Membrane Transition Temperature Determines Cisplatin Response

Cisplatin is a classical chemotherapeutic agent used in treating several forms of cancer including head and neck. However, cells develop resistance to the drug in some patients through a range of mechanisms, some of which are poorly understood. Using isolated plasma membrane vesicles as a model system, we present evidence suggesting that cisplatin induced resistance may be due to certain changes in the bio-physical properties of plasma membranes. Giant plasma membrane vesicles (GPMVs) isolated from cortical cytoskeleton exhibit a miscibility transition between a single liquid phase at high temperature and two distinct coexisting liquid phases at low temperature. The temperature at which this transition occurs is hypothesized to reflect the magnitude of membrane heterogeneity at physiological temperature. We find that addition of cisplatin to vesicles isolated from cisplatin-sensitive cells result in a lowering of this miscibility transition temperature, whereas in cisplatin-resistant cells such treatment does not affect the transition temperature. To explore if this is a cause or consequence of cisplatin resistance, we tested if addition of cisplatin in combination with agents that modulate GPMV transition temperatures can affect cisplatin sensitivity. We found that cells become more sensitive to cisplatin when isopropanol, an agent that lowers GPMV transition temperature, was combined with cisplatin. Conversely, cells became resistant to cisplatin when added in combination with menthol that raises GPMV transition temperatures. These data suggest that changes in plasma membrane heterogeneity augments or suppresses signaling events initiated in the plasma membranes that can determine response to cisplatin. We postulate that desired perturbations of membrane heterogeneity could provide an effective therapeutic strategy to overcome cisplatin resistance for certain patients.     

Elucidating membrane structure and protein behavior using giant plasma membrane vesicles

The observation of phase separation in intact plasma membranes isolated from live cells is a breakthrough for research into eukaryotic membrane lateral heterogeneity, specifically in the context of membrane rafts. These observations are made in giant plasma membrane vesicles (GPMVs), which can be isolated by chemical vesiculants from a variety of cell types and microscopically observed using basic reagents and equipment available in any cell biology laboratory. Microscopic phase separation is detectable by fluorescent labeling, followed by cooling of the membranes below their miscibility phase transition temperature. This protocol describes the methods to prepare and isolate the vesicles, equipment to observe them under temperature-controlled conditions and three examples of fluorescence analysis: (i) fluorescence spectroscopy with an environment-sensitive dye (laurdan); (ii) two-photon microscopy of the same dye; and (iii) quantitative confocal microscopy to determine component partitioning between raft and nonraft phases. GPMV preparation and isolation, including fluorescent labeling and observation, can be accomplished within 4 h.     

Liquid General Anesthetics Lower Critical Temperatures in Plasma Membrane Vesicles

A large and diverse array of small hydrophobic molecules induce general anesthesia. Their efficacy as anesthetics has been shown to correlate both with their affinity for a hydrophobic environment and with their potency in inhibiting certain ligand-gated ion channels. In this study we explore the effects that n-alcohols and other liquid anesthetics have on the two-dimensional miscibility critical point observed in cell-derived giant plasma membrane vesicles (GPMVs). We show that anesthetics depress the critical temperature (T_c) of these GPMVs without strongly altering the ratio of the two liquid phases found below T_c. The magnitude of this affect is consistent across n-alcohols when their concentration is rescaled by the median anesthetic concentration (AC₅₀) for tadpole anesthesia, but not when plotted against the overall concentration in solution. At AC₅₀ we see a 4°C downward shift in T_c, much larger than is typically seen in the main chain transition at these anesthetic concentrations. GPMV miscibility critical temperatures are also lowered to a similar extent by propofol, phenylethanol, and isopropanol when added at anesthetic concentrations, but not by tetradecanol or 2,6 diterbutylphenol, two structural analogs of general anesthetics that are hydrophobic but have no anesthetic potency. We propose that liquid general anesthetics provide an experimental tool for lowering critical temperatures in plasma membranes of intact cells, which we predict will reduce lipid-mediated heterogeneity in a way that is complimentary to increasing or decreasing cholesterol. Also, several possible implications of our results are discussed in the context of current models of anesthetic action on ligand-gated ion channels.     

