Mechanism of cryoprotection by extracellular polymeric solutes.

To elucidate the means by which polymer solutions protect cells from freezing injury, we cooled human monocytes to -80 degrees C or below in the presence of various polymers. Differential scanning calorimetric studies showed that those polymers which protect cells best have a limiting glass transition temperature (T'g) of approximately -20 degrees C; those with a T'g significantly higher or lower did not protect. Freeze-etch electron micrographs indicated that intracellular ice crystals had formed during this freezing procedure, but remained smaller than approximately 300 nm in the same proportion of cells as survived rapid thawing. We propose that cryoprotection of slowly frozen monocytes by polymers is a consequence of a T'g of -20 degrees C in the extracellular solution. In our hypothesis, the initial concentration and viscosity of protective polymer solutions reduce the extent and rate of cell water loss to extracellular ice and limit the injurious osmotic stress, which cells face during freezing at moderate rates to -20 degrees C. Below -20 degrees C, glass formation prevents further osmotic stress by isolating cells from extracellular ice crystals, virtually eliminating cell water loss at lower temperatures. On the other hand, the protective polymer solutions will allow some diffusion of water away from cells at temperatures above T'g. If conditions are correct, cells will concentrate the cytoplasm sufficiently during the initial cooling to T'g to avoid lethal intracellular freezing between T'g and the intracellular Tg, which has been depressed to low temperatures by that concentration. Thus, when polymers are used as cryoprotective agents, cell survival is contingent upon maintenance of osmotic stress within narrow limits.     

Seasonal variation in freeze tolerance and ice content of the tree frog Hyla versicolor

Freeze tolerance and ice content of Hyla versicolor showed pronounced variation between summer (June) and winter (December). Summer frogs survived freezing at -3 degrees C for up to 9 hr and ice accumulation up to 50% of their total body water. A time course of ice formation indicated that an equilibrium level was reached in approximately 15 hr. Thus, the lethal ice content was less than the equilibrium ice content for these conditions (63.1%). A second group was induced to enter an overwintering condition by holding them through the summer and then subjecting them to a progressive reduction in temperature and photoperiod for 2 months. These frogs survived freezing for 48 hr at -3 degrees C. Their equilibrium ice content at this temperature was significantly lower (52.5%) than comparably treated summer animals. In the winter acclimatized group, frozen frogs had substantially higher blood glucose levels than unfrozen frogs (22.7 mumol/ml vs. 1.33 mumol/ml), but glycerol levels were not elevated after freezing. Freezing frogs conditioned for overwintering at -7 degrees C resulted in a higher equilibrium ice content (62.6%), but none survived. It is evident that in preparation for overwintering, frogs reduce the amount of ice formed at a given subzero temperature, but there is little indication of a substantial change in the total amount of ice tolerated.     

Mutagenic effect of freezing on mitochondrial DNA of Saccharomyces cerevisiae

Although suggested in some studies, the mutagenic effect of freezing has not been proved by induction and isolation of mutants. Using a well-defined genetic model, we supply in this communication evidence for the mutagenic effect of freezing on mitochondrial DNA (mtDNA) of the yeast Saccharomyces cerevisiae. The cooling for 2 h at +4 degrees C, followed by freezing for 1 h at -10 degrees C and 16 h at -20 degrees C resulted in induction of respiratory mutations. The immediate freezing in liquid nitrogen was without mutagenic effect. The study of the stepwise procedure showed that the induction of respiratory mutants takes place during the freezing at -10 and -20 degrees C of cells pre-cooled at +4 degrees C. The genetic crosses of freeze-induced mutants evidenced their mitochondrial rho- origin. The freeze-induced rho- mutants are most likely free of simultaneous nuclear mutations. The extracellular presence of cryoprotectants did not prevent the mutagenic effect of freezing while accumulation of cryoprotectors inside cells completely escaped mtDNA from cryodamage. Although the results obtained favor the notion that the mutagenic effect of freezing on yeast mtDNA is due to formation and growth of intracellular ice crystals, other reasons, such as impairment of mtDNA replication or elevated levels of ROS production are discussed as possible explanations of the mutagenic effect of freezing. It is concluded that: (i) freezing can be used as a method for isolation of mitochondrial mutants in S. cerevisiae and (ii) given the substantial development in cryopreservation of cells and tissues, special precautions should be made to avoid mtDNA damage during the cryopreservation procedures.     

