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PubMedDate not supplied

Studies on cryoprotectant toxicity to zebrafish (Danio rerio) oocytes.

Cryopreservation of fish germ cells is an important measure in conservation of fish genetic material. Although investigations on cryopreservation of fish sperm and embryos have been carried out extensively, cryopreservation of fish oocytes has not been studied systematically. In the present study the toxicity of cryoprotectants to zebrafish (Danio rerio) oocytes was investigated. Commonly used cryoprotectants dimethyl sulfoxide (DMSO), methanol, ethylene glycol (EG), propylene glycol (PG), sucrose and glucose were studied. Stage III (vitellogenic), stage IV (maturation) and stage V (mature egg) zebrafish oocytes were incubated in Hank's medium containing different concentrations of cryoprotectants (0.25-4M) for 30 min at room temperature. Three different tests were used to assess oocyte viability: trypan blue (TB) staining, thiazolyl blue (MTT) staining and in vitro maturation followed by observation of germinal vesicle breakdown (GVBD). Results showed that the toxic effect of cryoprotectant on oocytes generally increased with increasing concentration. MTT test was shown to be the least sensitive testing method and gave poor correlation to subsequent GVBD results. Sensitivity of vital tests increases in the order of MTT, TB and GVBD. GVBD test showed that cryoprotectant toxicity to stage III zebrafish oocytes increased in the order of methanol, PG, DMSO, EG, glucose and sucrose. No Observed Effect Concentrations (NOECs) for stage III oocytes were 2M, 1M, 1M, 0.5M, less than 0.25M and less than 0.25M for methanol, PG, DMSO, EG, glucose and sucrose respectively. TB test also showed that the toxicity of tested cryoprotectants increased in the same order. The sensitivity of oocytes to cryoprotectants appeared to increase with development stage with stage V oocytes being the most sensitive.

Alcohols↗
PubMed1994

Incorporation of penetrating cryoprotectants in diluents for pellet-freezing ram spermatozoa.

Glycerol may be toxic to frozen-thawed ram spermatozoa and reduce their fertilizing capacity. This study examined the cryoprotective effects of dimethyl sulphoxide (DMSO), ethylene glycol, glycerol and propanediol alone and in combinations with each other in Triscitrate-glucose diluents on the post-thaw motility and acrosome integrity of pellet-frozen ram spermatozoa. The 4 cryoprotectants were examined in diluents at 5 concentrations (0, 1.5, 3.0, 6.0, 12.0% v/v). Post-thaw motility of spermatozoa was higher in diluents containing ethylene glycol (1.5 to 6.0% v/v), glycerol (at all levels tested) and propanediol (1.5 and 3.0% v/v) than in diluents without cryoprotectant (P<0.001), but there was no effect of DMSO on post-thaw motility. Motility of spermatozoa was higher in diluents containing ethylene glycol or glycerol than DMSO or propanediol (P<0.001). In diluents containing the 4 cryoprotectants at 3 concentrations (1.5, 3.0, 6.0% v/v), better recovery of spermatozoa was found with the addition of 18.0 than 4.5% v/v egg yolk. Combinations of ethylene glycol and/or propanediol (0 to 6.0% v/v) with glycerol (0 to 6.0% v/v) in diluents were also examined. In the presence of glycerol at all levels tested, increasing levels of ethylene glycol and/or propanediol decreased motility and acrosome integrity of spermatozoa (P<0.001). We conclude that the compounds examined exert a cryoprotective effect on pellet-frozen ram spermatozoa, except for DMSO which had no effect. In this study, glycerol remained the single most effective cryoprotectant, and there was no enhancement of this cryoprotection by addition of the other compounds.

Journal Article↗
PubMedDate not supplied

Osmotic tolerance of equine spermatozoa and the effects of soluble cryoprotectants on equine sperm motility, viability, and mitochondrial membrane potential.

