Osmotic responses of preimplantation mouse and bovine embryos and their cryobiological implications

Cells subjected to the events occurring before, during, and after freezing and thawing are exposed to major changes in the osmotic pressure of the surrounding medium; i.e., the osmolalities can exceed 30. An important question in understanding the mechanisms of injury is whether cells respond as ideal osmometers to these strongly anisotonic solutions. Mouse and bovine embryos from eight-cell to blastocyst stage were used to investigate the question. They were found to behave as ideal osmometers at room temperature over a wide range of tonicities; i.e., from four times isotonic to almost 1/3 times isotonic, ideality being defined by a Boyle-van't Hoff equation. Embryo volumes increased from 40 to 200% of isotonic over this range and survivals of mouse embryos were unaffected. However, outside this range the membrane apparently becomes leaky and the survival of mouse embryos drops sharply. Osmolalities rise to high values during freezing and the paper develops the thermodynamic equations to show how computed cell volumes as a function of subzero temperature can be translated into the Boyle-van't Hoff format of cell volume as a function of the reciprocal of osmolality.     

Volume Control by Muscle Fibers of the Blue Crab : Volume readjustment in hypotonic salines

Single isolated muscle fibers from the walking legs of the blue crab, Callinectes sapidus act as Boyle-van't Hoff osmometers with an osmotically inactive volume of 33 %. Fibers in hypotonic salines undergo a spontaneous volume readjustment toward the initial volumes of the cells found in isotonic salines. The volume readjustment is initiated by the increase in cell volume in hypotonic salines and appears to be dependent on the duration of exposure of the fiber to external sodium, the sodium concentration, and the pH of the external medium. The volume-readjusted cells continue to behave as osmometers, but with an increased relative osmotically inactive volume and a decreased internal resistivity. The decreases in cell volumes appear to be, in large part, due to losses of osmotically active nonelectrolytes from the cells.     

The Erythrocyte Ghost Is a Perfect Osmometer

The osmotic swelling of intact erythrocytes in hypotonic solutions was measured using microhematocrit tubes, Van Allen tubes, and a calibrated Coulter counter. In agreement with earlier workers the intact cells did not behave as perfect osmometers, the cells swelling less than predicted by the Boyle-van't Hoff law. Erythrocyte ghosts were prepared from fresh intact erythrocytes by one-step hemolysis in 0.25% NaCl at an extremely dilute concentration of cells and the membranes were sealed at 37°. The ghosts were mixed with NaCl solutions of different osmolarities and the MCV (mean cell volume) of the shrunken cells immediately monitored by a calibrated Coulter counter. It was found that the MCV values of the shrunken ghosts were accurately predicted by the Boyle-van't Hoff law. These results indicate that these erythrocyte ghosts behaved as perfect osmometers.     

Relative influence of unfrozen fraction and salt concentration on the survival of slowly frozen eight-cell mouse embryos

This laboratory has previously reported that the survival of frozen-thawed human erythrocytes is determined more by the fraction of the extracellular solution that remains unfrozen than by the salt concentration in that fraction, especially when the cells are frozen at low hematocrit. To determine the extent to which these findings are applicable to nucleated mammalian cells, we have studied the survival of some 3300 mouse embryos as a function of the unfrozen fraction and the concentration of salt in that unfrozen fraction. Also varied in the study was the weight percentage ratio of glycerol to salt. The concentration of embryos in these experiments (i.e., the cytocrit) was so low that embryo-embryo contacts should have been rare during the freezing. As in the case of the red cells at low hematocrit, we find that the survival of slowly frozen eight-cell embryos is not affected by the high concentrations of salt produced by freezing, at least up to 3.3 molal NaCl, and therefore is not affected by the extent to which the cells shrink below their isotonic volume, nor in general is survival influenced by the temperature at which given salt concentrations and unfrozen fractions are attained or by the glycerol concentration at those temperatures. On the other hand, the attainment of low values of the unfrozen fraction (U) is damaging, but the damage appears in part to be due to the fact that low values of U had to be achieved by placing embryos in solutions hypotonic with respect to NaCl, which caused their volume to be greater than isotonic prior to freezing.     