Contribution of extracellular ice formation and the solution effects to the freezing injury of PC-3 cells suspended in NaCl solutions

The mechanism of cell injury during slow freezing was examined using PC-3 human prostate adenocarcinoma cells suspended in NaCl solutions. The objective was to evaluate contribution of extracellular ice and the 'solution effects' to freezing injury separately. The solution effects that designate the influence of elevated concentration were evaluated from a pseudo-freezing experiment, where cells were subjected to the milieu that simulated a freeze-thaw process by changing the NaCl concentration and the temperature at the same time. The effect of extracellular ice formation on cell injury was then estimated from the difference in cell survival between the pseudo-freezing experiment and a corresponding freezing experiment. When cells were frozen to a relatively higher freezing temperature at -10 degrees C, about 30% of cells were damaged mostly due to extracellular ice formation, because the concentration increase without ice formation to 2.5-M NaCl, i.e., the equilibrium concentration at -10 degrees C, had no effect on cell survival. In contrast, in the case of the lower freezing temperature at -20 degrees C, about 90% of cells were injured by both effects, particularly 60-80% by the solution effects among them. The present results suggested that the solution effects become more crucial to cell damage during slow freezing at lower temperatures, while the effect of ice is limited to some extent.     

Avoidance of intracellular freezing by the freezing-tolerant New Zealand Alpine weta Hemideina maori (Orthoptera: Stenopelmatidae)

Calorimetric analysis indicates that 82% of the body water of Hemideina maori is converted into ice at 10 degrees C. This is a high proportion and led us to investigate whether intracellular freezing occurs in H. maori tissue. Malpighian tubules and fat bodies were frozen in haemolymph on a microscope cold stage. No fat body cells, and 2% of Malpighian tubule cells froze during cooling to -8 degrees C. Unfrozen cells appeared shrunken after ice formed in the extracellular medium. There was no difference between the survival of control tissues and those frozen to -8 degrees C. At temperatures below -15 degrees C (lethal temperatures for weta), there was a decline in survival, which was strongly correlated with temperature, but no change in the appearance of tissue. It is concluded that intracellular freezing is avoided by Hemideina maori through osmotic dehydration and freeze concentration effects, but the reasons for low temperature mortality remain unclear. The freezing process in H. maori appears to rely on extracellular ice nucleation, possibly with the aid of an ice nucleating protein, to osmotically dehydrate the cells and avoid intracellular freezing. The lower lethal temperature of H. maori (-10 degrees C) is high compared to organisms that survive intracellular freezing. This suggests that the category of 'freezing tolerance' is an oversimplification, and that it may encompass at least two strategies: intracellular freezing tolerance and avoidance.     

Enhanced survival of yeast expressing an antifreeze gene analogue after freezing.

Yeast, like most organisms, survives poorly under freezing conditions. It has been proposed that after rapid cooling yeast suffers a loss in viability from the recrystallization of intracellular ice. Antifreeze proteins found in the blood of certain polar fishes have been shown to be potent inhibitors of ice recrystallization at very low concentrations. We have examined the feasibility of protecting rapidly cooled yeast cells from freezing damage by inhibiting the recrystallization of intracellular ice through in vivo expression of an antifreeze analogue gene. A chemically synthesized gene encoding a protein similar to but differing from the antifreeze proteins of the fish Pseudopleuronectes americanus (winter flounder) was genetically fused to the 3' end of a truncated staphylococcal Protein A gene. When the fused gene was expressed in the budding yeast Saccharomyces cerevisiae, its cells were shown to produce a new chimeric protein that inhibited the recrystallization of ice in vitro. Yeast cells expressing the chimeric antifreeze protein showed a twofold increase in survival after rapid freezing (95 degrees C/min to -196 degrees C) and moderate rates of warming (26 to 64 degrees C/min) compared to cells lacking the chimeric protein.     

