Thylakoid membrane stability in drought-tolerant and drought-sensitive plants

The stress stability of membranes from two drought-tolerant plants (Craterostigma plantagineum andCeterach officinarum) was compared with that of a drought-sensitive plant (Spinacia oleracea) in model experiments. Thylakoids from these plants were exposed to excessive sugar or salt concentrations or to freezing. All stresses caused loss of membrane function as indicated by the loss of cyclic photophosphorylation or the inability of the membranes to maintain a large proton gradient in the light. However, loss of membrane functions caused by osmotic dehydration in the presence of sugars was reversible. Irreversible membrane damage during freezing or exposure to salt was attributed mainly to chaotropic solute effects. The sensitivity to different stresses was comparable in thylakoid membranes from tolerant and sensitive plants indicating that the stress tolerance of a plant can hardly be attributed to specific membrane structures which would increase membrane stability. Levels of membrane-compatible solutes such as sugars or amino acids, among them proline, were much higher in the drought-tolerant plants than in spinach. Isolated thylakoids suspended in solutions containing an excess of sugars remained functional after dehydration by freeze-drying. This indicates that membrane-compatible solutes are important in preventing membrane damage during dehydration of poikilohydric plants.Abbreviation BSA bovine serum albumin     

Methods and concepts in quantifying resistance to drought, salt and freezing, abiotic stresses that affect plant water status

The abiotic stresses of drought, salinity and freezing are linked by the fact that they all decrease the availability of water to plant cells. This decreased availability of water is quantified as a decrease in water potential. Plants resist low water potential and related stresses by modifying water uptake and loss to avoid low water potential, accumulating solutes and modifying the properties of cell walls to avoid the dehydration induced by low water potential and using protective proteins and mechanisms to tolerate reduced water content by preventing or repairing cell damage. Salt stress also alters plant ion homeostasis, and under many conditions this may be the predominant factor affecting plant performance. Our emphasis is on experiments that quantify resistance to realistic and reproducible low water potential (drought), salt and freezing stresses while being suitable for genetic studies where a large number of lines must be analyzed. Detailed protocols for the use of polyethylene glycol-infused agar plates to impose low water potential stress, assay of salt tolerance based on root elongation, quantification of freezing tolerance and the use of electrolyte leakage experiments to quantify cellular damage induced by freezing and low water potential are also presented.     

Differential destabilization of membranes by tryptophan and phenylalanine during freezing: the roles of lipid composition and membrane fusion

The stability of cellular membranes during dehydration can be strongly influenced by the partitioning of amphiphilic solutes from the aqueous phase into the membranes. The effects of partitioning on membrane stability depend in a complex manner on the structural properties of the amphiphiles and on membrane lipid composition. Here, we have investigated the effects of the amphiphilic aromatic amino acids Trp and Phe on membrane stability during freezing. Both amino acids were cryotoxic to isolated chloroplast thylakoid membranes and to large unilamellar liposomes, but Trp had a much stronger effect than Phe. In liposomes, both amino acids induced solute leakage and membrane fusion during freezing. The presence of the chloroplast galactolipids monogalactosyldiacylglycerol or digalactosyldiacylglycerol in egg phosphatidylcholine (EPC) membranes reduced leakage from liposomes during freezing in the presence of up to 5 mM Trp, as compared to membranes composed of pure EPC. The presence of the nonbilayer-forming lipid phosphatidylethanolamine increased leakage. Membrane fusion followed a similar trend, but was dramatically reduced when the anthracycline antibiotic daunomycin was incorporated into the membranes. Daunomycin has been shown to stabilize the bilayer phase of membranes in the presence of nonbilayer lipids and was therefore expected to reduce fusion. Surprisingly, this had only a small influence on leakage. Collectively, these data indicate that Trp and Phe induce solute leakage from liposomes during freezing by a mechanism that is largely independent of fusion events.     