Osmotic adjustment and the accumulation of organic solutes in whole cells and protoplasts of Saccharomyces cerevisiae

In the presence of a suitable carbon source, whole cells and protoplasts of Saccharomyces cerevisiae synthesized glycerol as a compatible organic solute in response to increased external osmotic pressure. Boyle-van't Hoff plots showed that protoplasts, and non-turgid cells, exhibited a linear relationship between volume and the external osmotic pressure (i.e. they behaved as near-ideal osmometers), and that both protoplasts and cells have a component which is not osmotically responsive--the non-osmotic volume (NOV). Glycerol levels in whole cells and protoplasts were elevated by increased external osmotic pressure over a similar time-scale to the period of exponential cell growth, reaching a maximum value at 6-12 h and declining thereafter. This suggests that the restoration of turgor pressure in whole cells was not the sole regulator of glycerol accumulation. Stationary phase whole cells had negligible levels of intracellular glycerol after growth in a medium of raised osmotic pressure. However, intracellular trehalose synthesis in these cells began earlier and reached a higher maximum level than in basal medium. Once exponential growth had stopped, cell turgor and internal osmotic pressure decreased somewhat. These new, lower values may be determined by the extent of trehalose accumulation in stationary phase cells.     

Osmotic shock of fertilized mouse ova.

The effect of osmotic changes on fertilized mouse ova was studied by measuring their survival, defined as development into hatching blastocysts, after exposure to various concentrations of ethanediol (ethylene glycol). In addition, a Boyle-van't Hoff plot was derived from exposing ova to hypotonic and hypertonic solutions ranging from 0.1 to 2.8 osmol. Volume of ova was inversely proportional to osmolality over this range. Extrapolation of this relationship yielded a nonosmotic volume of the ova of 22.5%. Eighty-five per cent or more of the ova survived exposure to this wide range of concentrations and developed into blastocysts. The rate of development of ova exposed to anisotonic solutions was the same as that of controls. Ova underwent osmotic shock when abruptly diluted out of concentrated solutions of ethanediol with an isotonic solution. Their survival was highly dependent on the ethanediol concentration with which they had equilibrated before dilution, and the manner, rate and temperature of dilution. The longer the exposure to ethanediol the greater was the sensitivity of the ova to osmotic shock, reflecting permeation of ethanediol into the ova. Osmotic shock could be alleviated by dilution at a high temperature, and prevented by the use of sucrose as an osmotic buffer at 37 degrees C. Identification of the variables that influence osmotic shock of ova will be helpful in the systematic study of their cryopreservation.     

Light scattering and cell volumes in osmotically stressed and frozen-thawed cells

Recent reports, indicating that under some conditions the intensity of light scattering from cells is a nonlinear function of cell volume, have led to the widespread generalization that intensity of low-angle light scattering indicates cell size. This study was performed to measure the relationships between light scattering and cell volumes in an-isotonic solutions and after a freeze-thaw stress. Cell volumes in isolated human lymphocytes, human granulocytes, and hamster fibroblasts were deliberately altered by exposure to anisotonic solutions. Boyle-vant Hoff plots of cell volume as a function of inverse osmotic pressure showed that the cells behaved as osmometers. Similar plots of right-angle and low-angle light scattering showed that the intensity of light scattering varied inversely with cell volume. In other experiments where cells were frozen without cryoprotectant at various sub zero temperatures to -25 degrees C and then thawed rapidly, cell viability decreased progressively with decreasing temperature, as did the intensity of both low-angle and right-angle light scattering, although cell volumes remained relatively constant. The intensity of both low- and high-angle light scattering varied inversely with cell volumes in hypertonic and hypotonic solutions, but cell damage induced by freezing and thawing resulted in significant reductions in the intensity of low-angle light scattering with little change in cell volume. These observations show that light scattering and cell volumes can vary independently, and they underline the need for a better understanding of the phenomenon of light scattering from living cells.     