Subzero water permeability parameters of mouse spermatozoa in the presence of extracellular ice and cryoprotective agents.

Optimization of techniques for cryopreservation of mammalian sperm is limited by a lack of knowledge regarding water permeability characteristics during freezing in the presence of extracellular ice and cryoprotective agents (CPAs). Cryomicroscopy cannot be used to measure dehydration during freezing in mammalian sperm because they are highly nonspherical and their small dimensions are at the limits of light microscopic resolution. Using a new shape-independent differential scanning calorimeter (DSC) technique, volumetric shrinkage during freezing of ICR mouse epididymal sperm cell suspensions was obtained at cooling rates of 5 and 20 degrees C/min in the presence of extracellular ice and CPAs. Using previously published data, the mouse sperm cell was modeled as a cylinder (122-microm long, radius 0.46 microm) with an osmotically inactive cell volume (V(b)) of 0.61V(o), where V(o) is the isotonic cell volume. By fitting a model of water transport to the experimentally obtained volumetric shrinkage data, the best-fit membrane permeability parameters (L(pg) and E(Lp)) were determined. The "combined best-fit" membrane permeability parameters at 5 and 20 degrees C/min for mouse sperm cells in solution are as follows: in D-PBS: L(pg) = 1.7 x 10(-15) m(3)/Ns (0.01 microm/min-atm) and E(Lp) = 94.1 kJ/mole (22.5 kcal/mole) (R(2) = 0.94); in "low" CPA media (consisting of 1% glycerol, 6% raffinose, and 15% egg yolk in D-PBS): L(pg)[cpa] = 1.7 x 10(-15) m(3)/Ns (0.01 microm/min-atm) and E(Lp)[cpa] = 122.2 kJ/mole (29.2 kcal/mole) (R(2) = 0.98); and in "high" CPA media (consisting of 4% glycerol, 16% raffinose, and 15% egg yolk in D-PBS): L(pg)[cpa] = 0.68 x 10(-15) m(3)/Ns (0.004 microm/min-atm) and E(Lp)[cpa] = 63.6 kJ/mole (15.2 kcal/mole) (R(2) = 0.99). These parameters are significantly different than previously published parameters for mammalian sperm obtained at suprazero temperatures and at subzero temperatures in the absence of extracellular ice. The parameters obtained in this study also suggest that damaging intracellular ice formation (IIF) could occur in mouse sperm cells at cooling rates as low as 25-45 degrees C/min, depending on the concentrations of the CPAs. This may help to explain the discrepancy between the empirically determined optimal cryopreservation cooling rates, 10-40 degrees C/min, and the numerically predicted optimal cooling rates, greater than 5000 degrees C/min, obtained using suprazero mouse sperm permeability parameters that do not account for the presence of extracellular ice. As an independent test of this prediction, the percentages of viable and motile sperm cells were obtained after freezing at two different cooling rates ("slow" or 5 degrees C/min; "fast," or 20 degrees C/min) in both the low and high CPA media. The greatest sperm motility and viability was found with the low CPA media under fast (20 degrees C/min) cooling conditions.     

Stabilization of frozen Lactobacillus delbrueckii subsp. bulgaricus in glycerol suspensions: Freezing kinetics and storage temperature effects

The interactions between freezing kinetics and subsequent storage temperatures and their effects on the biological activity of lactic acid bacteria have not been examined in studies to date. This paper investigates the effects of three freezing protocols and two storage temperatures on the viability and acidification activity of Lactobacillus delbrueckii subsp. bulgaricus CFL1 in the presence of glycerol. Samples were examined at -196 degrees C and -20 degrees C by freeze fracture and freeze substitution electron microscopy. Differential scanning calorimetry was used to measure proportions of ice and glass transition temperatures for each freezing condition tested. Following storage at low temperatures (-196 degrees C and -80 degrees C), the viability and acidification activity of L. delbrueckii subsp. bulgaricus decreased after freezing and were strongly dependent on freezing kinetics. High cooling rates obtained by direct immersion in liquid nitrogen resulted in the minimum loss of acidification activity and viability. The amount of ice formed in the freeze-concentrated matrix was determined by the freezing protocol, but no intracellular ice was observed in cells suspended in glycerol at any cooling rate. For samples stored at -20 degrees C, the maximum loss of viability and acidification activity was observed with rapidly cooled cells. By scanning electron microscopy, these cells were not observed to contain intracellular ice, and they were observed to be plasmolyzed. It is suggested that the cell damage which occurs in rapidly cooled cells during storage at high subzero temperatures is caused by an osmotic imbalance during warming, not the formation of intracellular ice.     

