Glycol Permeability Research Articles

Effect of cryoprotectant concentration on bovine oocyte permeability and comparison of two membrane permeability modelling approaches

The plasma membrane permeability to water and cryoprotectant (CPA) significantly impacts vitrification efficiency of bovine oocytes. Our study was designed to determine the concentration-dependent permeability characteristics for immature (GV) and mature (MII) bovine oocytes in the presence of ethylene glycol (EG) and dimethyl sulphoxide (Me2SO), and to compare two different modeling approaches: the two parameter (2P) model and a nondilute transport model. Membrane permeability parameters were determined by consecutively exposing oocytes to increasing concentrations of Me2SO or EG. Higher water permeability was observed for MII oocytes than GV oocytes in the presence of both Me2SO and EG, and in all cases the water permeability was observed to decrease as CPA concentration increased. At high CPA concentrations, the CPA permeability was similar for Me2SO and EG, for both MII and GV oocytes, but at low concentrations the EG permeability of GV oocytes was substantially higher. Predictions of cell volume changes during CPA addition and removal indicate that accounting for the concentration dependence of permeability only has a modest effect, but there were substantial differences between the 2P model and the nondilute model during CPA removal, which may have implications for design of improved methods for bovine oocyte vitrification.

Enhanced water and cryoprotectant permeability of porcine oocytes after artificial expression of human and zebrafish aquaporin‐3 channels

One of the major obstacles for the vitrification of mature porcine oocytes with ethylene glycol is their low permeability to this cryoprotectant, which results in osmotic stress-induced cell damage and low survival. Pig blastocysts, on the other hand, show enhanced water and cryoprotectant permeability, which has been related to the transcriptional activation of aquaporin-3 (AQP3) channels at this stage of development. In this study, we asked if expression of cRNAs encoding two aquaglyceroporins, human AQP3 (hAQP3) or the zebrafish Aqp3b-T85A mutant, in porcine oocytes can increase their permeability. Microinjection of germinal-vesicle-stage oocytes with enhanced green fluorescent protein (EGFP) or AQP3 cRNAs resulted in the expression of the corresponding proteins in ∼26% of the metaphase-II stage oocytes at 40-44 hr of in vitro culture; co-injection of EGFP cRNA appeared to be a suitable marker for oocyte selection since all EGFP-positive oocytes also expressed the corresponding aquaporin. Using this method, we found that mature oocytes co-expressing EGFP and hAQP3 or EGFP and Aqp3b-T85A showed approximately a twofold increase of the hydraulic conductivity (Lp ) with respect non-injected or EGFP alone-injected oocytes in a 0.43 M sucrose or 1.3 M ethylene glycol solution, whereas the ethylene glycol permeability (PEG ) of EGFP + hAQP3 and EGFP + Aqp3b-T85A oocytes was 6.7- and 12-fold higher, respectively, than control oocytes. These data demonstrate that the artificial expression of aquaglyceroporins in porcine metaphase-II oocytes improves their permeability, and that the zebrafish Aqp3b-T85A mutant is more efficient than the human channel at increasing the oocyte permeability to ethylene glycol.

Design and characterization of genetically engineered zebrafish aquaporin-3 mutants highly permeable to the cryoprotectant ethylene glycol

BackgroundIncreasing cell membrane permeability to water and cryoprotectants is critical for the successful cryopreservation of cells with large volumes. Artificial expression of water-selective aquaporins or aquaglyceroporins (GLPs), such as mammalian aquaporin-3 (AQP3), enhances cell permeability to water and cryoprotectants, but it is known that AQP3-mediated water and solute permeation is limited and pH dependent. To exploit further the possibilities of using aquaporins in cryobiology, we investigated the functional properties of zebrafish (Danio rerio) GLPs.ResultsWater, glycerol, propylene glycol and ethylene glycol permeability of zebrafish Aqp3a, -3b, -7, -9a, -9b, -10a and -10b, and human AQP3, was examined. Expression in Xenopus laevis oocytes indicated that the permeability of DrAqp3a and -3b to ethylene glycol was higher than for glycerol or propylene glycol under isotonic conditions, unlike other zebrafish GLPs and human AQP3, which were more permeable to glycerol. In addition, dose-response experiments and radiolabeled ethylene glycol uptake assays suggested that oocytes expressing DrAqp3b were permeated by this cryoprotectant more efficiently than those expressing AQP3. Water and ethylene glycol transport through DrAqp3a and -3b were, however, highest at pH 8.5 and completely abolished at pH 6.0. Point mutations in the DrAqp3b amino acid sequence rendered two constructs, DrAqp3b-T85A showing higher water and ethylene glycol permeability at neutral and alkaline pH, and DrAqp3b-H53A/G54H/T85A, no longer inhibited at acidic pH but less permeable than the wild type. Finally, calculation of permeability coefficients for ethylene glycol under concentration gradients confirmed that the two DrAqp3b mutants were more permeable than wild-type DrAqp3b and/or AQP3 at neutral pH, resulting in a 2.6- to 4-fold increase in the oocyte intracellular concentration of ethylene glycol.ConclusionBy single or triple point mutations in the DrAqp3b amino acid sequence, we constructed one mutant with enhanced ethylene glycol permeability and another with reduced pH sensitivity. The DrAqp3b and the two mutant constructs may be useful for application in cryobiology.