A two-step method for permeabilization of Drosophila eggs

As a first step in developing a procedure for the cryopreservation of Drosophila melanogaster embryos, we have established a method for permeabilization of the eggcase and have initiated studies of the hydraulic conductivity of permeabilized embryos and the permeation of selected cryoprotective agents. The eggcase of D. melanogaster embryos has a wax layer that precludes any flux of water. A two-step procedure employing organic solvents was developed to effect removal of the wax layer with minimal deleterious effects on the embryos. Dechorionated embryos (Oregon-R strain P2, 12 to 13 hr old) were rinsed sequentially in isopropanol and hexane. After removal of solvent, embryos were held in a modified cell culture medium for further manipulation. This procedure routinely yielded 80 to 95% of the eggs permeabilized (as determined by osmotic contraction in 1 M sucrose) and 75 to 90% survival (incidence of hatching). Hydraulic conductivity of permeabilized embryos and permeation of cryoprotectants were determined using a microdiffusion chamber and computerized video microscopy. Regression analysis of the volumetric data from individual embryos yielded the Boyle-van't Hoff function FVeq = 0.124 (osm-1) + 0.541 with the standard deviations of slope and intercept (Vb) being 0.010 and 0.040, respectively. Permeabilized embryos exhibited ideal osmotic behavior over the range of 0.265 to 2.00 osm. The mean hydraulic conductivity coefficient (Lp) was 0.722 +/- 0.366 micron/(min.atm) at 20 degrees C, based on observations of contraction following a step change in concentration of Ringer's solution.(ABSTRACT TRUNCATED AT 250 WORDS)     

Osmometric behavior of mouse oocytes in the presence of different intracellular sugars

In order to successfully cryopreserve oocytes using low concentrations of intracellular sugars, it is important to characterize their osmotic response in the presence of these intracellular sugars. In the present study, murine (B6D2F1) oocytes were microinjected with 0.8M glucose, trehalose or stachyose solutions to achieve an intracellular concentration equivalent to 0.1M, and then exposed to hypertonic solutions of increasing strength by supplementing an isotonic solution with 0.2, 0.3, 0.5, and 0.7 M of trehalose. Analysis of volumetric response of microinjected oocytes showed that the oocytes behaved as ideal osmometers in the presence of intracellular sugars and satisfied the Boyle van't Hoff relationship. Extrapolation of the osmotically inactive fraction (V macro b) from the Boyle van't Hoff relationship yielded values of 0.188+/-0.028, 0.212+/-0.042, 0.197+/-0.044, and 0.211+/-0.042 for control, glucose, trehalose and stachyose-injected oocytes, respectively. The present data revealing osmometric behavior of mouse oocytes in the presence of different intracellular sugars are important for the optimization of cryopreservation protocols using sugars.     

Transient breakdown in the selective permeability of the plasma membrane ofChlorella emersonii in response to hyperosmotic shock: Implications for cell water relations and osmotic adjustment

Summary In osmotic experiments involving cells of the euryhaline unicellular green algaChlorella emersonii exposed to hyperosmotic stress by immersion in a range of low molecular weight organic and inorganic solutes, a temporary breakdown in the selective permeability of the plasma membrane was observed during the initial phase of transfer to media of high osmotic strength (up to 2000 mosmol kg^–1). Thus, although the cells appeared to obey the Boyle-van't Hoff relationship in all cases, showing approximately linear changes in volume (at high salinity) as a function of the reciprocal of the external osmotic pressure, the extent of change was least for the triitols, propylene glycol and glycerol, intermediate for glucose, sorbitol, NaCl and KCl, with greatest changes in media containing the disaccharides sucrose and maltose. In NaCl-treated cells, uptake of external solute and loss of internal ions was observed in response to hyperosmotic treatment while sucrose-treated cells showed no significant uptake of external solute, although loss of intracellular K⁺ was observed. These observations suggest that the widely used technique of estimating cellular turgor, and osmotic/nonosmotic volume by means of the changes in volume that occur upon transfer to media containing increasing amounts of either a low molecular weight organic solute or an inorganic salt may be subject to error. The assumption that all algal cells behave as ideal osmometers, with outer membranes that are permeable to water but not to solutes, during the course of such experiments is therefore incorrect, and the data need to be adjusted to take account of hyperosmotically induced external solute penetration and/or loss of intracellular osmotica before meaningful estimates of cell turgor and osmotic volume can be obtained.     