Depression of the ice-nucleation temperature of rapidly cooled mouse embryos by glycerol and dimethyl sulfoxide.

The temperature at which ice formation occurs in supercooled cytoplasm is an important element in predicting the likelihood of intracellular freezing of cells cooled by various procedures to subzero temperatures. We have confirmed and extended prior indications that permeating cryoprotective additives decrease the ice nucleation temperature of cells, and have determined some possible mechanisms for the decrease. Our experiments were carried out on eight-cell mouse embryos equilibrated with various concentrations (0-2.0 M) of dimethyl sulfoxide or glycerol and then cooled rapidly. Two methods were used to assess the nucleation temperature. The first, indirect, method was to determine the in vitro survival of the rapidly cooled embryos as a function of temperature. The temperatures over which an abrupt drop in survival occurs are generally diagnostic of the temperature range for intracellular freezing. The second, direct, method was to observe the microscopic appearance during rapid cooling and note the temperature at which nucleation occurred. Both methods showed that the nucleation temperature decreased from - 10 to - 15 degrees C in saline alone to between - 38 degrees and - 44 degrees C in 1.0-2.0 M glycerol and dimethyl sulfoxide. The latter two temperatures are close to the homogeneous nucleation temperatures of the solutions in the embryo cytoplasm, and suggest that embryos equilibrated in these solutions do not contain heterogeneous nucleating agents and are not accessible to any extracellular nucleating agents, such as extracellular ice. The much higher freezing temperatures of cells in saline or in low concentrations of additive indicate that they are being nucleated by heterogeneous agents or, more likely, by extracellular ice.     

Protective effect of intracellular ice during freezing?

Injury results during freezing when cells are exposed to increasing concentrations of solutes or by the formation of intracellular ice. Methods to protect cells from the damaging effects of freezing have focused on the addition of cryoprotective chemicals and the determination of optimal cooling rates. Based on other studies of innocuous intracellular ice formation, this study investigates the potential for this ice to protect cells from injury during subsequent slow cooling. V-79W Chinese hamster fibroblasts and Madin-Darby Canine Kidney (MDCK) cells were cultured as single attached cells or confluent monolayers. The incidence of intracellular ice formation (IIF) in the cultures at the start of cooling was pre-determined using one of two different extracellular ice nucleation temperatures (-5 or -10 degrees C). Samples were then cooled at 1 degrees C/min to the experimental temperature (-5 to -40 degrees C) where samples were warmed rapidly and cell survival assessed using membrane integrity and metabolic activity. For single attached cells, the lower ice nucleation temperature, corresponding to increased incidence of IIF, resulted in decreased post-thaw cell recovery. In contrast, confluent monolayers in which IIF has been shown to be innocuous, show higher survival after cooling to temperatures as low as -40 degrees C, supporting the concept that intracellular ice confers cryoprotection by preventing cell dehydration during subsequent slow cooling.     

Ultrastructure before freezing,while frozen,and after thawing in assessing cryoinjury of mouse epididymal spermatozoa

Tails of mouse epididymides were treated as follows: control, unfrozen with and without cryoprotective agents (CPA); frozen (to below ?80 °C), slowly (8 °C/min), and rapidly (18 °C/sec), with and without CPA. Intracellular and/or extracellular location of CPA, at least glycerol, was influenced, respectively, by high (22 °C) or low (0 °C) exposure temperature. Standard procedures in electron microscopy were employed and the frozen state preserved by freeze-substitution. Motility before freezing and after thawing was the criterion of cryosurvival.Results showed no evidence of deleterious ultrastructural effects of freezing at rates compared, or of benefits of CPA, regardless of their cellular location. Differences were noted, however, in the appearance of spermatozoa in the frozen state, as a function of the rate of freezing but not as a function of the presence, absence, or location of either glycerol of DMSO. Rapidly frozen cells showed intracellular ice formation in the acrosome, neck, midpiece, and tail regions; there was no intranuclear ice, and extracellular ice artifacts were small. Slowly frozen cells showed large extracellular ice artifacts with evidence of shrinkage distortion due to the dehydration induced by extracellular ice. No spermatozoa survived any of the freezing treatments, showing the lethal effect of both extracellular ice during slow freezing and of intracellular and/or extracellular ice during rapid freezing.     

