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)