Differential destabilization of membranes by tryptophan and phenylalanine during freezing: the roles of lipid composition and membrane fusion

The stability of cellular membranes during dehydration can be strongly influenced by the partitioning of amphiphilic solutes from the aqueous phase into the membranes. The effects of partitioning on membrane stability depend in a complex manner on the structural properties of the amphiphiles and on membrane lipid composition. Here, we have investigated the effects of the amphiphilic aromatic amino acids Trp and Phe on membrane stability during freezing. Both amino acids were cryotoxic to isolated chloroplast thylakoid membranes and to large unilamellar liposomes, but Trp had a much stronger effect than Phe. In liposomes, both amino acids induced solute leakage and membrane fusion during freezing. The presence of the chloroplast galactolipids monogalactosyldiacylglycerol or digalactosyldiacylglycerol in egg phosphatidylcholine (EPC) membranes reduced leakage from liposomes during freezing in the presence of up to 5 mM Trp, as compared to membranes composed of pure EPC. The presence of the nonbilayer-forming lipid phosphatidylethanolamine increased leakage. Membrane fusion followed a similar trend, but was dramatically reduced when the anthracycline antibiotic daunomycin was incorporated into the membranes. Daunomycin has been shown to stabilize the bilayer phase of membranes in the presence of nonbilayer lipids and was therefore expected to reduce fusion. Surprisingly, this had only a small influence on leakage. Collectively, these data indicate that Trp and Phe induce solute leakage from liposomes during freezing by a mechanism that is largely independent of fusion events.     

Cryobehavior of the plasma membrane in protoplasts isolated from cold-acclimated Arabidopsis leaves is related to surface area regulation

Extracellular freezing in plants results in dehydration and mechanical stresses upon the plasma membrane. Plants that acquire enhanced freezing tolerance after cold acclimation can withstand these two physical stresses. To understand the tolerance to freeze-induced physical stresses, the cryobehavior of the plasma membrane was observed using protoplasts isolated from cold-acclimated Arabidopsis thaliana leaves with the combination of a lipophilic fluorescent dye FM 1-43 and cryomicroscopy. We found that many vesicular structures appeared in the cytoplasmic region near the plasma membrane just after extracellular freezing occurred. These structures, referred to as freeze-induced vesicular structures (FIVs), then developed horizontally near the plasma membrane during freezing. There was a strong correlation between the increase in individual FIV size and the decrease in the surface area of the protoplasts during freezing. Some FIVs fused with their neighbors as the temperature decreased. Occasionally, FIVs fused with the plasma membrane, which may be necessary to relax the stress upon the plasma membrane during freezing. Vesicular structures resembling FIVs were also induced when protoplasts were mechanically pressed between a coverslip and slide glass. Fewer FIVs formed when protoplasts were subjected to hyperosmotic solution, suggesting that FIV formation is associated with mechanical stress rather than dehydration. Collectively, these results suggest that cold-acclimated plant cells may balance membrane tension in the plasma membrane by regulating the surface area. This enables plant cells to withstand the direct mechanical stress imposed by extracellular freezing.     

The effects of solutes on the freezing properties of and hydration forces in lipid lamellar phases.

Quantitative deuterium nuclear magnetic resonance is used to study the freezing behavior of the water in phosphatidylcholine lamellar phases, and the effect upon it of dimethylsulfoxide (DMSO), sorbitol, sucrose, and trehalose. When sufficient solute is present, an isotropic phase of concentrated aqueous solution may coexist with the lamellar phase at freezing temperatures. We determine the composition of both unfrozen phases as a function of temperature by using the intensity of the calibrated free induction decay signal (FID). The presence of DMSO or sorbitol increases the hydration of the lamellar phase at all freezing temperatures studied, and the size of the increase in hydration is comparable to that expected from their purely osmotic effect. Sucrose and trehalose increase the hydration of the lamellar phase, but, at concentrations of several molal, the increase is less than that which their purely osmotic effect would be expected to produce. A possible explanation is that very high volume fractions of sucrose and trehalose disrupt the water structure and thus reduce the repulsive hydration interaction between membranes. Because of their osmotic effect, all of the solutes studied reduced the intramembrane mechanical stresses produced in lamellar phases by freezing. Sucrose and trehalose at high concentrations produce a greater reduction than do the other solutes.     

Membrane phase behavior during cooling of stallion sperm and its correlation with freezability

Stallion sperm exhibits great male-to-male variability in survival after cryopreservation. In this study, we have investigated if differences in sperm freezability can be attributed to membrane phase and permeability properties. Fourier transform infrared spectroscopy (FTIR) was used to determine supra and subzero membrane phase transitions and characteristic subzero membrane hydraulic permeability parameters. Sperm was obtained from stallions that show differences in sperm viability after cryopreservation. Stallion sperm undergoes a broad and gradual phase transition at suprazero temperatures, from 30-10°C, whereas freezing-induced dehydration of the cells causes a more severe phase transition to a highly ordered gel phase. Sperm from individual stallions showed significant differences in post-thaw progressive motility, percentages of sperm with abnormal cell morphology, and chromatin stability. The biophysical membrane properties evaluated in this study, however, did not show clear differences amongst stallions with differences in sperm freezability. Cyclodextrin treatment to remove cholesterol from the cellular membranes increased the cooperativity of the suprazero phase transition, but had little effects on the subzero membrane phase behavior. In contrast, freezing of sperm in the presence of protective agents decreased the rate of membrane dehydration and increased the total extent of dehydration. Cryoprotective agents such as glycerol decrease the amount of energy needed to transport water across cellular membranes during freezing.     

