FESA Frozen Embryo

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Freezing mouse sperm

• FESA (Frozen Embryo Sperm Archive)
• Medical Research Council
• Harwell, UK
• Martin Fray

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FESA

• Worldwide Genetic Resource
• 1300 stocks, gt450,000 embryos
• Includes transgenic, mutants, chromosome
  anomalies inbred strains
• Sole UK archiving centre
• http//www.mgu.har.mrc.ac.uk
• EMMA (European Mouse Mutant Archive)
• IMSR (International Mouse Strain Resource)
• FIMRe (Federation of International Mouse
  Resources)

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Mary Lyon Centre
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What can be frozen?

• Pre-implantation embryos
• Oocytes
• Ovarian tissue
• Whole testis
• Spermatozoa

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Benefits of freezing

• Reduce number of GA mice on the shelf
• Safety from disease, fire, genetic contamination
  and breeding failure
• Larger range of stocks available
• Simplified exchange of stocks, nationally and
  internationally
• Economy
• Stocks remain viable indefinitely

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Storage of sperm stocks

• Store samples in duplicate
• Safe storage in vapour phase freezers
• Place cryo-boxes in LN2 bath immediately after
  removing them from the freezer

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Landmarks 1

• 1949 Parkes, Smith Polge
• Demonstrated cryoprotective properties of
  glycerol on fowl and human sperm
• 1952 Polge and Rowson
• Fertilizing capacity of bull sperm frozen at
  -79ºC
• 1953 Therapeutic use of frozen thawed human
  sperm started
• 1972 Ian Wilmut
• Survival of mouse embryos frozen in 1.5M DMSO in
  LN2
• 1972 Whittingham, Leibo Mazur
• Many live mice from embryos frozen in 1M DMSO in
  LN2

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Freezing mouse sperm

• 1990 Yokoyama et al used various combinations
  of raffinose, glycerol, DMSO and skim milk
• 1990 Tada et al used 18 raffinose in saline.
  Also glycerol and DMSO
• 1990 Okuyama et al used 18 raffinose plus 3
  skim milk
• 1991 Takeshima Nakagata refined Okuyamas
  method
• 1992 Nakagata Takeshima modified
  cryoprotectant elimination

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Types of cryoprotectants

• Alcohols (glycerol, propanediol)
• Aldehydes (formamide)
• Macromolecules (skim milk)
• Sugars (sucrose, raffinose, trehalose)
• Dimethylsulphoxide
• Sugars appear to minimise membrane damage
• Sztein et al (2001) Cryobiology

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Freezing mouse sperm

• Low tech in comparison with embryo freezing

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Sperm freezing equipment
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Sperm freezing apparatus
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Sperm freezing applications

• Archiving, plus DNA library
• Emergency cryo-preservation of sick males
• Export/import mutants
• Cheap, easy and reliable
• Small number of donors required
• Rapidly freeze down stocks

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Cryoprotective agent

• Preparation of 40ml of CPA
• 7.2g raffinose 1.2g skimmed milk powder 36 ml
  sterile water
• Place 36ml of sterile water into a 50ml
  centrifuge tube and equilibrate to 60?C in a
  water bath.
• Add 7.2g of raffinose and dissolve by gentle
  inversion return to water bath.
• Add 1.2g of skimmed milk powder to tube and mix
  by gentle inversion. Make up to 40ml if
  necessary.
• Aliquot into sterile microfuge tubes and
  centrifuge at 14,000rpm for 10 mins.
• Carefully tip off the supernatant and discard the
  pellets.
• Filter (0.45?m) the solution into a clean 50ml
  centrifuge tube.
• Aliquot 5-10ml CPA into 15ml tubes or 1.2ml into
  1.5 microfuge tubes.
• Store at -20?C.

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CPA - notes

• CPA is stable for 2 to 3 months
• The CPA will appear turbid
• You can make the CPA clearer by centrifuging
  longer and/or at higher speeds
• Alternatively - centrifuge the skimmed milk
  solution before adding the raffinose. The milk
  solids are pelleted through less dense solution
  and producing a clearer CPA (more involved
  protocol).
• CPA is essentially milk and sugar and an ideal
  bacterial culture medium.
• Discard any unused solution.