Nonequilibrium freezing of one-cell mouse embryos. Membrane integrity and developmental potential.

A thermodynamic model was used to evaluate and optimize a rapid three-step nonequilibrium freezing protocol for one-cell mouse embryos in the absence of cryoprotectants (CPAs) that avoided lethal intracellular ice formation (IIF). Biophysical parameters of one-cell mouse embryos were determined at subzero temperatures using cryomicroscopic investigations (i.e., the water permeability of the plasma membrane, its temperature dependence, and the parameters for heterogeneous IIF). The parameters were then incorporated into the thermodynamic model, which predicted the likelihood of IIF. Model predictions showed that IIF could be prevented at a cooling rate of 120 degrees C/min when a 5-min holding period was inserted at -10 degrees C to assure cellular dehydration. This predicted freezing protocol, which avoided IIF in the absence of CPAs, was two orders of magnitude faster than conventional embryo cryopreservation cooling rates of between 0.5 and 1 degree C/min. At slow cooling rates, embryos predominantly follow the equilibrium phase diagram and do not undergo IIF, but mechanisms other than IIF (e.g., high electrolyte concentrations, mechanical effects, and others) cause cellular damage. We tested the predictions of our thermodynamic model using a programmable freezer and confirmed the theoretical predictions. The membrane integrity of one-cell mouse embryos, as assessed by fluorescein diacetate retention, was approximately 80% after freezing down to -45 degrees C by the rapid nonequilibrium protocol derived from our model. The fact that embryos could be rapidly frozen in the absence of CPAs without damage to the plasma membrane as assessed by fluorescein diacetate retention is a new and exciting finding. Further refinements of this protocol is necessary to retain the developmental competence of the embryos.     

Osmotic significance of glycerol accumulation in exponentially growing yeasts.

Natural-abundance 13C-nuclear magnetic resonance spectroscopy has shown glycerol to be the major osmotically significant low-molecular-weight solute in exponentially growing, salt-stressed cells of the yeasts Saccharomyces cerevisiae, Zygosaccharomyces rouxii, and Debaromyces hansenii. Measurement of the intracellular nonosmotic volume (i.e., the fraction of the cell that is osmotically unresponsive) by using the Boyle-van't Hoff relationship (for nonturgid cells, the osmotic volume is directly proportional to the reciprocal of the external osmotic pressure) showed that the nonosmotic volume represented up to 53% of the total cell volume; the highest values were recorded in media with maximum added NaCl. Determinations of intracellular glycerol levels with respect to cell osmotic volumes showed that increases in intracellular glycerol may counterbalance up to 95% of the external osmotic pressure due to added NaCl. The lack of other organic osmotica in 13C-nuclear magnetic resonance spectra indicates that inorganic ions may constitute the remaining component of intracellular osmotic pressure.     

Osmotic significance of glycerol accumulation in exponentially growing yeasts

Natural-abundance 13C-nuclear magnetic resonance spectroscopy has shown glycerol to be the major osmotically significant low-molecular-weight solute in exponentially growing, salt-stressed cells of the yeasts Saccharomyces cerevisiae, Zygosaccharomyces rouxii, and Debaromyces hansenii. Measurement of the intracellular nonosmotic volume (i.e., the fraction of the cell that is osmotically unresponsive) by using the Boyle-van't Hoff relationship (for nonturgid cells, the osmotic volume is directly proportional to the reciprocal of the external osmotic pressure) showed that the nonosmotic volume represented up to 53% of the total cell volume; the highest values were recorded in media with maximum added NaCl. Determinations of intracellular glycerol levels with respect to cell osmotic volumes showed that increases in intracellular glycerol may counterbalance up to 95% of the external osmotic pressure due to added NaCl. The lack of other organic osmotica in 13C-nuclear magnetic resonance spectra indicates that inorganic ions may constitute the remaining component of intracellular osmotic pressure.     