Pulsed-laser creation and characterization of giant plasma membrane vesicles from cells

Femtosecond-pulsed laser irradiation was found to initiate giant plasma membrane vesicle (GPMV) formation on individual cells. Laser-induced GPMV formation resulted from intracellular cavitation and did not require the addition of chemical stressors to the cellular environment. The viscosity, structure, and contents of laser-induced GPMVs were measured with fluorescence microscopy and single-particle tracking. These GPMVs exhibit the following properties: (1) GPMVs grow fastest immediately after laser irradiation; (2) GPMVs contain barriers to free diffusion of incorporated fluorescent beads; (3) materials from both the cytoplasm and surrounding media flow into the growing GPMVs; (4) the GPMVs are surrounded by phospholipids, including phosphatidylserine; (5) F-actin is incorporated into the vesicles; and (6) caspase activity is not essential for GPMV formation. The effective viscosity of 65 nm polystyrene nanoparticles within GPMVs ranged from 32 to 434 cP. The nanoparticle diffusion was commonly affected by relatively large, macromolecular structures within the bleb.     

Cell-Derived Plasma Membrane Vesicles Are Permeable to Hydrophilic Macromolecules

Giant plasma membrane vesicles (GPMVs) are a widely used experimental platform for biochemical and biophysical analysis of isolated mammalian plasma membranes (PMs). A core advantage of these vesicles is that they maintain the native lipid and protein diversity of the PM while affording the experimental flexibility of synthetic giant vesicles. In addition to fundamental investigations of PM structure and composition, GPMVs have been used to evaluate the binding of proteins and small molecules to cell-derived membranes and the permeation of drug-like molecules through them. An important assumption of such experiments is that GPMVs are sealed, i.e., that permeation occurs by diffusion through the hydrophobic core rather than through hydrophilic pores. Here, we demonstrate that this assumption is often incorrect. We find that most GPMVs isolated using standard preparations are passively permeable to various hydrophilic solutes as large as 40 kDa, in contrast to synthetic giant unilamellar vesicles. We attribute this leakiness to stable, relatively large, and heterogeneous pores formed by rupture of vesicles from cells. Finally, we identify preparation conditions that minimize poration and allow evaluation of sealed GPMVs. These unexpected observations of GPMV poration are important for interpreting experiments utilizing GPMVs as PM models, particularly for drug permeation and membrane asymmetry.     

MPP1 as a Factor Regulating Phase Separation in Giant Plasma Membrane-Derived Vesicles

The existence of membrane-rafts helps to conceptually understand the spatiotemporal organization of membrane-associated events (signaling, fusion, fission, etc.). However, as rafts themselves are nanoscopic, dynamic, and transient assemblies, they cannot be directly observed in a metabolizing cell by traditional microscopy. The observation of phase separation in giant plasma membrane-derived vesicles from live cells is a powerful tool for studying lateral heterogeneity in eukaryotic cell membranes, specifically in the context of membrane rafts. Microscopic phase separation is detectable by fluorescent labeling, followed by cooling of the membranes below their miscibility phase transition temperature. It remains unclear, however, if this lipid-driven process is tuneable in any way by interactions with proteins. Here, we demonstrate that MPP1, a member of the MAGUK family, can modulate membrane properties such as the fluidity and phase separation capability of giant plasma membrane-derived vesicles. Our data suggest that physicochemical domain properties of the membrane can be modulated, without major changes in lipid composition, through proteins such as MPP1.     

Miscibility of phosphatidylcholine binary mixtures in unilamellar vesicles: Phase equilibria

Summary Miscibility among phospholipids with different lipid chain-lengths or with different head groups has attracted a number of research efforts because of its significance in biological membrane structure and function. The general consensus about the miscibility of phosphatidylcholines with varying lipid chainlengths appears to be that binary mixtures of phospholipids with a difference of two carbon atoms in the lipid chain mix well at the main phase transition. Miscibility between phosphatidylcholines with differences of four carbon atoms appears to be inconclusive. Previous reports on the phase transition of binary phospholipid mixtures are concerned mainly with multilamellar vesicles and are mostly limited to the main transition. In the present study, unilamellar vesicles were used and miscibility in binary systems between dimyristoyl-, dipalmitoyl- and distearoyl-phosphatidylcholines at pretransition, as well as main transition temperatures was evaluated by constructing phase diagrams. Two methods were used to monitor the phase transitions: differential scanning microcalorimetry and optical absorbance methods. The optical method has the advantage that unilamellar vesicles of dilute phospholipid concentrations can be used. The liquidus and solidus phase boundaries were determined by the onset temperature of heating and cooling scans, respectively, because the completion temperature of a phase transition has no meaning in binary solutions. Dimyristoyl- and distearoyl-phosphatidylcholines. where the difference in the, lipid chain-length is four carbon atoms, mixed well even at pretransition temperature.     