Osmotic stress attributed to differences in the relative permeability of cryoprotectants, such as glycerol and water, appears to be an important factor in cryodamage. The objective of this study was to characterize the osmotic tolerance of equine spermatozoa, and to evaluate the effects of addition and removal of cryoprotectants from equine spermatozoa on their motility, and membrane and acrosomal integrity, as well as their mitochondrial membrane potential. Equine spermatozoa had a limited osmotic tolerance to anisosmotic conditions. Although the addition of increasing concentrations of glycerol decreased the motility and viability of equine spermatozoa, the rapid removal of glycerol by dilution in isosmotic media resulted in an even greater decline in motility and viability compared with spermatozoa maintained under anisosmotic conditions. Likewise, the addition and rapid removal of 1.0 M glycerol, ethylene glycol, dimethylsulfoxide, or propylene glycol resulted in a significant decline in sperm motility and viability. Among these cryoprotectants, ethylene glycol had the least detrimental effect on either viability or motility of spermatozoa following the rapid addition and removal of these cryoprotectants. These data demonstrate that equine spermatozoa have a limited osmotic tolerance compared with published reports for mouse or human spermatozoa, and appear to be more similar to boar spermatozoa in their osmotic tolerance. Of the 4 cryoprotectants evaluated in equine spermatozoa, the addition and removal of glycerol resulted in a more marked osmotic stress as indicated by alterations in motility, viability, and acrosomal integrity. These data suggest that alternative cryoprotectants should be considered for cryopreservation of equine spermatozoa in order to reduce osmotic stress associated with the addition of these agents during semen freezing.

Animals↗
PubMed1993

Survival rate of frozen-thawed bovine IVF embryos in relation to exposure time using various cryoprotectants.

The relationship between cryoprotectant and exposure time on the development of IVF-derived embryos in culture after freezing and thawing was examined. Good to excellent quality Day 7 to 8 blastocysts and expanded blastocysts were suspended in 1.6 M propylene glycol (PG), 1.8 M ethylene glycol (EG), 1.1 M diethylene glycol, and 1.3 M ethylene glycol monomethyl ether (EME). In Experiment I, after 30 and 120 min of exposure, embryos were placed directly into culture medium and washed three times. Embryos were cocultured with cumulus cells in culture medium. After a 48-h culture, there were no significant differences in fully expanded, hatching, and hatched rates between the 30- and 120-min exposure to all four cryoprotectants except for the fully expanded rate with PG (55 vs 83%). In Experiment II, to compare the cryoprotective effects of the four cryoprotectants, embryos were exposed to each cryoprotectant for variable times from 10 to 120 min, seeded, and cooled to -30 degrees C at 0.3 degree C/min, plunged into liquid nitrogen for storage, and then thawed. Survival was measured by coculturing the embryos with cumulus cells in culture medium. There were no significant differences in development to hatched blastocysts of embryos exposed to either PG (27-42%) or EG (37-56%). The number of embryos developing after exposure to EME was similar to that in PG or EG when exposure time did not exceed 40 min (43-49%). Our findings suggest that the toxicity of different cryoprotectants is related to the exposure time and that PG and EG were relatively nontoxic as is exposure to EME for short periods.

Animals↗
PubMed1994

Cryoprotection of yeast by alcohols during rapid freezing.

We have investigated the cryoprotective effect of alcohols on Saccharomyces cerevisiae and other yeast under various freezing conditions. For S. cerevisiae, at a cooling rate of 3 degrees C min-1 methanol and ethanol acted as cryosensitizers. However, at a cooling rate of 200 degrees C min-1, both methanol and ethanol proved superior to all other cryoprotectants tested, including glycerol, dimethyl sulfoxide, lactose, trehalose, polyethylene glycol, and polyvinyl pyrrolidone. Propan-2-ol also demonstrated a small but measurable effect although propan-1-ol and butan-1-ol demonstrated no cryoprotective effect. A minimum cooling rate of 25 degrees C min-1 to elicit the cryoprotective effect of ethanol was necessary; below this rate it acted as a cryosensitizer. At cooling rates up to 650 degrees C min-1 substantial cryoprotective effect was still evident. Although the effect of ethanol was variable for other yeast genera tested, ethanol acted positively for all strains of S. cerevisiae. We hypothesize that the cryoprotective effect of alcohols during rapid cooling is a result of their ability to induce increased membrane permeability, allowing rapid water equilibration during extracellular freezing and avoidance of intracellular ice crystal formation.

Alcohols↗
PubMed1997

Chill sensitivity and cryoprotectant permeability of dechorionated zebrafish embryos, Brachydanio rerio.