Estimates of mouse oviductal fluid tonicity based on osmotic responses of embryos

Zygotes and early cleavage-stage embryos are very sensitive to increased osmolality in vitro, although the tonicity of their in vivo environment, oviductal fluid, is unknown. A preference for low osmolality in vitro might imply similar conditions in vivo or be specific to culture. Previous electron probe x-ray microanalysis measurements of total ion content predicted oviductal fluid osmolalities of 310-360 mOs/kg, higher than osmolalities tolerated by mouse zygotes in vitro. However, such indirect estimates may not reflect the tonicity experienced by embryos. We have now used embryos themselves as osmosensors to determine the tonicity of mouse oviductal fluid. In one method, we measured the mean volume of zygotes in undiluted oviductal fluid and compared this to the mean volumes measured for zygotes in media spanning a range of osmolalities. The osmolality corresponding to the measured mean volume in oviductal fluid was taken to be isotonic. In another, independent method, the sizes of zygotes and two-cell embryos were measured as a function of time beginning immediately after removal from oviducts. The osmolality in which the embryos neither swelled nor shrank was taken to be isotonic. Both methods yielded approximately the same range for the tonicity of oviductal fluid: around 290-300 mOs/kg.     

Osmotic properties of the rabbit corneal endothelium and their relevance to cryopreservation

The process of cryopreservation subjects cells to gross changes in the composition of the solution that surrounds them, changes that cause the cells first to shrink and then to swell by an osmotic mechanism. Empirical methods have been developed that permit many cells to survive freezing and thawing, but the cornea, which is crucially dependent upon the function of its endothelial monolayer, has proved quite refractory. In this paper we explore the osmotic response of the corneal endothelium of the rabbit to solutions ranging in osmolality from 0.25 to 8.6 × isotonic. Boyle van't Hoff behavior was observed between 0.43 and 8.6 × isotonic, and there was an apparent nonosmotic volume of 33.6%. However, ultrastructural damage was observed at the limits of this range, and it appeared that the tolerated range was 0.64–4.4 × isotonic. We show the extent to which dimethyl sulfoxide (Me₂SO) would be expected to moderate changes in volume during freezing and suggest that its initial concentration should be at least 2M to prevent excessive shrinkage. We also show that cell swelling during removal of Me₂SO is especially likely to be hazardous.     

Osmotic response of mammalian cells: Effects of permeating cryoprotectants on nonsolvent volume

The nonsolvent volume, b, of a cell permits calculation of cell water volume from measurements of total cell volume, and, consequently, it is used extensively in the determination of membrane permeability coefficients for water and solutes and also in simulations of water and solute fluxes during freezing of cells. The nonsolvent volume is most commonly determined from the ordinate intercept of plots of cell volume as a function of the reciprocal of extracellular nonpermeating solute concentration (so-called Boyle-van't Hoff plots). Once derived, b is often assumed to be constant even under conditions that may differ markedly from those under which it was determined. Our aim was to investigate whether this assumption was valid when cells were exposed to the cryoprotectants glycerol, dimethyl sulphoxide (Me₂SO), or propane-1,2-diol. Rabbit corneal keratocytes, a fibroblastic cell type, were exposed to 10% (v/v) cryoprotectant for 30 min at 22°C in solutions containing a range of nonpermeating solute concentrations. Cell volumes were determined by an electronic particle sizer and mode volume plotted as an inverse function of the concentration of nonpermeating solute. The cells behaved as osmometers under all conditions studied, but we found no evidence to suggest that the nonsolvent volume of cells was altered by Me₂SO or propane-1,2-diol. Glycerol, however, reduced the slope of the Boyle-van't Hoff plot, but this could be ascribed to the failure of the cells to equilibrate fully with the glycerol over the 30 min exposure time; thus, b was unaffected by glycerol. It may be assumed, therefore, that the nonsolvent volume was not influenced by the presence inside cells of any of these nonelectrolyte cryoprotectants. © 1996 Wiley-Liss, Inc.     