Membrane permeability parameters for freezing of stallion sperm as determined by Fourier transform infrared spectroscopy

Cellular membranes are one of the primary sites of injury during freezing and thawing for cryopreservation of cells. Fourier transform infrared spectroscopy (FTIR) was used to monitor membrane phase behavior and ice formation during freezing of stallion sperm. At high subzero ice nucleation temperatures which result in cellular dehydration, membranes undergo a profound transition to a highly ordered gel phase. By contrast, low subzero nucleation temperatures, that are likely to result in intracellular ice formation, leave membrane lipids in a relatively hydrated fluid state. The extent of freezing-induced membrane dehydration was found to be dependent on the ice nucleation temperature, and showed Arrhenius behavior. The presence of glycerol did not prevent the freezing-induced membrane phase transition, but membrane dehydration occurred more gradual and over a wider temperature range. We describe a method to determine membrane hydraulic permeability parameters (E_Lp, Lpg) at subzero temperatures from membrane phase behavior data. In order to do this, it was assumed that the measured freezing-induced shift in wavenumber position of the symmetric CH₂ stretching band arising from the lipid acyl chains is proportional to cellular dehydration. Membrane permeability parameters were also determined by analyzing the H₂O-bending and -libration combination band, which yielded higher values for both E_Lp and Lpg as compared to lipid band analysis. These differences likely reflect differences between transport of free and membrane-bound water. FTIR allows for direct assessment of membrane properties at subzero temperatures in intact cells. The derived biophysical membrane parameters are dependent on intrinsic cell properties as well as freezing extender composition.     

Membrane phase behavior during cooling of stallion sperm and its correlation with freezability

Abstract

Stallion sperm exhibits great male-to-male variability in survival after cryopreservation. In this study, we have investigated if differences in sperm freezability can be attributed to membrane phase and permeability properties. Fourier transform infrared spectroscopy (FTIR) was used to determine supra and subzero membrane phase transitions and characteristic subzero membrane hydraulic permeability parameters. Sperm was obtained from stallions that show differences in sperm viability after cryopreservation. Stallion sperm undergoes a broad and gradual phase transition at suprazero temperatures, from 30–10°C, whereas freezing-induced dehydration of the cells causes a more severe phase transition to a highly ordered gel phase. Sperm from individual stallions showed significant differences in post-thaw progressive motility, percentages of sperm with abnormal cell morphology, and chromatin stability. The biophysical membrane properties evaluated in this study, however, did not show clear differences amongst stallions with differences in sperm freezability. Cyclodextrin treatment to remove cholesterol from the cellular membranes increased the cooperativity of the suprazero phase transition, but had little effects on the subzero membrane phase behavior. In contrast, freezing of sperm in the presence of protective agents decreased the rate of membrane dehydration and increased the total extent of dehydration. Cryoprotective agents such as glycerol decrease the amount of energy needed to transport water across cellular membranes during freezing.     

Specific interactions of tryptophan with phosphatidylcholine and digalactosyldiacylglycerol in pure and mixed bilayers in the dry and hydrated state

Amphiphilic solutes play an important role in the desiccation tolerance of plant cells, because they can reversibly partition into cellular membranes during dehydration. Their effects on membrane stability depend on their chemical structure, but also on the lipid composition of the host membrane. We have shown recently that tryptophan destabilizes liposomes during freezing. The degree of destabilization depends on the presence of glycolipids in the membranes, but not on the phase preference (bilayer or non-bilayer) of the lipids in mixtures with the bilayer lipid phosphatidylcholine. Here, we have investigated the influence of tryptophan on the phase behavior and intermolecular interactions in dry and hydrated bilayers made from the phospholipid egg phosphatidylcholine and the plant chloroplast glycolipid digalactosyldiacylglycerol, or from a mixture (1:1) of these lipids, using Fourier-transform infrared spectroscopy. To distinguish effects of the hydrophobic ring structure of tryptophan from those of the amino acid moiety, we also performed experiments with the hydrophilic amino acid glycine. Our data show that there are specific interactions between tryptophan and either phospholipid or glycolipid in the dry state, as well as H-bonding interactions between the lipids and both solutes. In the rehydrated state, the H-bonding interactions between amino acids and lipids are mostly replaced by interactions between water and lipids, while the hydrophobic interactions between lipids and tryptophan mostly persist.     