The zebrafish (Brachydanio rerio) was used as a model for basic studies of the chilling sensitivity, permeability and toxicity of cryoprotectants. In both intact and dechorionated embryos, early-stage embryos (1.25, 1.5, 1.75, and 2 h) were more susceptible (P < 0.05) to chilling injury at 0 degrees C than late-stage embryos (50, 75, and 100% epiboly and three-somite stage). Moreover, enzymatic removal of the chorion did not alter (P > 0.05) this pattern of sensitivity to chilling. Eight-hour zebrafish embryos tolerated short-term exposures to temperatures ranging from 4 to 23 degrees C for 3.5 h with no detrimental developmental effects. The permeability of dechorionated embryos to cryoprotectants was examined by measuring the kinetics of volumetric change at various developmental stages (16 cells to six somites or ca. 1.25 to 14 h postfertilization) at 28.5 degrees C. The dechorionated zebrafish embryo is composed of two complex cellular compartments (i.e., a large yolk and the developing blastoderm). From 40 to 100% epiboly, the volumes of yolk and blastoderm remained constant, ca. 82 and 18%, respectively. However, these volumes changed rapidly after epiboly. For example, at the six-somite stage, the yolk composed 61% of the total volume, whereas the blastoderm composed 39%. When three- and six-somite embryos were placed in 1.5 and 2.0 M cryoprotectants (dimethyl sulfoxide and propylene glycol), osmometric measurement of volume changes indicated no permeation of the cryoprotectants. However, some permeation was observed for six-somite embryos immersed in a 2.0 M methanol solution, but not for 3-somite embryos. For up to 30 min at room temperature, these cryoprotectant solutions were toxic to zebrafish embryos; however, 1.5 M glycerol and ethylene glycol solutions were. We conclude that the complex nature of the zebrafish embryo reduces the effectiveness and predictive value of light microscopical measurements for cryoprotectant permeability studies.

Animals↗
PubMed1998

The effects of cryoprotection on the structure and activity of p21 ras: implications for electron spin-echo envelope modulation spectroscopy.

Electron spin-echo envelope modulation (ESEEM) spectroscopy is widely used to investigate the active sites of biological molecules in frozen solutions. Various cryoprotection techniques, particularly the addition of co-solvents, are commonly employed in the preparation of such samples. In conjunction with ESEEM studies of Mn(II) guanosine nucleotide complexes of p21 ras, we have investigated the effects of cryoprotection on the spectroscopy, the structure, and the activity of this protein. Echo decay times, which typically govern ESEEM spectral resolution, were found to vary linearly with the concentration of glycerol or methyl alpha-D-glucopyranoside (MG), with both additives equally effective on a per-mole basis. The effect of glycerol and MG on the ESEEM amplitudes of various protein nucleiwas studied in ras p21.Mn(II). 5'guanylylimido-diphosphate(p21.Mn(II)-GMPPNP) complexes: these additives did not alter the distances of these nuclei from the Mn(II) ion. In particular, in p21 incorporating [2H-3]Thr, the Mn(II)-[2H-3]Thr35 distance was found to be unaffected by the concentration of cryoprotectant or the rate of freezing. The proximity of the cryoprotectants to the Mn(II) ion was probed by 2H ESEEM in solutions made with d5-glycerol and d7-methyl alpha-D-glucopyranoside (d7-MG). In p21.Mn(II)GMPPNP, the large deuterium modulations from the d5-glycerol exhibit saturation behavior with increasing d5-glycerol concentration, implying that glycerol, a widely used cryoprotectant, replaces the aquo ligands of the Mn(II) ion. The interaction between the Mn(II) ion of p21 and MG, however, is less intimate: the deuterium ESEEM amplitudes are much smaller for samples prepared with d7-MG than with d5-glycerol. Several polyhydroxylic compounds were found to have essentially no effect on the ability of the guanosine 5'-triphosphate (GTP) hydrolysis activating protein, GAP334, to catalyze hydrolysis of p21. guanosine 5'-triphosphate. This observation implies that the introduction of cryoprotectant does not significantly perturb the structure of p21 and gives insight into the mechanism of the GTPase reaction.

Cryoprotective Agents↗
PubMed1991

Insights into the cryoprotective mechanism of dimethyl sulfoxide for phospholipid bilayers.