The effects of ultrarapid freezing on meiotic and mitotic spindles of mouse oocytes and embryos

Preovulatory mouse oocytes and 2-cell embryos were frozen with dimethyl sulfoxide and propanediol by an ultrarapid method. The survival of frozen oocytes was low (33–34%) compared to that of 2-cell embryos (78–79%) with either cryoprotectant. Development to blastocysts after postthaw culture was about 7–15% for oocytes and 79–80% for the embryos. Ultrarapid freezing preserves cell structure quite well as revealed by electron microscopy, but meiotic oocytes and late 2-cell embryos undergoing mitosis showed evidence of spindle disorganization involving loss or clumping of microtubules resulting in some scattering of chromosomes. Embryos developed from frozen eggs showed clear evidence of micronuclear formation and incomplete incorporation of chromosomal material into main nuclei. These experiments confirm our observations on freezing of human oocytes and show that spindle microtubules are sensitive to freeze-thawing and that cryopreservation could cause chromosomal aberrations during early development. A cautious approach to the introduction of oocyte freezing in human in vitro fertilization (IVF) programs is advocated.     

Cytological effects of prefixation treatment

The effects produced by prefixation treatments on cells in metaphase from 10-day mouse fetuses and from several embryonic stages of the frog were investigated. The technical value of some of these pretreatments is noted. Pretreatment with isotonic solutions (both ionic and non-ionic in the case of the mouse, ionic only in the frog) generally produced a similar effect, viz., chromosomal swelling with little effect on the spindle. A notable exception is provided by frog embryos preceding the neurula stage; spindle disorganization without chromosomal swelling was produced by pretreatment in isotonic modified Niu-Twitty solution, containing no divalent cations. Pretreatment with hypotonic solutions (both ionic and non-ionic in the case of the mouse, ionic only in the frog) generally produced several major effects, viz., despiralization of chromosomes, chromatid separation, and spindle disorganization. The conclusion is drawn from the mouse data that, in order to produce these effects, pretreating solutions must be of low osmotic pressure. Low ionic strength alone (e.g., isotonic sucrose solutions) is not sufficient. As differentiation of frog embryos progressed, pretreatments either of longer duration or with solutions of increasing degrees of hypotonicity were required to produce comparable intensities of the same effects. Many of the effects on metaphases produced by hypotonic pretreatment of frog embryos were reversible by subsequent exposure to isotonic solutions. The significance of results presented here is discussed briefly with respect to some current considerations of the macromolecular structure of chromosomes.     

Osmotic properties of the rabbit corneal endothelium and their relevance to cryopreservation

The process of cryopreservation subjects cells to gross changes in the composition of the solution that surrounds them, changes that cause the cells first to shrink and then to swell by an osmotic mechanism. Empirical methods have been developed that permit many cells to survive freezing and thawing, but the cornea, which is crucially dependent upon the function of its endothelial monolayer, has proved quite refractory. In this paper we explore the osmotic response of the corneal endothelium of the rabbit to solutions ranging in osmolality from 0.25 to 8.6 X isotonic. Boyle van't Hoff behavior was observed between 0.43 and 8.6 X isotonic, and there was an apparent nonosmotic volume of 33.6%. However, ultrastructural damage was observed at the limits of this range, and it appeared that the tolerated range was 0.64-4.4 X isotonic. We show the extent to which dimethyl sulfoxide (Me2SO) would be expected to moderate changes in volume during freezing and suggest that its initial concentration should be at least 2M to prevent excessive shrinkage. We also show that cell swelling during removal of Me2SO is especially likely to be hazardous.