Roles of the plasma membrane and the cell wall in the responses of plant cells to freezing

In an effort to clarify the responses of a wide range of plant cells to freezing, we examined the responses to freezing of the cells of chilling-sensitive and chilling-resistant tropical and subtropical plants. Among the cells of the plants that we examined, those of African violet ( Saintpaulia grotei Engl.) leaves were most chilling-sensitive, those of hypocotyls in mungbean [ Vigna radiata (L.) R. Wilcz.] seedlings were moderately chilling-sensitive, and those of orchid [ Paphiopedilum insigne (Wallich ex Lindl.) Pfitz.] leaves were chilling-resistant, when all were chilled at -2 degrees C. By contrast, all these plant cells were freezing-sensitive and suffered extensive damage when they were frozen at -2 degrees C. Cryo-scanning electron microscopy (Cryo-SEM) confirmed that, upon chilling at -2 degrees C, both chilling-sensitive and chilling-resistant plant cells were supercooled. Upon freezing at -2 degrees C, by contrast, intracellular freezing occurred in Saintpaulia leaf cells, frost plasmolysis followed by intracellular freezing occurred in mungbean seedling cells, and extracellular freezing (cytorrhysis) occurred in orchid leaf cells. We postulate that chilling-related destabilization of membranes might result in the loss of the ability of the plasma membrane to act as a barrier against the propagation of extracellular ice in chilling-sensitive plant cells. We also examined the role of cell walls in the response to freezing using cells in which the plasma membrane had been disrupted by repeated freezing and thawing. In chilling-sensitive Saintpaulia and mungbean cells, the cells with a disrupted plasma membrane responded to freezing at -2 degrees C by intracellular freezing. By contrast, in chilling-resistant orchid cells, as well as in other cells of chilling-resistant and freezing-resistant plant tissues, including leaves of orchard grass ( Dactylis glomerata L.), leaves of Arabidopsis thaliana (L.) Heynh. and cortical tissues of mulberry ( Morus bombycis Koids.), cells with a disrupted plasma membrane responded to freezing by extracellular freezing. Our results indicate that, in the chilling-sensitive plants cells that we examined, not only the plasma membrane but also the cell wall lacked the ability to serve as a barrier against the propagation of extracellular ice, whereas in the chilling-resistant plant cells that we examined, not only the plasma membrane but also the cell wall acted as a barrier against the propagation of extracellular ice. It appears, therefore, that not only the plasma membrane but also the cell wall greatly influences the freezing behavior of plant cells.     

Vitrification in plants as a natural form of cryoprotection

A small group of woody plants from the far northern hemisphere can, while in the dormant state, tolerate freezing and thawing to and from any subzero temperature at rates less than 30 degrees C/hr. In addition, the hardiest of them can tolerate cooling and warming between -20 degrees C and any colder temperature at virtually any combination of rates subsequent to cooling to -20 degrees C at rates less than 5 degrees C/hr. We term this latter capability "quench hardiness." I and my colleagues have shown that the limits of this quench hardiness can be closely correlated to the stability of intracellular glasses formed during the slow cooling of hardy tissues in the presence of extracellular ice. In this paper, I briefly review the evidence for intracellular glass formation and present data indicating that major components of the glass forming solutions are raffinose and stachyose. Evidence from differential scanning calorimetry that sugar-binding soluble proteins are also important is presented. Finally, I correlate data from our work with that of other workers to make the case that, even when most of a cytoplasmic solution is vitrified, fluid microdomains remain which can lead to long-term biodegradation during storage at high subzero temperatures.     