Dimethyl sulfoxide (Me2SO) is a widely used cryoprotectant for biological structures such as membranes. Despite hundreds of studies on the effects of this molecule, surprisingly little is known about its cryoprotective mechanism. This study investigates the ability of various Me2SO analogs to serve as cryoprotectants for liposomes. The data show that an increase in hydrophobicity progressively reduces the cryoprotective effect of sulfoxides. Additional experiments using phospholipid vesicles of varying composition demonstrate the Me2SO is markedly less effective on liposomes carrying a net negative charge. In fact, cryoprotection by Me2SO was virtually eliminated in vesicles composed of 30% phosphatidylserine (a negatively charged lipid). Based on these results, we suggest that the polar sulfoxide moiety of Me2SO interacts electrostatically with phospholipid membranes and that this interaction is critical for Me2SO's cryoprotective effect for membranes.

Binding Sites↗
PubMed2005

Cryoprotectant equilibration in tissues.

The first step in the cryopreservation of cells or tissues is often the movement of a permeating cryoprotectant into the cells or tissues from the solution into which they have been placed. The cryoprotectant enters the cells or tissues by thermodynamic equilibration with the surroundings. In the reverse case, thermodynamic equilibration also drives the removal of permeating cryoprotectants by a dilution solution at the end of the preservation process when the cells or tissues are being readied for use. There have been reports of tissues having equilibrium cryoprotectant concentrations lower than that of the surrounding carrier solution. For various tissues, the equilibrium concentration of cryoprotectant inside the tissue is either equal to, or lower than the cryoprotectant concentration of the surrounding solution. A simple thermodynamic treatment of the solution-tissue equilibrium shows that an equilibrium concentration difference can exist between a tissue and the surrounding solution if a pressure difference can be maintained.

Animals↗
PubMed2006

Expression of aquaporin-3 improves the permeability to water and cryoprotectants of immature oocytes in the medaka (Oryzias latipes).

The permeability of the plasma membrane plays a crucial role in the successful cryopreservation of oocytes and embryos. Several efforts have been made to facilitate the movement of water and cryoprotectants across the plasma membrane of fish oocytes/embryos because of their large size. Aquaporin-3 is a water/solute channel that can also transport various cryoprotectants. In this study, we tried to improve the permeability of immature medaka (Oryzias latipes) oocytes to water and cryoprotectants by artificially expressing aquaporin-3. The oocytes were injected with aquaporin-3 cRNA and cultured for 6-7 h. Then, hydraulic conductivity (L(P)) and cryoprotectant permeability (P(S)) were determined from volume changes in a hypertonic sucrose solution and various cryoprotectant solutions, respectively, at 25 degrees C. The L(P) value of the cRNA-injected oocytes was 0.22+/-0.04 microm/min/atm, nearly twice larger than that of intact or water-injected oocytes (0.14+/-0.02 and 0.14+/-0.03 microm/min/atm, respectively). P(S) values of intact oocytes for ethylene glycol, propylene glycol, and DMSO were 1.36+/-0.34, 1.97+/-0.20, and 1.17+/-0.52 x 10(-3) cm/min, respectively. The permeability to glycerol could not be calculated because oocytes remained shrunken in the glycerol solution. On the other hand, cRNA-injected oocytes had significantly higher P(S) values (glycerol, 2.20+/-1.29; ethylene glycol, 2.98+/-0.36; propylene glycol, 3.93+/-1.70; DMSO, 3.11+/-0.74 x 10(-3) cm/min) than intact oocytes. When cRNA-injected oocytes were cultured for 12-14 h, 51% matured to the metaphase II stage, and 43% of the matured oocytes were fertilized and hatched following in vitro fertilization and 14 days of culture. Thus, the permeability of medaka oocytes to water and cryoprotectants was improved by the artificial expression of aquaporin-3, and the oocytes retained the ability to develop to term.

Animals↗
PubMed2004

Glycine betaine as a cryoprotectant for prokaryotes.

