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The influence of cooling on fertilization of human oocytes
ABSTRACTS,
26th ANNUAL
constitutional supercooling during plane front freezing of single crystals might give a set of boundary conditions-temperature gradients in the solid and liquid, interface velocity-for the use of directional freezing of cryobiological systems. A single crystal ice plane front would zone refine out all the individual cells in a suspension of, for example, erythrocytes, if the cells were pushed ahead of the advancing ice front. The commercial single crystal techniques, Czochralski, Bridgemann, and Floating Zone, all employ a heat source as well as a heat sink during freezing. For unidirectional dendritic freezing, only a heat sink is employed. An upper bound for this type of freezing of cryobiological systems might occur when transverse heat flow becomes prevalent enough to cause significant equiaxed grains in an otherwise columnar structure. Again, assuming that the cells are pushed ahead of the ice dendrites, individual cells would experience the solute microsegregation which occurs in a volume element of interdendritic liquid. Depending on the ternary liquidus surface, this segregation may be lethal to the cells, if the intracellular liquid follows the segregation of the intercellular liquid. However, through this same mechanism, intracellular ice nucleation may be prevented. However, if the exchange across the cell membrane is too flow, the intracellular liquid may lag the intercellular liquid solute segregation, and lethal intracellular ice may nucleate. Hopefully, a window of survivability exists between the above limits for single crystal and unidirectional growth. SESSION I--GAMETE
PRESERVATION
15. The Influence of Cooling on Fertilization of Human Oocytes. B.J. FULLER, A. BERNARD, J. HUNTER, C. IFFLAND, W. REID, AND R. W. SHAW (Academic Department of Surgery and Obstetrics & Gynaecology, The Royal Free Hospital School of Medicine, London, England). Injury to living cells resulting from rapid cooling to temperature at or near O”C, in the absence of ice, has long been recognized. The phenomenon, which is termed cold shock, has been known to occur in some mammalian gametes (e.g., pig) and certain bacteria. It has also been shown that the meiotic spindle of human preovulatory oocytes are very sensitive to simple cooling and depolymerize even at 0°C. Although human embryos have been successfully stored at low temperatures, thawed, and transferred to recipients with resulting pregnancies, cryopreservation of the human oocyte has not shown such encouraging results. Whether or not this lack of success is a direct result of cellular injury brought about by cold shock is the purpose of our investigations. Six freshly donated human preovulatory oocytes were used in this series of experiments, and although in some terms this is a small
MEETING
539
number, ethically we were unable to justify a larger sample of very precious human material. After rapid cooling to and holding at 0°C for 30 min and rewarming to 37”C, five of the six eggs showed evidence of fertilization (two pronuclei) 16 hr postinsemination. Thus 30 min of cooling per se does not appear to inhibit subsequent fertilization, although other effects on spindle-chromosome integrity must be investigated. 16. Theoretical Modeling of the Kinetics of Internal Ice Formation during Freezing of Mouse 0ocytes.M. TONER,E. G. CRAVALHO,AND M. KAREL (Harvard-MIT Division of Health Sciences & Technology, and Department of Mechanical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139). The heterogeneous nucleation theory was modified to accommodate simultaneous temperature, volume, and concentration changes occurring during freezing. The concentration dependence of the viscosity of the cytoplasm was estimated based on Vand’s viscosity equation (J. Phys. Chem. 52, 277, 1947). The temperature dependence of the viscosity was estimated using a power law temperature dependence (Bengtzelius and Sjolander, J. Phys. ~17, 5915, 1984). The increase in the cytoplasmic viscosity during freezing-induced dehydration at various cooling rates was shown to affect the ice-nucleation rate. The exact temperature dependence of the Gibbs free energy was evaluated using the measurement of the specific heat of undercooled water (Angel1 et al., .I. Phys. Chem. 77,3092, 1973; Rasmussen and MacKenzie, J. Chem. Phys. 59,5003, 1973).It ’ was shown that the exact solution was in good agreement with the classical prediction of Hoffman (J. Chem. Phys. 29, 1192, 1958). It was also shown that the variation of the surface free energy with the size of the cluster could be ignored during freezing of biological cells without cryoadditives. Two active nucleating sites, namely, the plasma membrane and the supramolecular structures in the cytoplasm, were assumed to be effective in initiating the ice-nucleation within oocytes during freezing depending on the freezing conditions. The plasma membrane-catalyzed nucleation was characterized by the ice-plasma membrane contact angle, and the supramolecular particles-catalyzed nucleation was characterized by the ice-particle contact angle and the radius of the particle, in addition to two parameters, Q, and K,,, describing the homogeneous nucleation kinetics. A mathematical model based on stochastic nucleation was suggested to determine the nucleation rate from experimental freezing data. It was also suggested that the best approach to the determination of ice-nucleation parameters was to correlate the theory with freezing experiments at rapid rates (>40”C/min) to decouple the water transport equation from the ice-nucleation equations.