Cold hardiness abilities vary with the size of the land snail Cornu aspersum

The land snail Cornu aspersum (syn. Helix aspersa) living in Brittany (France) can be considered partially freezing tolerant as it possesses a low ability to supercool and a limited capacity to bear freezing of its body tissues. The absence of a marked cold hardiness strategy permits the emphasis of the role of parameters such as individual size or water mass (W(M)) contained by the organism. Adult snails (shell diameter 30-32 mm) had a supercooling ability, about 1-1.5 degrees C lower than that of immatures (shell diameter 12-20 mm) and survived longer to an exposure to -5 degrees C, with an Lt(50) comprised between 6.0 and 9.8 h against 2.6 to 4.2 h for immature snails. This better ability to bear freezing was explained by the faster dynamic of body ice formation observed in small individuals, which attained ice lethal quantity more rapidly. At the species level, large snails will then tend to be more tolerant to freezing and small ones to be freezing avoidant, a statement also observable at the phylum level.     

Intracellular freezing of glycerolized red cells.

The response of glycerolized human red blood cells to freezing has been evaluated in terms of the thermodynamic state of the frozen intracellular medium. The physiochemical conditions requisite for intracellular freezing, characterized by the cooling rate and the degree of extracellular supercooling, are altered appreciably by the prefreezing addition of glycerol to the cells.Fresh human erythrocytes were suspended in an isotonic glycerol solution yielding a final cryophylactic concentration of either 1.5 or 3.0 m. Subsequently the cell suspension was frozen on a special low temperature stage, mounted on a light microscope, at controlled constant cooling rates with varying degrees of extracellular supercooling (ΔT_sc). The formation of a pure intracellular ice phase was detected by direct observation of the cells.The addition of glycerol produced several significant variations in the freezing characteristics of the blood. As in unmodified cells, the incidence of intracellular freezing increased with the magnitudes of both the cooling rate and the extracellular supercooling. However, the glycerolized cells exhibited a much greater tendency to supercool prior to the initial nucleation of ice. Values of ΔT_sc > ?20 °C were readily obtained. Also, the transition from 0 to 100% occurrence of intracellular ice covered a cooling rate spectrum in excess of 300 to 600 °K/min, as compared with 10 °C/min for unmodified cells. Thus, the incidence of intracellular ice formation was significantly increased in glycerolized cells.     

Low temperature preservation and space medicine

Cold maintenance may be an option for compromised space-borne astronauts. Contemporary aneurysm surgery can involve cooling below 20 degrees C for nearly one hour. Dogs and baboons have survived blood-substituted hypothermia for 1-3 hours. Hamsters have recovered from partial-freezing below -1 degree C, and supercooling at -5 degrees C. Laboratory frogs have survived partial-freezing from -9 degrees C, while in nature, frogs may overwinter in these states. While some invertebrates can tolerate freezing to cryogenic temperatures, no vertebrate has survived complete freezing. The following studies (hypothermia and sub-zero experiments) were conducted to explore low temperature preservation of rodents, dogs and baboons.     

Effects of hold time after extracellular ice formation on intracellular freezing of mouse oocytes

MII mouse oocytes in 1 and 1.5M ethylene glycol(EG)/phosphate buffered saline have been subjected to rapid freezing at 50 degrees C/min to -70 degrees C. When this rapid freezing is preceded by a variable hold time of 0-3 min after the initial extracellular ice formation (EIF), the duration of the hold time has a substantial effect on the temperature at which the oocytes subsequently undergo intracellular ice formation (IIF). For example, in 1M EG, the IIF temperatures are -23.7 and -39.2 degrees C with 0 and 2 min hold times; in 1.5M EG, the corresponding IIF temperatures are -29.1 and -40.8 degrees C.     

Intracellular freezing, viability, and composition of fat body cells from freeze-intolerant larvae of Sarcophaga crassipalpis.