Osmoprotectants are low molecular weight, hydrophilic, nontoxic molecules that assist a cell under osmotic stress to stabilize its concentration of internal solutes. These properties are similar to compounds used as cryoprotectants for the preservation of prokaryotic cells during freezing. This study tested the ability of a common compatible solute, glycine betaine (GB), to act as a cryoprotectant. In a series of freeze-drying studies using a variety of prokaryotes, GB performed as well, or better than, two commonly used cryoprotectants, sucrose/bovine serum albumin (S/BSA) and trehalose/dextran (T/D). GB did especially well maintaining cell viability after long-term storage (simulated equivalent of 20 years) for microorganisms like Neisseria gonorrhoeae and Streptococcus pneumoniae. GB was tested for its ability to preserve members of the genus Acidothiobacillus, a difficult genus to preserve. For two strains of Acidithiobacillus ferrooxidans that were preserved using liquid drying, GB performed as well as S/BSA. Results were more mixed for two strains of Acidithiobacillus thiooxidans; one strain could be preserved with S/BSA but not GB, the other strain gave low recoveries with both cryoprotectants. GB also proved to be a useful cryoprotectant for liquid nitrogen preservation yielding equivalent results to the cryopreservative, glycerol for halophilic archaea, and neutrophilic Fe-oxidizing bacteria. These results indicate that GB is a simple and useful cryoprotectant that works for a wide range of prokaryotic organisms under different cryopreservation regimens.

Betaine↗
PubMed2003

Spawn viability in edible mushrooms after freezing in liquid nitrogen without a cryoprotectant.

Five strains of edible mushrooms (Lentinula boryana, Lentinula edodes, Pleurotus djamor, Pleurotus pulmonarius, and Volvariella volvacea) were studied. Spawn were prepared from sorghum seeds and then incubated for 14 days under optimum conditions for each species. Once covered by mycelia, the sorghum seeds were placed in polycarbonate vials for freezing in liquid nitrogen. The effect of adding a cryoprotective solution before freezing (either 10% glycerol v/v or 5% dimethylsulfoxide v/v) was evaluated as a function of mycelial growth and percent viability. Three main treatments were undertaken: (1) freezing with a glycerol or dimethylsulfoxide cryoprotectant, (2) freezing with water and (3) freezing without cryoprotectant or water. Samples were maintained frozen for a week, after which time they were thawed (10 min at 30 degrees C) and the seeds placed in Petri dishes with a culture medium. A recovery rate of 96.8% was obtained for the total number of samples summed over all strains and treatments. In contrast, 99.2% of the samples frozen without cryoprotectant were recovered. The recovery of frozen mycelia was delayed with respect to a control group, which was not frozen. However, no difference was observed in percent recovery and mycelial diameter when a new series of spawn was prepared from mycelia that had been previously frozen. Results obtained from this experiment demonstrate that an adequate recovery of mycelia can be obtained without using a cryoprotectant. This capacity might enable large quantities of commercial mushroom strains to be handled at reduced production costs. It is suggested that the mycelia survived freezing without cryoprotectants because they were embedded and protected within the sorghum seeds used to elaborate the spawn.

Agaricales↗
PubMed1998

Chilling sensitivity of carp (Cyprinus carpio) embryos at different developmental stages in the presence or absence of cryoprotectants: work in progress.

The unsolved problem of cryopreservation of the yolk-rich teleost embryos may be related, in part, to their sensitivity to chilling and cryoprotective agents. The aim of this study was to gain data on the sensitivity of carp embryos to low temperatures at different developmental stages and on the possible protective and toxic effects of cryoprotectants. A total of 86,400 morulae, half-epiboly and heartbeat-stage embryos was selected and then placed in water or in 1 M methanol, dimethyl sulfoxide (Me2SO), glycerol or 0.1 M sucrose solution at 0, 4 or 24 degrees C for 5 min or 1 h. Following these treatments, the embryos were held in a 24 degrees C water bath until the evaluation of hatching rates. In every developmental stage a significant decrease of hatching rates following exposure to 4 or 0 degree C was detected. Sensitivity to chilling changed significantly with development (heartbeat < morula < half-epiboly). Half-epiboly stage embryos were less sensitive to a short period of exposure to cryoprotectants than morula and heartbeat stages. A 1-h exposure to cryoprotectants revealed a stage dependent sensitivity. Toxicity increased in the order of methanol < Me2SO < glycerol in morula and half-epiboly stages, and methanol < glycerol < Me2SO in the heartbeat stage. The results show morulae are partially protected against chilling in Me2SO and sucrose, half-epiboly in Me2SO, sucrose and methanol, and heartbeat-stage in methanol and glycerol. The results further suggest that carp embryos are sensitive to chilling and that toxicity and protective effects against chilling of cryoprotectants are stage-dependent. The finding on the low chilling sensitivity of heartbeat-stage embryos and the protective effect of certain cryoprotectants may be useful in designing cryopreservation protocols.

