CRYOPRESERVATIONOFMOUSESPERMATOZOA

Note:
MethodfromPeterH.Glenister&ClaireE.Thornton(1998)(MRCMammalianGenetics
Unit, Harwell, OX11 0RD, UK) adapted from original descriptions of N. Nakagata
(UniversityofTokyo,Japan)andJ.Sztein(TheJacksonLaboratory,USA).Thefollowing
protocolispartofaonedayintensivecourseon"Spermcryopreservation"givenatMRCin
Harwell(UK)byPeterH.GlenisterandClaireE.Thornton.

Internetlinks:
http://www.mgu.har.mrc.ac.uk/sperm.html
sperm@har.mrc.ac.uk

References:
ThorntonC.E.,BrownS.D.M,GlenisterP.H.(1999).Largenumbersofmiceestablishedbyin
vitrofertilizationwithcryopreservedspermatozoa:implicationsandapplicationsfor
geneticresourcebanks,mutagenesisscreens,andmousebackcrosses. Mammalian
Genome10:987992.

NakagataN.(1994).Cryopreservationofembryosandgametesinmice.Exp.Anim.43:11
18.

MarschallS.,HrabdeAngelis,M.(1999)Cryopreservationofmousespermatozoa:double
yourmousespace.TrendsinGenetics15(4):128131.

Materials:

Cryoprotectantsolution
1.8mlNunccryotubes
Cryotuberack
Eppendorfcentrifuge5415Cand1.5mlEppendorftubes
35mmcellculturePetridishes(e.g.Falcon3001)
Deeppolystyreneboxwithlidsuitableforholdingliquidnitrogen
SmallDewarflaskofliquidnitrogen
CO2incubator(37oC,5%CO2)
Hotblockat37oC
Sexuallymaturemalemiceatleast8weeksold(preferablynotrecentlymated)

CryoprotectiveAgent(CPA):

18%raffinose(SigmaR7630),3%skimmilk(DifcoBetalab003217)

Place9mlofSigmawater(SigmaW1503)inascrewtop15mlFalcontube(#2097)and
equilibrateto60 oCinawaterbath.Add1.8gofraffinoseanddissolvebygentleinversion.
Add0.3gofskimmilkanddissolvebygentleinversion.Makeupto10mlifnecessary.
AliquotintoEppendorftubesandcentrifugueat14,000rpmfor10min.Tipoffsupernatant

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andMilliporefilter(0.20.45m)intocryotubesorEppendorftubes.Storeat20 oC(1.1ml
aliquots).

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CryopreservationMethod:

1).Preparethecoolingapparatus.Placeaplatform,(forexample,theinsertfromaGilson
yellow tip box), into the polystyrene box. This acts as a support for the cryotube rack.
Carefullypourliquidnitrogenintothepolystyreneboxtojustcovertheplatform.Placea
cryotuberackontopoftheplatformsothatitissuspendedinliquidnitrogenvapour.Replace
thelidofthepolystyreneboxandallowittofillwithvapour.Replenishtheliquidnitrogenas
necessaryduringthefreezingsession,butdonotallowtheleveltoriseabovetheplatform.

2).Thawonealiquotofcryoprotectantsolutionforeachmalemouseandbringto37oCinthe
incubatororhotblock.Mixbyinversionifthereisanyprecipitation.

3).Pipette1.1mlCPAintoasmallFalcon35mmdish(#3001)onthehotblockat37 oC.
Dissectthevasdeferensandcaudaepididymesfromthemouseandcleanoffallfatandblood.
This is best achieved by placing the organs on a tissue and examining them under a
microscope.Usingwatchmaker'sforceps,micethecaudaeandsqueezethespermgentlyout
ofthevasdeferens.Todispersethesperm,tapandshakethedishgentlyforabout30sec.
PlaceinaCO2incubatorat37oCfor10minutes,restingthespermdishatanangleonthelid.

4).Keepingthedishatanangle,removetheepididymalandvastissuefromthesuspension
byscrapingthemtoonesideofthedish.Aliquot100lintoeachof10cryotubes(or200l
aliquotsin5cryotubes).Replacethescrewcapandtightentosealthecryotube.

5).Placethecryotubesintotheprecooledfreezingapparatus(polystyrenebox)andleavefor
10minutes.

6). Remove and plunge into liquid nitrogen. Store in liquid nitrogen refrigerator until
required.

P.H.Glenister&C.E.Thornton(1998)
MRCMammalianGeneticsUnit,Harwell,OX110RD,UK

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 THAWINGMOUSESPERMATOZOAANDINVITRO
FERTILISATION(IVF)
Materials:

Aliquot(s)offrozensperm
Waterbathat37oC
Eppendorfcentrifuge5415Cand1.5mlEppendorftubes
CO2incubator(37oC,5%CO2)
Superovulatedfemalemice
MediumforIVF
Mineraloil(SigmaM8410)
35mmcellculturePetridishes(e.g.Falcon3001)
60mmcellculturePetridishes(e.g.Falcon3004)
MediumM2containing0.4%w/vBSA
+0.5d.p.c.pseudopregnantfemalemice

MEMmediumforIVF:

WeuseSigmaMEM(SigmaM4655)withadditionsasfollowsper100ml:

2.5mg NaPyruvate(SigmaP2256)
0.38mg EDTA(SigmaE5134)
7mg Penicillin(SigmaP4687)
5mg Streptomycin(SigmaS1277)
300mg BSA(SigmaA3311)

DissolvetheBSAwithoutshaking.Filtersterilise~10mlaliquotsintoFalcontubes(#2001).
Themediumcanbefrozenat20oC.Iffrozenmediumisused,refilteronthawing.