26th ANNUAL
constitutional supercooling during plane front freezing of single crystals might give a set of boundary conditions-temperature gradients in the solid and liquid, interface velocity-for the use of directional freezing of cryobiological systems. A single crystal ice plane front would zone refine out all the individual cells in a suspension of, for example, erythrocytes, if the cells were pushed ahead of the advancing ice front. The commercial single crystal techniques, Czochralski, Bridgemann, and Floating Zone, all employ a heat source as well as a heat sink during freezing. For unidirectional dendritic freezing, only a heat sink is employed. An upper bound for this type of freezing of cryobiological systems might occur when transverse heat flow becomes prevalent enough to cause significant equiaxed grains in an otherwise columnar structure. Again, assuming that the cells are pushed ahead of the ice dendrites, individual cells would experience the solute microsegregation which occurs in a volume element of interdendritic liquid. Depending on the ternary liquidus surface, this segregation may be lethal to the cells, if the intracellular liquid follows the segregation of the intercellular liquid. However, through this same mechanism, intracellular ice nucleation may be prevented. However, if the exchange across the cell membrane is too flow, the intracellular liquid may lag the intercellular liquid solute segregation, and lethal intracellular ice may nucleate. Hopefully, a window of survivability exists between the above limits for single crystal and unidirectional growth. SESSION I--GAMETE
PRESERVATION
15. The Influence of Cooling on Fertilization of Human Oocytes. B.J. FULLER, A. BERNARD, J. HUNTER, C. IFFLAND, W. REID, AND R. W. SHAW (Academic Department of Surgery and Obstetrics & Gynaecology, The Royal Free Hospital School of Medicine, London, England). Injury to living cells resulting from rapid cooling to temperature at or near O”C, in the absence of ice, has long been recognized. The phenomenon, which is termed cold shock, has been known to occur in some mammalian gametes (e.g., pig) and certain bacteria. It has also been shown that the meiotic spindle of human preovulatory oocytes are very sensitive to simple cooling and depolymerize even at 0°C. Although human embryos have been successfully stored at low temperatures, thawed, and transferred to recipients with resulting pregnancies, cryopreservation of the human oocyte has not shown such encouraging results. Whether or not this lack of success is a direct result of cellular injury brought about by cold shock is the purpose of our investigations. Six freshly donated human preovulatory oocytes were used in this series of experiments, and although in some terms this is a small
MEETING
539
number, ethically we were unable to justify a larger sample of very precious human material. After rapid cooling to and holding at 0°C for 30 min and rewarming to 37”C, five of the six eggs showed evidence of fertilization (two pronuclei) 16 hr postinsemination. Thus 30 min of cooling per se does not appear to inhibit subsequent fertilization, although other effects on spindle-chromosome integrity must be investigated. 16. Theoretical Modeling of the Kinetics of Internal Ice Formation during Freezing of Mouse 0ocytes.M. TONER,E. G. CRAVALHO,AND M. KAREL (Harvard-MIT Division of Health Sciences & Technology, and Department of Mechanical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139). The heterogeneous nucleation theory was modified to accommodate simultaneous temperature, volume, and concentration changes occurring during freezing. The concentration dependence of the viscosity of the cytoplasm was estimated based on Vand’s viscosity equation (J. Phys. Chem. 52, 277, 1947). The temperature dependence of the viscosity was estimated using a power law temperature dependence (Bengtzelius and Sjolander, J. Phys. ~17, 5915, 1984). The increase in the cytoplasmic viscosity during freezing-induced dehydration at various cooling rates was shown to affect the ice-nucleation rate. The exact temperature dependence of the Gibbs free energy was evaluated using the measurement of the specific heat of undercooled water (Angel1 et al., .I. Phys. Chem. 77,3092, 1973; Rasmussen and MacKenzie, J. Chem. Phys. 59,5003, 1973).It ’ was shown that the exact solution was in good agreement with the classical prediction of Hoffman (J. Chem. Phys. 29, 1192, 1958). It was also shown that the variation of the surface free energy with the size of the cluster could be ignored during freezing of biological cells without cryoadditives. Two active nucleating sites, namely, the plasma membrane and the supramolecular structures in the cytoplasm, were assumed to be effective in initiating the ice-nucleation within oocytes during freezing depending on the freezing conditions. The plasma membrane-catalyzed nucleation was characterized by the ice-plasma membrane contact angle, and the supramolecular particles-catalyzed nucleation was characterized by the ice-particle contact angle and the radius of the particle, in addition to two parameters, Q, and K,,, describing the homogeneous nucleation kinetics. A mathematical model based on stochastic nucleation was suggested to determine the nucleation rate from experimental freezing data. It was also suggested that the best approach to the determination of ice-nucleation parameters was to correlate the theory with freezing experiments at rapid rates (>40”C/min) to decouple the water transport equation from the ice-nucleation equations.