Animals↗
PubMed2002

Effect of antioxidants, citrate, and cryoprotectants on protein denaturation and texture of frozen cod (Gadus morhua).

To investigate the role of antioxidants and cryoprotectants in minimizing protein denaturation in frozen lean fish, cod fillets were treated with either antioxidants (vitamin C (500 mg kg(-1)) or vitamin C (250 mg kg(-1)) + vitamin E (250 mg kg(-1))), antioxidants (vitamins C + E 250 mg kg(-1)each) with citrate (100 mg kg(-1)), cryoprotectants (4% (w/w) sucrose + 4% (w/w) sorbitol), or a mixture of antioxidants (vitamins C + E 250 mg kg(1)), citrate (100 mg kg(-1)), and cryoprotectants (sucrose 40 g kg(-1) + sorbitol 40 g kg(-1)). Untreated and treated fish samples were stored at -10 degrees C; cod fillets stored at -30 degrees C were used as a control. Stored frozen samples were analyzed at intervals for up to 210 days for changes in protein extractability, thermodynamic parameters (transition temperature T(m) and enthalpy DeltaH), structure by FT-Raman spectroscopy, and rheological properties by large and small deformation tests. Results indicated that protein denaturation and texture changes were minimized in the presence of cryoprotectants, as well as in the presence of antioxidants with citrate, antioxidants alone, or the mixture of antioxidants, citrate, and cryoprotectants. In the presence of increased formaldehyde levels in fish treated with vitamin C, toughening was still lower compared to that of the -10 degrees C control due to the antioxidant property of vitamin C. Thus, ice crystal formation and lipid oxidation products are the major factors that cause protein denaturation in lean frozen fish, and antioxidants in addition to cryoprotectants can be used to minimize toughness.

Animals↗
PubMed2003

Improved sperm cryopreservation using cold cryoprotectant.

It has generally been assumed that very rapid cooling above freezing point would be deleterious to human sperm because it would result in cold shock. Consequently, most routine cryopreservation protocols involve the use of warm (20-30 degrees C) cryoprotectant and slow cooling above the freezing point in order to minimise the risk of cold shock. In order to test this assumption, we added an equal volume of cold (4 degrees C) cryoprotectant in a single aliquot to warm (20, 30 or 37 degrees C) semen to induce rapid cooling. The results of this procedure were compared with those obtained using warm cryoprotectant or with the routine cryopreservation protocol used in this laboratory. The use of cold cryoprotectant resulted in a significant (P = 0.016) improvement (mean 63%, range 42%-79%) in post-thaw motility recovery compared with a standard procedure(mean 47%, range 35%-67%) and a significant (P = 0.016) improvement in post-thaw sperm velocity. A cold glycerol/egg yolk/citrate (GEYC) mixture also gave significantly higher motility recovery than GEYC equilibrated to either room temperature (20 degrees C) or body temperature (37 degrees C). Sperm frozen using the cold cryoprotectant protocol were as efficient at binding to and penetrating the human zona pellucida as sperm frozen with a standard protocol. The modified cryopreservation procedure may lead to improved pregnancy rates in donor insemination and in vitro fertilisation. Further investigation is required to determine how the cold cryoprotectant improves the cryopreservation outcome.

Cryopreservation↗
PubMed1996

Magnetic resonance microscopy and spectroscopy reveal kinetics of cryoprotectant permeation in a multicompartmental biological system.

Successful cryopreservation of most multicompartmental biological systems has not been achieved. One prerequisite for success is quantitative information on cryoprotectant permeation into and amongst the compartments. This report describes direct measurements of cryoprotectant permeation into a multicompartmental system using chemical shift selective magnetic resonance (MR) microscopy and MR spectroscopy. We used the developing zebrafish embryo as a model for studying these complex systems because these embryos are composed of two membrane-limited compartments: (i) a large yolk (surrounded by the yolk syncytial layer) and (ii) differentiating blastoderm cells (each surrounded by a plasma membrane). MR images of the spatial distribution of three cryoprotectants (dimethyl sulfoxide, propylene glycol, and methanol) demonstrated that methanol permeated the entire embryo within 15 min. In contrast, the other cryoprotectants exhibited little or no permeation over 2.5 h. MR spectroscopy and microinjections of cryoprotectants into the yolk inferred that the yolk syncytial layer plays a critical role in limiting the permeation of some cryoprotectants throughout the embryo. This study demonstrates the power of MR technology combined with micromanipulation for elucidating key physiological factors in cryobiology.