Structural determinants for partitioning of lipids and proteins between coexisting fluid phases in giant plasma membrane vesicles

The structural basis for organizational heterogeneity of lipids and proteins underlies fundamental questions about the plasma membrane of eukaryotic cells. A current hypothesis is the participation of liquid ordered (Lo) membrane domains (lipid rafts) in dynamic compartmentalization of membrane function, but it has been difficult to demonstrate the existence of these domains in live cells. Recently, giant plasma membrane vesicles (GPMVs) obtained by chemically induced blebbing of cultured cells were found to phase separate into optically resolvable, coexisting fluid domains containing Lo-like and liquid disordered (Ld)-like phases as identified by fluorescent probes. In the present study, we used these GPMVs to investigate the structural bases for partitioning of selected lipids and proteins between coexisting Lo-like/Ld-like fluid phases in compositionally complex membranes. Our results with lipid probes show that the structure of the polar headgroups, in addition to acyl chain saturation, can significantly affect partitioning. We find that the membrane anchor of proteins and the aggregation state of proteins both significantly influence their distributions between coexisting fluid phases in these biological membranes. Our results demonstrate the value of GPMVs for characterizing the phase preference of proteins and lipid probes in the absence of detergents and other perturbations of membrane structure.     

Structural determinants for partitioning of lipids and proteins between coexisting fluid phases in giant plasma membrane vesicles

The structural basis for organizational heterogeneity of lipids and proteins underlies fundamental questions about the plasma membrane of eukaryotic cells. A current hypothesis is the participation of liquid ordered (Lo) membrane domains (lipid rafts) in dynamic compartmentalization of membrane function, but it has been difficult to demonstrate the existence of these domains in live cells. Recently, giant plasma membrane vesicles (GPMVs) obtained by chemically induced blebbing of cultured cells were found to phase separate into optically resolvable, coexisting fluid domains containing Lo-like and liquid disordered (Ld)-like phases as identified by fluorescent probes. In the present study, we used these GPMVs to investigate the structural bases for partitioning of selected lipids and proteins between coexisting Lo-like/Ld-like fluid phases in compositionally complex membranes. Our results with lipid probes show that the structure of the polar headgroups, in addition to acyl chain saturation, can significantly affect partitioning. We find that the membrane anchor of proteins and the aggregation state of proteins both significantly influence their distributions between coexisting fluid phases in these biological membranes. Our results demonstrate the value of GPMVs for characterizing the phase preference of proteins and lipid probes in the absence of detergents and other perturbations of membrane structure.     

Adhesion Stabilizes Robust Lipid Heterogeneity in Supercritical Membranes at Physiological Temperature

Regions of contact between cells are frequently enriched in or depleted of certain protein or lipid species. Here, we explore a possible physical basis that could contribute to this membrane heterogeneity using a model system of a giant vesicle tethered to a planar supported bilayer. Vesicles contain coexisting liquid-ordered (L_o) and liquid-disordered (L_d) phases at low temperatures and are tethered using trace quantities of adhesion molecules that preferentially partition into one liquid phase. We find that the L_d marker DiI-C₁₂ is enriched or depleted in the adhered region when adhesion molecules partition into L_d or L_o phases, respectively. Remarkably, adhesion stabilizes an extended zone enriched or depleted of DiI-C₁₂ even at temperatures >15°C above the miscibility phase transition when membranes have compositions that are in close proximity to a critical point. A stable adhesion zone is also observed in plasma membrane vesicles isolated from living RBL-2H3 cells, and probe partitioning at 37°C is diminished in vesicles isolated from cells with altered cholesterol levels. Probe partitioning is in good quantitative agreement with predictions of the two-dimensional Ising model with a weak applied field for both types of model membranes. These studies experimentally demonstrate that large and stable domain structure can be mediated by lipids in single-phase membranes with supercritical fluctuations.     

