vitrified-warmed,andculturedinvitrowasverylow.Initially,theywonderedifthepolarizedmaterialleftincontactwiththeblastomereduringvitrificationmayhavehadsomeinhibitoryeffectfollowingwarmingandrecovery.Subsequentexperimentsindicatedthatthepresenceofthelipidmaterialintheperivitellinespacewasnotthereasonfordifficultyinhatching.Eitherthezonapellucidawasalteredbytheconditions,ortheembryowascompromisedsuchthatitcouldnothatch.Removalorthinningofthezonapellucidaafterwarmingimprovedthesurvivalrateofcyopreservedembryo,andresultedinlivepiglets[30].Ourresultsindicatethatbothtrypsintreatmentandcryopreservationitselfcanhardenthezonapellucida,sotheremovalofthezonapellucidaafterwarmingandbeforetransferisnecessaryforbothtrypsinandhigh-osmolaritytreatments.
Theenzymedigestionmethodusedapartialdigestionofthezonapellucidawithtrypsin[14]orpronase[15].Pronasehasbeenshowntonotharmporcineembryosifusedappropriately
No.ofembryostransferred
838090
Zonaremovalaftervitrificationandwarming
YesYesYes
Outcome
1Piglet(male)
ReturnedonDay22ofthecycleReturnedonDay19ofthecycle
*Onegiltwasnotincludedintheabovedataduetoareproductivetractinfection.
570
FIG.7.Nucleartransferembryosafterhigh-osmolalitytreatmentandcentrifuga-tion.aandb)Afterhigh-osmolalitytreat-mentandseveral-hourculture.candd)Afterhigh-osmolalitytreatmentandculturetoblastocyststage.e)Afterwarming.f)ReexpandedafterinvitrocultureinBRLcell-conditionedmedium.Bar¼50lm(b,c,e,andf)and150lm(aandd).
LIETAL.
[31,32],andhasbeenusedforassistedhatchingofembryos,butwefoundthatitwasveryharsh,andresultedinrapidzonapellucidadigestion.Previouswork[14]inadditiontoourstudiesindicatesthattrypsinisalsocompatiblewithpigembryodevelopment,aswewereabletoobtainblastocyst-stageembryosthatresultedinpiglets.However,trypsintreatmenthassomedisadvantageswhenitisusedforlipidseparation.Wefoundthattrypsintreatmentcanelicitparthenogeneticactivationofoocytes;thus,toavoidpartheno-geneticactivation,treatmentofoocyteswithtrypsincannotbedonebeforeorimmediatelyafterfertilization.Inaddition,thetrypsintreatmentdidnotworkconsistently.Theeffectofthetrypsintreatmentdependedonthebatchoftrypsinpowder.Assuch,theembryoshadtobeobservedwhentheywerebeingexposedtothetrypsinsolution,andthetreatmentworkedbestiftheoocytesweretreatedinsmallgroupssothattheycouldbeobservedandthedigestionmonitored.However,evenwiththesecaveats,pregnanciesandoffspringcanbeproducedwiththeseprocedures.
Theproceduresdescribedhererequirethatthezonapellucidaberemovedpriortoembryotransfer.Anintactzonapellucidaisbeneficialforpreventingdiseasetransmission.Thehigh-osmolalitytreatmentsresultinanintactzonapellucidathroughoutthecryopreservationprocess;thus,aftercryopres-ervation,itcanbeconfirmedthatthezonapellucidaisintact.Trypsintreatmentresultsinaswellingofthezonapellucida;additionalexperimentsarerequiredtodetermineifthezonapellucidaiscompromisedtosuchanextentastoallowpathogenentry.Ineithercase—high-osmolalityortrypsintreatment—theintegrityofthezonapellucidacanbedeterminedjustafterorpriortotransfer.
Inpreliminaryexperiments,BSAwasaddedat3mg/mltothemediawithhighosmolalities.However,afterseveraldaysofstorageat48C,themediadidnotworkwellforlipidseparation(i.e.,thepercentlipidseparationwasdecreased).ApossiblereasoncouldbethatthehighlychargedproteinsfromtheBSAchelateionsinthesolutionaltertheosmolality[33].Next,wedecreasedtheBSAconcentrationto0.1mg/mlorreplacedBSAwith0.1mg/mlPVA.BothPVAandalowerconcentrationofBSAworkedstablyforseparatingthelipids.Theembryoscouldbetreatedinlargegroups,severalhundredatatime.
In2006,wereportedtwolittersoftransgenicpigletsproducedfromcryopreservedNTembryos[10].Subsequently,Nagashimaetal.[11]reportedpigletsproducedfromcryopreservedIVPembryos.Here,wereportmorepracticalnoninvasivedelipationmethods(e.g.,nomicromanipulationisrequired),thusleavingthezonapellucidaintactbyhigh-osmolalitytreatmentpluscentrifugation.Theresultsreportedhereestablishaprocedurethatmayhaveimportantimplica-tionsforthecryopreservationofporcineIVPembryosonacommercialscale.ACKNOWLEDGMENTS
WewouldliketoacknowledgeLonnieDowellforhelpingtomanagethepigsatthefarm,JianguoZhaoforhelpingwiththenucleartransfer,andthepeoplewhodrovetotheslaughterhousetoretrieveovaries.
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