Animals↗
PubMed1995

Effect of cryoprotectant solutes on water permeability of human spermatozoa.

Osmotic permeability characteristics and the effects of cryoprotectants are important determinants of recovery and function of spermatozoa after cryopreservation. The primary purpose of this study was to determine the osmotic permeability parameters of human spermatozoa in the presence of cryoprotectants. A series of experiments was done to: 1) validate the use of an electronic particle counter for determining both static and kinetic changes in sperm cell volume; 2) determine the permeability of the cells to various cryoprotectants; and 3) test the hypothesis that human sperm water permeability is affected by the presence of cryoprotectant solutes. The isosmotic volume of human sperm was 28.2 +/- 0.2 microns3 (mean +/- SEM), 29.0 +/- 0.3 microns3, and 28.2 +/- 0.4 microns3 at 22, 11, and 0 degrees C, respectively, measured at 285 mOsm/kg via an electronic particle counter. The osmotically inactive fraction of human sperm was determined from Boyle van't Hoff (BVH) plots of samples exposed to four different osmolalities (900, 600, 285, and 145 mOsm/kg). Over this range, cells behaved as linear osmometers with osmotically inactive cell percentages at 22, 11, and 0 degrees C of 50 +/- 1%, 41 +/- 2%, and 52 +/- 3%, respectively. Permeability of human sperm to water was determined from the kinetics of volume change in a hyposmotic solution (145 mOsm/kg) at the three experimental temperatures. The hydraulic conductivity (Lp) was 1.84 +/- 0.06 microns.min-1.atm-1, 1.45 +/- 0.04 microns.min-1.atm-1, and 1.14 +/- 0.07 microns.min-1.atm-1 at 22, 11, and 0 degrees C, respectively, yielding an Arrhenius activation energy (Ea) of 3.48 kcal/mol. These biophysical characteristics of human spermatozoa are consistent with findings in previous reports, validating the use of an electronic particle counter for determining osmotic permeability parameters of human sperm. This validated system was then used to investigate the permeability of human sperm to four different cryoprotectant solutes, i.e., glycerol (Gly), dimethylsulfoxide (DMSO), propylene glycol (PG), and ethylene glycol (EG), and their effects on water permeability. A preloaded, osmotically equilibrated cell suspension was returned to an isosmotic medium while cell volume was measured over time. A Kedem-Katchalsky model was used to determine the permeability of the cells to each solute and the resulting water permeability. The permeabilities of human sperm at 22 degrees C to Gly, DMSO, PG, and EG were 2.07 +/- 0.13 x 10(-3) cm/min, 0.80 +/- 0.02 x 10(-3) cm/min, 2.3 +/- 0.1 x 10(-3) cm/min, and 7.94 +/- 0.67 x 10(-3) cm/min, respectively. The resulting Lp values at 22 degrees C were reduced to 0.77 +/- 0.08 micron.min-1.atm-1, 0.84 +/- 0.07 micron.min-1.atm-1, 1.23 +/- 0.09 microns.min-1.atm-1, and 0.74 +/- 0.06 micron.min-1.atm-1, respectively. These data support the hypothesis that low-molecular-weight, nonionic cryoprotectant solutes affect (decrease) human sperm water permeability.

Cell Membrane Permeability↗
PubMed1993

Cryoprotective effect of gelatin and albumin on recombinant human tumor necrosis factor liposome.

A search for a cryoprotective agent of a liposome containing recombinant human tumor necrosis factor (rHuTNF) was undertaken. Various additives were added and liposomes were freeze-thawed. As a result of these studies, albumin and gelatin were found to suppress the leakage of rHuTNF from liposome. This cryoprotective effect was only observed when the additives were added to the inside of the liposomal membrane; their addition to the outside of the membrane stimulated the leakage. This characteristic was clearly different from well-known cryoprotective agents, such as saccharides which have the cryoprotective effect on both sides of the liposomal membrane. Although this cryoprotective effect was also observed for recombinant human interleukin-1 alpha (rHuIL-1 alpha), it was not observed for fluorescein sodium. These effects of gelatin and albumin were restricted to macromolecular proteins such as rHuTNF and rHuIL-1 alpha, but both substances were very effective cryoprotectants for a liposome containing protein.

Albumins↗