Hemagglutinin of Influenza Virus Partitions into the Nonraft Domain of Model Membranes

The HA of influenza virus is a paradigm for a transmembrane protein thought to be associated with membrane-rafts, liquid-ordered like nanodomains of the plasma membrane enriched in cholesterol, glycosphingolipids, and saturated phospholipids. Due to their submicron size in cells, rafts can not be visualized directly and raft-association of HA was hitherto analyzed by indirect methods. In this study, we have used GUVs and GPMVs, showing liquid disordered and liquid ordered domains, to directly visualize partition of HA by fluorescence microscopy. We show that HA is exclusively (GUVs) or predominantly (GPMVs) present in the liquid disordered domain, regardless of whether authentic HA or domains containing its raft targeting signals were reconstituted into model membranes. The preferential partition of HA into ld domains and the difference between lo partition in GUV and GPMV are discussed with respect to differences in packaging of lipids in membranes of model systems and living cells suggesting that physical properties of lipid domains in biological membranes are tightly regulated by protein-lipid interactions.     

Gel to Liquid-Crystalline Phase Transitions of Lipids and Membranes Isolated from Escherichia coli Cells

Differential scanning calorimetry (DSC) was used to examine the relationship of the gel to liquid-crystalline phase transition of lipids to fatty acid composition with membrane lipids and spheroplast membranes isolated from cells of a wild strain and an unsaturated fatty acid auxotroph of Escherichia coli grown under various conditions. These lipids and membranes underwent thermotropic phase transitions at different temperatures depending on the thermal properties of their constituent fatty acids. The lipid phase transition occurred at higher temperatures in biomembranes than in extracted lipids. DSC thermograms of lipids synthesized by bacterial cells which were observed at a temperature scanning rate as slow as 0.3 K min^-1 were characterized by a distinctly plain peak summit. Endothermic peaks given by samples derived from elaidic acid-enriched cells were relatively narrow and asymmetric. The discrepancy between the transition temperatures measured with extracted lipids and with membraneous fractions, and the shape of the endothermic peaks, are discussed.     

Temperature-dependent phase behavior and protein partitioning in giant plasma membrane vesicles

Liquid-ordered (Lo) and liquid-disordered (Ld) phase coexistence has been suggested to partition the plasma membrane of biological cells into lateral compartments, allowing for enrichment or depletion of functionally relevant molecules. This dynamic partitioning might be involved in fine-tuning cellular signaling fidelity through coupling to the plasma membrane protein and lipid composition. In earlier work, giant plasma membrane vesicles, obtained by chemically induced blebbing from cultured cells, were observed to reversibly phase segregate at temperatures significantly below 37 °C. In this contribution, we compare the temperature dependence of fluid phase segregation in HeLa and rat basophilic leukemia (RBL) cells. We find an essentially monotonic temperature dependence of the number of phase-separated vesicles in both cell types. We also observe a strikingly broad distribution of phase transition temperatures in both cell types. The binding of peripheral proteins, such as cholera toxin subunit B (CTB), as well as Annexin V, is observed to modulate phase transition temperatures, indicating that peripheral protein binding may be a regulator for lateral heterogeneity in vivo. The partitioning of numerous signal protein anchors and full length proteins is investigated. We find Lo phase partitioning for several proteins assumed in the literature to be membrane raft associated, but observe deviations from this expectation for other proteins, including caveolin-1.     

Coupling of Membrane Nanodomain Formation and Enhanced Electroporation near Phase Transition

Biological cells are enveloped by a heterogeneous lipid bilayer that prevents the uncontrolled exchange of substances between the cell interior and its environment. In particular, membranes act as a continuous barrier for salt and macromolecules to ensure proper physiological functions within the cell. However, it has been shown that membrane permeability strongly depends on temperature and, for phospholipid bilayers, displays a maximum at the transition between the gel and fluid phase. Here, extensive molecular dynamics simulations of dipalmitoylphosphatidylcholine bilayers were employed to characterize the membrane structure and dynamics close to phase transition, as well as its stability with respect to an external electric field. Atomistic simulations revealed the dynamic appearance and disappearance of spatially related nanometer-sized thick ordered and thin interdigitating domains in a fluid-like bilayer close to the phase transition temperature (T_m). These structures likely represent metastable precursors of the ripple phase that vanished at increased temperatures. Similarly, a two-phase bilayer with coexisting gel and fluid domains featured a thickness minimum at the interface because of splaying and interdigitating lipids. For all systems, application of an external electric field revealed a reduced bilayer stability with respect to pore formation for temperatures close to T_m. Pore formation occurred exclusively in thin interdigitating membrane nanodomains. These findings provide a link between the increased membrane permeability and the structural heterogeneity close to phase transition.     

