Fundamental aspects of cryobiology: sample, individual and species differences Bill Holt

Fundamental aspects of cryobiology: sample, individual and species differences Bill Holt

Fundamental aspects of cryobiology:
sample, individual and species
differences
Bill Holt
Which type of cell?
• For animal breeding
– Spermatozoa
– Oocytes
– Embryos
– Primordial germ cells
– Embryonic stem cells
– Somatic cells (as sources of genetics for nuclear transfer)
• For research
– Skin Fibroblasts (renewable sources of DNA, RNA, etc)
– Hepatocytes (for toxicology)
– Reproductive cells (e.g. for testing oestrogen-like activity)
What causes
cryoinjury?
Causes of cryoinjury
ATP depletion
Gain in cell Ca++
Loss of K+
Intracellular acidosis; pH 4.0
Stress
Intracellular
ice formation
Necrosis
Apoptosis
CELL LYSIS
Controlled cell
disassembly
Cryoinjury mechanisms
Cell
Lipids and proteins
randomly distributed
High extracellular CPA
concentration extracts water
pH7  4
Lipid phase
transitions
Membrane surface
Cells shrink during freezing
Temperature
-20 C
Cells shrink because
water is removed
during freezing
-80 C
Water
flows out
of the cell
during
freezing
TIME
Unshrunken cell, dead
on thawing
Shrunken cell, little or no
internal ice: live on thawing
Ice crystals and pockets of high salt
Sperm in ice
Frozen sperm freeze-substitution
Suggested mechanism of cooling effect
Rho proteins
RhoA. RhoB Rac1 and Cdc42
ROCK = Rho associated kinase
ROS
Generation
Death
receptor
Death inducing
signalling complex
ROCK activation…affecting actin
dynamics and membrane
permeability
Mitochondria
Caspase activation
Caspase activation
ROCK inhibitor Y-27632
Improves survival of hES cells
Apoptosis
New directions in cryopreservation
Novel media that use “intracellular fluid-like” solutions
ViaSpan (organ transplant media)
The guiding principles for the development of Viaspan were

osmotic concentration maintained by the use of metabolically inert substances like
lactobionate and raffinose rather than with glucose


Hydroxyethyl starch (HES) is used to prevent oedema

Other media; BioLife, CryoStor, Unisol 4, Adesta, Celsior
Substances are added to scavenge free radicals, along with steroids and insulin.
Targeted apoptotic control

Inhibition of Rho-Associated Kinases; used with hepatocytes, cord blood,
stem cells, fibroblasts, keratinocytes
Requirements of sperm cryopreservation
• For successful semen cryopreservation;
– Majority of spermatozoa should retain an intact plasma membrane; typically
~50% do not
– Cell functions in the “live” population should not be impaired. They usually
have a shortened lifespan
– All organelles should be intact and functional
– Sperm DNA should be intact and able to support development
Is there a unifying theoretical framework?
Can we answer these questions?
•
•
•
•
•
How do cryoprotectants really interact with cells?
Can we choose cryoprotectants with minimal DNA damage in mind?
How do we know when to use egg yolk (and why?)
Should we use antioxidants?
Which buffer solutions should we use?
Currently these questions cannot be answered but
presumably there is an explanation waiting to be found
some time
Example
Which cryoprotectant should be used?
Glycerol?
Egg yolk?
DMSO?
Methanol?
Acetamide?
Milk proteins?
%?
Raffinose?
Cholesterol?
How do we decide on the best technique?
• Ideally there should be some basic measurements of
sperm composition
– Plasma membrane lipids composition, cholesterol; phospholipid ratio,
saturated:unsaturated fatty acids
– Protein biochemistry
– Acrosome structure and composition
– Chromatin and DNA organisation
• Followed by predicting the optimal cryopreservation
technique
What really happens?
• Someone used to cattle, sheep or human sperm tries Glycerol + Egg
yolk
• If that doesn’t work we try DMSO
• Change the egg yolk concentration
• Or leave it out altogether!
• AND THEN: which cooling rate should be used?
Short-lifespan causes poor fertility
When are eggs present?
Frozen sperm
Fresh sperm
0
6
Sperm lifespan after insemination (h)
Insemination timing has to be accurate
24-36
Eventually a protocol is developed. E.g.
• Bull and ram sperm, typically using 6% glycerol and 20% EY
• Bull sperm in straws but ram sperm in pellets
•
•
•
•
•
Boar sperm in <3% glycerol + EY
Mouse sperm in 1.5% glycerol + Raffinose
Eagle sperm in dimethylacetamide
Fish sperm (medaka); 10% dimethyl formamide works well
Various frog sperm protocols usually use DMSO
Species differences
Species differences in sperm preservation
• Cryoprotectant differences
– Bull sperm….6-8% glycerol
– Pig sperm <3% glycerol
– Mouse sperm 1.5% glycerol
– Brush-tailed possum 4-8% glycerol
– African and Asian elephant 5% glycerol of DMSO
• Xenopus, Cane toad, Puerto Rican frog
– 5 – 15% DMSO with sucrose, FBS, Amphibian Ringer, etc.
Hypothesis for species differences
• The most established explanation (Darin-Bennett et al)
• Sperm with higher cholesterol; phospholipid ratios are
more robust
– e.g. Human and chicken > boar or sheep
COLD SHOCK RESISTANT v cold sensitive
This is a highly unsatisfactory; i.e. the lipid measurement is a
complex and intricate mean value and does not represent any
part of the cell
What causes between-male
differences?
Male-male differences have been observed in:






Humans
Rhesus monkey
pigs
dogs
Sheep
Iberian red deer
Between-male differences
• Within any given species the sperm lipid content should be
similar
– i.e. the sperm membrane fluidity has to be physiologically suitable
for fertilisation
• Between strain differences have been observed in mice
– These must be based on genetic differences
• Therefore we argued that male-male variation in other
species may also be genetically based
Between-male differences




129 boars screened for sperm cryosurvival
Tested for 5 x repeat ejaculates x 5 straws
Assessed for motility and viability
Genomic DNA assessed by AFLP
Thurston LM, Siggins K, Mileham AJ, Watson PF, Holt WV. 2002. Identification of
amplified restriction fragment length polymorphism markers linked to genes
controlling boar sperm viability following cryopreservation. Biol Reprod 66:545554.
Between-male differences
Data were combined and analysed using
multivariate cluster analysis
Three groups of boars were identified
Split into three groups
Poor (42 boars)
Medium (63 boars)
Good (24 boars)
Analysis of genomic DNA was undertaken
(AFLP) to search for differences between
“Poor” and “Good” boars
16 markers used
What might explain these results?
• Differences in membrane lipid composition?
– Known to differ between species
• Differences in age?
– Not in the pig study
• Differences in intrinsic ROS production?
– Possibly….one of the loci identified was close to the Glutathione
Peroxidase site
DNA
The main cargo of the sperm
DNA quality assessment objectives
• Some sperm may contain DNA that was:
– Inadequately modified during spermiogenesis
– Perhaps from spermatids in the process of apoptosis
• Some sperm may have been damaged by treatments
during processing (i.e. cooling and freezing)
• These processes produce single and double strand breaks
Fluorescence or Bright Field Microscopy
Ram
Dynamics of Sperm DNA Fragmentation
Sperm Samples
Incubated at 37ºC
Sperm fragmentation dynamics
Conclusions and observations
• We need to check the performance of our media
– Do the cryomedia induce apoptosis?
– Does the culture medium induce apoptosis?
– Are the media cell- or species-specific?
• Apoptosis requires involvement of gene expression and
enzyme activation, so this may be less important for sperm
• However, sperm cannot repair DNA, so this may be the
most important issue in gamete preservation
Conclusions and observations
• I have not discussed oocyte freezing as this has so far
proved completely unsuccessful.
• The impermeable outer layers prevent water efflux or entry
of cryoprotectant solutions
• There may be some merit in preheating cells and tissues
before cryopreservation to induce heat shock protein
production
Future directions
• Vitrification
– The use of high cryoprotectant concentrations and complete
avoidance of ice crystallisation
• Freeze-drying
– Freeze dried mouse sperm nuclei have been used for ICSI
– Freeze-dried somatic cell nuclei have been used in nuclear transfer
Source of cells Cultured
2-8 cells
Morula Blastocyst
Granulosa control
129
43
27 (20.9%)
Granulosa Freeze-dried
160
52
25 (15.6%)
Lino Loi et al (2008)