Note:MediumM16canbeusedaswellforIVF.

ThawingmethodandIVF:

1). The day before IVF, prepare an appropriate number of IVF dishes and preincubate
overnighttoequilibrate.AsetofdishesforIVFconsistsof:

A35mmPetridishcontaining~3mlIVFmediumforoocytecollection.
Afertilisationdish(35mm)containinga200ldropofIVFmediumoverlaidwithmineral
oil.
Awashdish(60mm)containingfour100ldropsofIVFmediumoverlaidwithmineraloil.
Aculturedish(60mm)containingfour100ldropsofIVFmediumoveralidwithmineral
oil.

2).Usingforceps,holdthecryotubeinairfor30sec,andthenthawrapidlybyplacingina
37oCwaterbath.Takespecialcarethatthetubehasnotfilledwithliquidnitrogenbefore

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plungingintothewaterbath(suchtubesmayexplode!).Ifliquidnitrogenispresentinthe
tube,waitforittoevaporateandescapefirst.

3).Whenthesampleisthawed,pipetteintoanEppendorftube.Centrifugueat3,000rpmfor
4minutes.Discardthesupernatantandresuspendthepelletin55 lofIVFmedium(pre
incubatedat37oC).Flickthetubetoliftthepelletoffthebottomandthenincubateat37 oC,
5%CO2for10minutestoallowresuspensionofsperm.Take10laliquotsandpipettegently
intothefertilisationdishes.Thereshouldbeenoughspermfor5x200lfertilisationdrops.
Donotpipettethespermupanddowninthedrop.

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4). Dissect oviducts from 5 superovulated female mice per fertilisation drop. Place the
oviductsintopreincubatedIVFmedium,pindownwithforceps,andtearoutthecumulus
masses.Usinga1mlGilsonsetat100ml,transferthe10cumulusmassesintooneofthe
fertilisationdropsbeingcarefultotransferaslittleIVFmediumaspossible.Repeatforeach
fertilisationdish.Incubateat37oC,5%CO2for~5hours(37hours).

5).Pickuptheeggsfromthefertilisationdropsandwashthrough4x100ldropsofpre
incubatedIVFmedium(onewashdishforeachfertilisationdish).Transferthewashedeggs
totheculturedish.

6).Incubateovernight.Nextdaytransfer2cellembryostoaholdingdishofM2mediumat
roomtemperatureandthentotheoviductsof+0.5d.p.c.pseudopregnantfostermothers(58
peroviduct).

P.H.Glenister&C.E.Thornton(1998)
MRCMammalianGeneticsUnit,Harwell,OX110RD,UK

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Hintsandtips:

ThetimingofthehCGhormoneinjectionandthecollectionoftheovulatedeggsisimportant.
If"old"eggsareusedmanybecomeresistanttofertilisationandmayundergoparthenogenic
activation.WefindthathCGadministeredapprox.18hoursfollowedbytheadditionofeggs
tothespermsuspensionbeforeapprox.8hoursthefollowingday,worksforourparticular
strainofmice.Otherstrainsmaywellreactdifferently.

MEMiscertainlynottheonlymediumthatcanbeusedforIVF.OtherpossibilitiesareHTF
andT6,recipesforwhicharewelldocumented.Differentmediamayworkbetterfordifferent
strains.

Dividinga100 lspermsamplebetween5fertilisationdishesora200 lspermsample

between10fertilisationdishesmaymaximisetheyieldoffertilisedeggs.Italsocreatedmore
work, uses a lot of mice and potentially reduces the concetration of sperm capable of
fertilisation.Thissaturationapproachisonlyrecommendedfor"good"strainsofmalesand
wherelargenumbersofoffspringsarerequired.Wehavesuccessfullyusedthewholeofone
spermsamplefroma"poor"straininone500lfertilisationdrop.Inthiscasethenumberof
offspringwasmuchreducedbutstillallowedreestablishmentofthestock.

ResultsobtainedatMGU,Harwell:

The following results have been obtained using frozen/thawed sperm from (C3H/HeH x
BALB/c)F1tofertilisefresh(C3H/HeHx101/H)F1eggsinvitro.

Spermvolume No.fertilisationdrops No.liveborn

50l 1 35
50l 1 28
50l 1 36
50l 2 82
50l 3 65
50l 5 108
100l 10 177

Sperm volume refers to the volume of thawed sperm recovered after centrifugation and
resuspensioninIVFmedium.Nothethatwherethenumberoffertilisationdropsis>1the
sperm suspension, after centrifugation, was divided equally between the drops. As we
normallyfreeze10x100lof5x200laliquotspermalewecanpredictforthisparticular
cross,thatispossibletorecoverapprox.1000livebornoffspringfromthefrozenspermofone
male.

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References

OkuyamaM,IsogaiS,SagaM,HamadaH,OgawaS(1990)J.Fert.Implant.(Tokyo),7,116.

NakagataN,TakeshimaT(1992)Theriogenology,37,1283.

SzteinJM,FarleyJS,YoungAF,MobraatenLE(1997)Cryobiology,35,46.

HuffstadtU,BallingR(personalcommunication,1997)

SzteinJM,FarleyJS,YoungAF(personalcommunication,1997)

WoodMJ,WhittinghamDG(personalcommunication,1997)