Infrared spectroscopic study of the gel to liquid-crystal phase transition in live Acholeplasma laidlawii cells

The temperature dependences of the infrared spectra of deuterium-labeled plasma membranes of live Acholeplasma laidlawii B cells and of the isolated plasma membranes demonstrate that the profiles of the gel to liquid-crystal phase transitions are very different. At temperatures within the range of the phase transition, the live mycoplasma is able to keep the "fluidity" of its plasma membrane at a much higher value than that of the isolated plasma membrane at the same temperature. The difference is particularly pronounced at and around the temperature of growth. Live Acholeplasma laidlawii, grown at 37 degrees C on a fatty acid depleted medium supplemented with myristic acid (C14:0), pentadecanoic acid (C15:0), or palmitic acid (C16:0), are highly "fluid"; i.e., at the temperature of growth, the fractional population of the liquid-crystalline phase is 95-100% at 37 degrees C, whereas in the case of the isolated plasma membranes the fractional population of the liquid-crystalline phase at 37 degrees C is only 58% (C14:0), 36% (C15:0), or 38% (C16:0).     

Infrared spectroscopic studies of Acholeplasma laidlawaii B membranes. Comparison of the gel to liquid-crystal phase transition in intact cells and isolated membranes

The physical state of the membrane lipids in the plasma membranes of intact, live Acholeplasma laidlawii B cells was probed by Fourier-transform infrared spectroscopy and compared with that in isolated membranes. Infrared spectra of live A. laidlawii B cells, enriched biosynthetically in the presence of avidin, with saturated deuterated and unsaturated non-deuterated fatty acids have been recorded at a variety of temperatures. The results indicate that within the temperature range of the gel to liquid-crystal phase transition, the live cells are able to keep the 'fluidity' of their plasma membranes at a considerably higher value compared to that in the isolated membranes at the same temperature. While this is a generally valid observation, the degree by which live and isolated membranes differ in their liquid-crystal-phase content at a given temperature depends on the nature of the exogenous fatty acid and the temperature of growth.     

Phase Transition of Thylakoid Membranes Modulates Photoinhibition in the Cyanobacterium Anabaena siamensis

A shift of the growth temperature from 40 degrees C to 18 degrees C promoted an increase in the degree of fatty acids unsaturation and a decrease, from 26 degrees C to 0 degrees C, of the phase transition temperature of thylakoid membranes in Anabaena siamensis. The pattern of photoinhibition of photosynthesis at distinct temperatures varied as a function of the phase transition temperature. In the absence of streptomycin, a pronounced photoinhibition at temperatures near the phase transition (26 degrees C) was observed in cells grown at 40 degrees C, while protection from photodamage was observed at chilling temperatures (15 degrees C to 5 degrees C). In this same range of temperature, such a protection was not verified if cells were grown at 18 degrees C. In both types of cells, however, the rate of photoinactivation in the presence of streptomycin was progressively decreased by lowering the temperature of photoinhibition. When recovery from photoinhibition was followed at the respective temperature in which cells were grown, the restoration profile of the photosynthetic O(2) evolution to initial levels was essentially the same in both types of cells. The protective effect of low temperatures against photoinhibition was attributed to a decreased solubility and diffusion of oxygen in the thylakoid membranes due to an increase of the membrane viscosity that would avoid the photogeneration of reactive oxygen species around PS II.     

The effect of alterations in the physical state of the membrane lipids on the ability of Acholeplasma laidlawii B to grow at various temperatures