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Urinogenital tract mouse
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Dissected cauda vas
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Releasing sperm - cauda
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Releasing sperm - vas
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Freezing sperm in vials

• Sperm can be frozen in either straws or vials
• Place 1ml CPA in 35mm culture warm to 37ºC
• Dissect out vas deferens and cauda edidymus
• Mince cauda and vas in CPA
• Incubate at 37ºC for 10 min
• Place 100?l aliquots into labelled screw-top
  cryotubes
• Place tubes in LN2 vapour for 10 min
• Freezing rate approximately -20ºC/min
• Plunge into LN2 and store until required

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Thawing vials of sperm

• Wear eye protection
• Remove vial from holding tank and place in LN2
  bath
• Hold vial in air for 30sec - using pre-cooled
  forceps
• Wait for any LN2 to evaporate
• Explosion risk take care
• Plunge into 37ºC water bath and wait for ice
  crystals to dissolve
• Add 10µl of sperm to pre-prepared IVF dishes
• On thawing frozen/thawed sperm will appear non
  motile be patient!
• The frozen thawed/sperm will retain approximately
  50 motility

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Recovery using IVF

• Rapidly build up new stocks
• Fast-track embryo freezing
• Colony rescue
• Can achieve gt100 offspring per IVF, enough to
  provide a map position to 10-20 cM
• Produce cohorts of age matched animals exhibiting
  age related or progressive phenotype e.g. late
  onset deafness models
• Gene driven screens

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IVF using frozen sperm

• 1 x 10?l aliquot of frozen (C3H/HeH x BALB/c)F1
  sperm used to fertilise 420 (C3H/HeH x 101/H)F1
  oocytes in vitro
• 239, 2-cell embryos obtained (57)
• 112 transferred to 7 recipient females, 67
  animals born (60 of transferred)
• If all frozen sperm used in similar IVFs, we
  predict 6,700 offspring from this male

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Sperm freezing limitations

• Theoretically possible to recover 5,000-10,000
  mice from the frozen sperm of one male
• Limiting Factors
• No. of eggs available for IVF
• No. of recipient females
• Genotype dependent
• Nakagata (2000) Mammalian Genome 11, 572
• Perform IVF viability tests on stocks
• CASA

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Genotype effects

• BALB/c sperm
• 811 (C3H/HeH x 101/H)F1 eggs
• 30 2-cell embryos transferred
• 4 live born offspring
• C57BL/6 sperm
• 1,132 (C3H/HeH x 101/H)F1 eggs
• 116 2-cell embryos transferred
• 0 live born offspring
• 129 sub-strains also freeze/thaw poorly

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Poor sperm samples

• Micro-insemination (Intra-cytoplasmic sperm
  injection)
• Partial zona-pellucida dissection
• Nakagata et al (1997) Biol. Reprod. 57, 1050
• Zona thinning with acid tyrodes solution (pH 3.0)
  
• Personal communication (A Doyle TJL)
• Selection of motile sperm, plus removal of cell
  debris
• Bath (2003) Biol. Reprod 68, 19
• All methods require removal of the cumulus cells.
  

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Freezing without CPA

• Freeze dried sperm stored at 4ºC
• Ward et al (2003) Biol. Reprod. 69, 2100
• Spermatozoa/spermatids retrieved from
  reproductive tissues
• Ogonuki et al (2006) PNAS, 103, 13098
• Freezing in EDTA /Tris-HCL buffered saline
• Ward et al (2003) Biol. Reprod. 69, 2100
• Micro-insemination is required to recover live
  mice ICSI

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Transporting samples

• Secure labelled cryo-vials onto a cane using
  LN2 bath
• Place in fully charged dry shipper
• Short term transport on dry-ice is possible
  Okamoto et al (2001) Exp Anim, 50, 83
• Extended transport within refrigerated
  epididymides before freezing has been reported

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Archiving Summary

• Sperm
• Newer technology, 15 years
• Success dependent on genetic background
• Only haploid genotype, requires oocytes for IVF
• Simple, rapid cheap
• IVF more skilful
• Dissemination more difficult

• Embryos
• Well proven, gt30 years
• Success not particularly strain dependent
• Requires large numbers of mice
• Requires skilled personnel
• Dissemination is relatively easy

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