Some emerging principles underlying the physical properties, biological actions, and utility of vitrification solutions

Vitrification solutions are aqueous cryoprotectant solutions which do not freeze when cooled at moderate rates to very low temperatures. Vitrification solutions have been used with great success for the cryopreservation of some biological systems but have been less successful or unsuccessful with other systems, and more fundamental knowledge about vitrification solutions is required. The purpose of the present survey is to show that a general understanding of the physical behavior and biological effects of vitrification solutions, as well as an understanding of the conditions under which vitrification solutions are required, is gradually emerging. Detailed nonequilibrium phase diagram information in combination with specific information on the tolerance of biological systems to ice and to cryoprotectant at subzero temperatures provides a quantitative theoretical basis for choosing between vitrification and freezing. The vitrification behavior of mixtures of cryoprotective agents during cooling is predictable from the behavior of the individual agents, and the behavior of individual agents is gradually becoming predictable from the details of their molecular structures. Progress is continuing concerning the elucidation of mechanisms and cellular sites of toxicity and mechanisms for the reduction of toxicity. Finally, important new information is rapidly emerging concerning the crystallization of previously vitrified cryoprotectant solutions during warming. It appears that vitrification tendency, toxicity, and devitrification all depend on subtle variations in the organization of water around dissolved substances.     

Validation of cryopreservation protocols for plant germplasm conservation: a pilot study using Ribes L.

Uniformly applicable techniques for germplasm preservation are important to the international genetic resources community and validation of techniques among working genebanks will enable the integration of new technologies into plant genetic resources programs. Apical meristems from micropropagated plants of Ribes nigrum L. cv. Ojebyn and R. aureum cv. Red Lake were used to test three cryopreservation protocols (controlled freezing, plant vitrification solution no. 2 (PVS2) vitrification and encapsulation–dehydration) at the USDA-ARS National Clonal Germplasm Repository (NCGR), Corvallis, OR, USA and the University of Abertay Dundee (UAD), Scotland. Similar results were obtained with PVS2 vitrification at both locations but meristem regrowth varied greatly for the other techniques. Variable results between the locations were noted for controlled freezing and were largely attributed to differences in ice crystal initiation by the controlled rate freezers. Low survival of `Red Lake' at UAD with all three techniques was attributed to poorly performing shoot cultures. Attention to protocol details is important for limiting variation between locations and step by step instructions for procedures and solution preparation aided protocol standardization. These studies suggest that source plant status, cryogenic facilities, and culture conditions may be the most likely causes of variation when validating cryopreservation methodologies in different locations. However, in-house optimization of standard procedures offers considerable potential in ensuring that cryopreservation methodologies can be transferred between international laboratories.     

High concentrations of the compatible solute glycinebetaine destabilize model membranes under stress conditions

Compatible solutes are accumulated by diverse organisms in response to environmental stresses such as drought, salt, or cold. Glycinebetaine (Bet) is such a solute that is accumulated by many plants and microorganisms to high concentrations under stress conditions. It is an osmoprotectant in bacteria and stabilizes both soluble and peripherally membrane-bound proteins in vitro. Here, the effects of Bet on the stability of model lipid membranes are compared to the effects of two other compatible solutes, sucrose and trehalose. Both in the presence of 1M NaCl and during freezing to -20 degrees C, Bet is highly destabilizing to liposomes containing nonbilayer lipids, while the disaccharides are either protective or, in some cases, much less destabilizing. The destabilizing effect of Bet is more pronounced in membranes containing the nonbilayer galactolipid monogalactosyldiacylglycerol from plant chloroplasts than in membranes containing the nonbilayer phospholipid phosphatidylethanolamine. The most dramatic differences between the sugars and Bet were observed in liposomes made from a combination of lipids resembling plant chloroplast thylakoid membranes. Measurements with the dye merocyanine 540 indicate that the water-membrane interface was affected in opposite directions by the presence of high concentrations of sucrose or Bet. The dynamics of the lipids, however, were not differentially affected by the solutes, making direct solute-lipid interactions an unlikely explanation for the different effects on stability. The data offer an explanation, why Bet at high concentrations achieved during exogenous feeding of leaf tissues can be detrimental to cellular stability and survival under stress, while bacterial membranes that contain phosphatidylethanolamine instead of monogalactosyldiacylglycerol, or cyanobacteria that contain highly saturated monogalactosyldiacylglycerol are less susceptible.     