Although it is often assumed that survival of freezing requires that ice formation must be restricted to extracellular compartments, fat body cells from freeze-tolerant larvae of the gall fly, Eurosta solidaginis (Diptera, Tephritidae) survive intracellular freezing. Furthermore, these cells are highly susceptible to inoculative freezing by external ice, undergo extensive lipid coalescence upon thawing, and survive freezing better when glycerol is added to the suspension medium. To determine whether these traits are required for intracellular freeze tolerance or whether they are incidental and possessed by fat body cells in general, we investigated the capacity of fat body cells from nondiapause-destined and diapause-destined (i.e., cold-hardy) larvae of the freeze-intolerant flesh fly Sarcophaga crassipalpis (Diptera, Sarcophagidae) to survive intracellular freezing. Fat body cells from both types of larvae were highly susceptible to inoculative freezing; all cells froze between -3.7 to -6.2 degrees C. The highest rates for survival of intracellular freezing occurred at -5 degrees C. The addition of glycerol to the media markedly increased survival rates. Upon thawing, the fat body cells showed little or no lipid coalescence. Fat body cells from E. solidaginis had a water content of only 35% compared to cells from S. crassipalpis larvae that had 52-55%; cells with less water may be less likely to be damaged by mechanical forces during intracellular freezing.     

Subzero water transport characteristics of boar spermatozoa confirm observed optimal cooling rates

Incomplete understanding of the water transport parameters (reference membrane permeability, L(pg), and activation energy, E(Lp)) during freezing in the presence of extracellular ice and cryoprotective agents (CPAs) is one of the main limiting factors in reconciling the difference between the numerically predicted value and the experimentally determined optimal rates of freezing in boar (and in general mammalian) gametes. In the present study, a shape-independent differential scanning calorimeter (DSC) technique was used to measure the water transport during freezing of boar spermatozoa. Water transport data during freezing of boar sperm cell suspensions were obtained at cooling rates of 5 and 20 degrees C/min in the presence of extracellular ice and 6% (v/v) glycerol. Using previously published values, the boar sperm cell was modeled as a cylinder of length 80.1 microm and a radius of 0.31 microm with an osmotically inactive cell volume, V(b), of 0.6 V(o), where V(o) is the isotonic cell volume. By fitting a model of water transport to the experimentally obtained data, the best-fit water transport parameters (L(pg) and E(Lp)) were determined. The "combined-best-fit" parameters at 5 and 20 degrees C/min for boar spermatozoa in the presence of extracellular ice are: L(pg) = 3.6 x 10(-15) m(3)/N. s (0.02 microm/min-atm) and E(Lp) = 122.5 kJ/mole (29.3 kcal/mole) (R(2) = 0.99); and the corresponding parameters in the presence of extracellular ice and glycerol are: L(pg)[cpa] = 0.90 x 10(-15) m(3)/N. s (0.005 microm/min-atm) and E(Lp)[cpa] = 75.7 kJ/mole (18.1 kcal/mole) (R(2) = 0.99). The water transport parameters obtained in the present study are significantly different from previously published parameters for boar and other mammalian spermatozoa obtained at suprazero temperatures and at subzero temperatures in the absence of extracellular ice. The theoretically predicted optimal rates of freezing using the new parameters ( approximately 30 degrees C/min) are in close agreement with previously published but experimentally determined optimal cooling rates. This analysis reconciles a long-standing difference between theoretically predicted and experimentally determined optimal cooling rates for boar spermatozoa.     

Nucleation and growth of ice in deeply undercooled erythrocytes

Previous studies of the mechanism of freezing of erythrocytes in the absence of intracellular ice have been extended to define the catalytic sites responsible for promoting nucleation. The following aspects have been investigated: (1) the freeze propagation between undercooled erythrocytes, (2) the nucleation of ice in undercooled erythrocyte ghosts, and (3) the freezing behavior of undercooled hemoglobin solutions. The main findings are: (1) no cross-nucleation occurs between individual cells packed within the same emulsified water droplet; (2) the differential scanning calorimetric power-time curves of intact cells and ghosts are identical, indicating that hemoglobin does not affect ice nucleation; (3) the nucleation temperature of ice in an aqueous solution of hemoglobin (isolated from the cells) is substantially lower than that for the same solution when contained in the intact cell; (4) the threefold freeze concentration which accompanies the freezing of a 25% hemoglobin solution does not cause denaturation of the protein.