The physical state of the membrane lipids, as determined by fatty acid composition and environmental temperature, has a marked effect on both the temperature range within which Acholeplasma laidlawii B cells can grow and on growth rates within the permissible temperature ranges. The minimum growth temperature of 8 °C is not defined by the fatty acid composition of the membrane lipids when cells are enriched in fatty acids giving rise to gel to liquid-crystalline membrane lipid phase transitions occurring below this temperature. The elevated minimum growth temperatures of cells enriched in fatty acids giving rise to lipid phase transitions occurring at higher temperatures, however, are clearly defined by the fatty acid composition of the membrane lipids. The optimum and maximum growth temperatures are also influenced indirectly by the physical state of the membrane lipids, being significantly reduced for cells supplemented with lower melting, unsaturated fatty acids. The temperature coefficient of growth at temperatures near or above the midpoint of the lipid phase transition is 16 to 18 $\text{kcal}\text{mol}$, but this value increases abruptly to 40 to 45 $\text{kcal}\text{mol}$ at temperatures below the phase transition midpoint. Both the absolute rates and temperature coefficients of cell growth are similar for cells whose membrane lipids exist entirely or predominantly in the liquid-crystalline state, but absolute growth rates decline rapidly and temperature coefficients increase at temperatures where more than half of the membrane lipids become solidified. Cell growth ceases when the conversion of the membrane lipid to the gel state approaches completion, but growth and replication can continue at temperatures where less than one tenth of the total lipid remains in the fluid state. An appreciable heterogeneity in the physical state of the membrane lipids can apparently be tolerated by this organism without a detectable loss of membrane function.     

Studies on Freezing Injury of Plant Cells: I. Relation between Thermotropic Properties of Isolated Plasma Membrane Vesicles and Freezing Injury

The thermotropic transition of plasma membrane of Dactylis glomerata was studied by using fluorescence polarization of embedded fluorophore, 1,6-diphenyl-1,3,5-hexatriene. Under the presence of 35% ethylene glycol, reversible thermotropic transitions were observed in isolated plasma membrane vesicles in nearly the same temperature range as the temperature of freezing injury to cells. In liposomes prepared from isolated plasma membranes, however, the thermotropic transitions occurred at much lower temperatures in comparison with those of intact membrane vesicles. Following treatment with pronase, the thermotropic transition also shifted downward.

Thus, the thermotropic properties of plasma membranes appeared to be dependent on the membrane proteins. In vitro freezing of the isolated plasma membrane vesicles without addition of any cryoprotectant, such as sorbitol, resulted in an irreversible alteration both in the fluorescence anisotropy values and the temperatures for the thermotropic transition, suggesting an irreversible alteration in the membrane structure, presumably changes in lipid-protein interactions and protein conformation.

     

Phase Transition Temperature and Chilling Sensitivity of Bovine Oocytes

A limiting factor for achieving cryopreservation of oocytes is direct chilling injury (DCI), which occurs during cooling. DCI, or cold shock, is defined as an irreversible damage expressed shortly after exposure to low, but not freezing, temperatures. The primary target of DCI is thought to be the plasma membrane. Recently, an association between DCI in sperm and the thermotropic phase transition of their membrane lipids was demonstrated. In the present study, we examined the phase transition of the membrane lipids of immature andin vitro-matured bovine oocytes during cooling, using Fourier transform infrared spectroscopy (FTIR). The phase transition of the membrane lipids of oocytes at the germinal vesicle (GV) stage occurred between 13 and 20°C, while a very broad phase transition, which centered around 10°C, was observed for mature oocytes (MII) stage. Thermotropic phase transitions were demonstrated to be related to the temperature at which DCI affected the integrity of the oocyte membranes. When immature oocytes were cooled to 13°C, fewer oocytes (40%) retained their membrane integrity than after exposure to 4°C (51%) or holding them at 38°C (78%), (as determined by the Fluorescein Diacetate-FDA test). This finding might suggest that holding immature oocytes at the phase transition temperature is more damaging to their membranes than exposure to lower temperatures. By contrast, no significant differences in membrane integrity were observed whenin vitro-matured oocytes were cooled to the same temperatures. Subsequently, GV oocytes were cooled to 4°C, and 26% underwent maturation and 19% underwent fertilizationin vitro. In vitro-matured oocytes that were cooled to 4°C displayed a slightly decreased rate of fertilization; the overall fertilization was 60% with 24% polyspermy, rather than the 76% fertilization rate with 12% polyspermy obtained with those not subjected to cooling. The high rate of polyspermy indicates that a site(s) other than the plasma membrane is affected during cooling of bovine oocytes. Nucleated bovine GV oocytes were electrofused within vitro-matured and enucleated oocytes, and then cooled to 4°C. Evaluation of the membrane integrity of the fused oocytes showed that these oocytes are chilling resistant, which strongly suggests that alteration of the membrane composition of an oocyte can change the cell's susceptibility to low temperatures. This finding led to an improvement in the survival of oocytes after cryopreservation.