Freezing injury and uncoupling of phosphorylation from electron transport in chloroplasts

Freezing of chloroplast membranes uncouples photophosphorylation from electron transport and inactivates the light-dependent and thiol-requiring ATPase, conformational changes and the light-dependent proton uptake. All of these energy requiring activities can be protected against inactivation by addition of sucrose prior to freezing. The direct relation to photophosphorylation is demonstrated by the quantitatively similar response of photophosphorylation and the other activities to sucrose protection. Salts interfere with the protection afforded by sucrose.

In contrast to the light-dependent ATPase, the ATPase activities which are unmasked by digestion with trypsin show no significant response to freezing. Similarly, the chloroplast coupling factor, which is released from the membranes by ethylenediamine tetraacetic acid treatment, survives freezing. The membranes, which are depleted of the factor, are damaged by freezing.

The results suggest that uncoupling of phosphorylation from electron transport is caused by interference of freezing with a structure involved in the formation of a non-phosphorylated high energy state of chloroplasts. They are best explained on the basis of Mitchell's theory of phosphorylation. Since freezing alters the permeability properties of chloroplast membranes—frozen membrane vesicles no longer function as osmometers—it may be assumed that freezing uncouples phosphorylation from electron transport by preventing the formation of a pH gradient across the vesicle membranes owing to proton leakage through the membranes. From the results, the basic injury caused by freezing appears to consist in the alteration of permeability properties of biological membranes due to the dehydration which accompanies freezing.

     

Cryopreservation of spinach chloroplast membranes by low-molecular-weight carbohydrates: II. Discrimination between colligative and noncolligative protection

Thylakoid membranes isolated from spinach leaves (Spinacia oleracea L. cv. Monatol) were subjected to a freeze-thaw cycle in the presence of various concentrations of sugars, polyhydric alcohols, and NaCl. Functional integrity of the membranes was assayed by means of cyclic photophosphorylation. From the nonideal activity—concentration profiles of the carbohydrates the effective NaCl concentrations in the surroundings of the membranes at the respective freezing temperatures were calculated.Comparison of the cryoprotective efficiency of the various polyols revealed that cryopreservation by low-molecular-weight compounds is predominantly due to colligative action of the solutes. In addition, specific effects of carbohydrates which cannot be explained by the colligative concept are involved in cryoprotection. At NaCl concentrations exceeding 15 mm, the relative contribution of noncolligative membrane protection of a given polyol to overall cryopreservation was independent of the salt concentration. However, during freezing in the presence of very low salt concentrations, for instance 1–4 mm NaCl, cryoprotection due to colligative phenomena is reduced in favor of other mechanisms.     

The results of freezing and dehydration of horseradish peroxidase

Explanations of the mechanism of freezing injury have included the one that freezing may result in dehydration of enzymes. This hypothesis has been examined by comparing the effects of freezing and dehydration on horseradish peroxidase.It was found that freezing and dehydration reduce the activity of peroxidase when compared with the native enzyme. Polyvinyl pyrrolidone and increased protein concentration protect the enzyme against loss of activity in both treatments, Protein-protein interactions exclude water, and this mechanism is suggested to stabilize peroxidase and protect against desiccation. Polyvinyl pyrrolidone may protect against freezing by reducing dehydration through reduced vapor-pressure difference or may stabilize by a protein-polymer interaction.     

Factors contributing to inactivation of isolated thylakoid membranes during freezing in the presence of variable amounts of glucose and NaCl.

During freezing of isolated spinach thylakoids in sugar/salt solutions, the two solutes affected membrane survival in opposite ways: membrane damage due to increased electrolyte concentration can be prevented by sugar. Calculation of the final concentrations of NaCl or glucose reached in the residual unfrozen portion of the system revealed that the effects of the solutes on membrane activity can be explained in part by colligative action. In addition, the fraction of the residual liquid in the frozen system contributes to membrane injury. During severe freezing in the presence of very low initial solute concentrations, membrane damage drastically increased with a decrease in the volume of the unfrozen solution. Freezing injury under these conditions is likely to be due to mechanical damage by the ice crystals that occupy a very high fraction of the frozen system. At higher starting concentrations of sugar plus salt, membrane damage increased with an increase in the amount of the residual unfrozen liquid. Thylakoid inactivation at these higher initial solute concentrations can be largely attributed to dilution of the membrane fraction, as freezing damage at a given sugar/salt ratio decreased with increasing the thylakoid concentration in the sample. Moreover, membrane survival in the absence of freezing decreased with lowering the temperature, indicating that the temperature affected membrane damage not only via alterations related to the ice formation. From the data it was evident that damage of thylakoid membranes was determined by various individual factors, such as the amount of ice formed, the final concentrations of solutes and membranes in the residual unfrozen solution, the final volume of this fraction, the temperature and the freezing time. The relative contribution of these factors depended on the experimental conditions, mainly the sugar/salt ratio, the initial solute concentrations, and the freezing temperature.     

Effect of freezing and dehydration on ion and cryoprotectant distribution and hemolymph volume in the goldenrod gall fly, Eurosta solidaginis

Extracellular freezing and dehydration concentrate hemolymph solutes, which can lead to cellular injury due to excessive water loss. Freeze tolerant larvae of the goldenrod gall fly, Eurosta solidaginis, may experience extreme cold and desiccation in winter. To determine whether larvae employ protective mechanisms against excessive cellular water loss we examined the effect of extracellular freezing and dehydration on hemolymph volume, and cryoprotectant and ion levels in the hemolymph. Dehydrated larvae or ones that had been frozen at −5 or −20 °C had a significantly smaller proportion of their body water as hemolymph (26.0-27.4%) compared to controls (30.5%). Even with this reduction in water content, hemolymph osmolality was similar or only slightly higher in frozen or dehydrated individuals than controls (908 mOsm kg⁻¹), indicating these stresses led to a reduction in hemolymph solutes. Hemolymph and intracellular content of ions remained largely unchanged between treatment groups; although levels of Mg⁺⁺ in the hemolymph were lower in larvae subjected to freezing (0.21 ± 0.01 μg mg⁻¹ dry mass) compared to controls (0.29 ± 0.01 μg mg⁻¹ dry mass), while intracellular levels of K⁺ were lower in groups exposed to low temperature (8.31 ± 0.21 μg mg⁻¹ dry mass). Whole body glycerol and sorbitol content was similar among all treatment groups, averaging 432 ± 25 mOsm kg⁻¹ and 549 ± 78 mOsm kg⁻¹ respectively. However, larvae subjected to dehydration and freezing at −20 °C had a much lower relative amount of cryoprotectants in their hemolymph (∼35%) compared to controls (54%) suggesting these solutes moved into intracellular compartments during these stresses. The correlation between reduced hemolymph volume (i.e. increased cellular water content) and intracellular movement of cryoprotectants may represent a link between tolerance of dehydration and cold in this species.     

The effect of internal and external cryoprotectants on zebrafish (Danio rerio) embryos

The study investigated the effects of internal (DMSO, 1,2-propanediol, glycerol, ethylene glycol, methanol, N,N-dimethylacetamide) and external cryoprotectants (glucose, sucrose) on the viability and on morphometric parameters of zebrafish embryos. From the tested internal cryoprotectants, DMSO had the lowest toxicity, followed by 1,2-propanediol and glycerol. The external cryoprotectants were less toxic then the internal ones. Early ontogenetic stages were more sensible to cryoprotectant exposure than advanced stages. Two-step incubation procedures in increasing concentrations of internal and external cryoprotectants were superior to multiple-step exposure procedures. All tested vitrification solutions exceeded the tolerance limit of embryos. The tolerance of zebrafish embryos to cryoprotectants was highly variable in a concentration range causing approximately 50% embryo mortality. The width of the perivitelline space showed significant morphometrical changes due to cryoprotectant exposure. In the germinative tissue non-significant changes occurred. The yolk did not change morphometrically after exposure to internal cryoprotectants and showed no sign of dehydration after exposure to external cryoprotectants. Based on these results the study comes to the following conclusions: as yolk dehydration was impossible and as vitrification solutions were over the tolerance limit it seems unlikely that successful vitrification of zebrafish embryos can be achieved. Under these considerations slow freezing methods would be a better option as lower cryoprotectant concentrations can be used and embryos can be dehydrated during freezing.