Determination of the membrane permeability characteristics of Pacific oyster, Crassostrea gigas, oocytes and development of optimized methods to add and remove ethylene glycol

In order for cryopreservation to become a practical tool for aquaculture, optimized protocols must be developed for each species and cell type. Knowledge of a cell's osmotic tolerance and membrane permeability characteristics can assist in optimized protocol development. In this study, these characteristics were determined for Pacific oyster oocytes and modified methods for loading and unloading ethylene glycol (EG) were tested. Oocytes were found to behave as ideal osmometers and their osmotically inactive fraction (V(b)) was calculated to be 0.48. Oocytes exposed to NaCl solutions of 0.6 to 2.3 Osm fertilized at rates equivalent to oocytes left in seawater. This corresponds to volume changes of +27.3 and -38.1+/-1.2%. The permeability of the oocytes to water (L(p)) was determined to be 3.8+/-0.4 x 10(-2), 5.7+/-0.8 x 10(-2), and 13.2+/-1.3 x 10(-2) microm min(-1)atm(-1), when measured at temperatures of 5, 10 and 20 degrees C. The respective EG permeability values (P(s)) were 9.5+/-0.1 x 10(-5), 14.6+/-1.2 x 10(-5), and 41.7+/-2.4 x 10(-5) cm min(-1). The activation energies for L(p) and P(s) were determined to be 14.5 and 17.5 kcal mol(-1), respectively. Different models for EG loading and unloading from oocytes were developed and tested. Post-thaw fertilization did not differ significantly between a published step addition method and single step addition at 20 degrees C. This represents a considerable reduction in handling. The results of this study demonstrate that the cryobiological characteristics of a given cell type should be taken into account when developing cryopreservation methods.

Human oocyte vitrification: the permeability of metaphase II oocytes to water and ethylene glycol and the appliance toward vitrification

To determine the permeability of human metaphase II oocytes to ethylene glycol and water in the presence of ethylene glycol, and to use this information to develop a method to vitrify human oocytes. An incomplete randomized block design. A university-affiliated assisted reproductive center. Women undergoing assisted reproduction in the Center for Reproductive Medicine at Shandong University. Oocytes were exposed to 1.0 molar ethylene glycol in a single step and photographed during subsequent volume excursions. A two-parameter model was employed to estimate the permeability to water and ethylene glycol. Water permeability ranged from 0.15 to 1.17 microm/(min.atm), and ethylene glycol permeability ranged from 1.5 to 30 microm/min between 7 degrees C at 36 degrees C. The activation energies for water and ethylene glycol permeability were 14.42 Kcal/mol and 21.20 Kcal/mol, respectively. Despite the lower permeability of human metaphase II oocytes to ethylene glycol compared with previously published values for propylene glycol and dimethylsulfoxide, methods to add and remove human oocytes with a vitrifiable concentration of ethylene glycol can be designed that prevent excessive osmotic stress and minimize exposure to high concentrations of this compound.

Determination of plasma membrane characteristics of boar spermatozoa and their relevance to cryopreservation.

The osmotic tolerance limits for boar spermatozoa were determined at 22 degrees C. These cells can swell to within 1.02 times and shrink to within 0.97 times their isosmotic volume and maintain > 70% motility. In the presence of an extender, cells can swell to within 1.1 times and shrink to within 0.97 times their isosmotic volume and maintain > 70% motility. Plasma membrane permeability coefficients were determined in the presence of 1 M dimethyl sulfoxide (DMSO), 1 M glycerol, and 2 M ethylene glycol (EG) at 22 degrees C. Hydraulic conductivity (Lp) was estimated to be 0.120+/-0.016 (mean+/-SEM), 0.138+/-0.006, and 0.204 +/-0.021 microm/min/atm in the presence of DMSO, glycerol, and EG, respectively, at 22 degrees C. Solute permeability (P[CPA]) was determined to be 0.930+/-0.118, 0.481+/-0.045, and 1.98+/-0.106 x 10(-3) cm/min, for DMSO, glycerol, and EG, respectively. Subsequent experiments were performed at 8 degrees C and 0 degrees C. Activation energies were calculated for Lp in the presence of glycerol and EG to be 7.20 and 11.51 Kcal/mol, respectively. The activation energies for P(CPA) were 4.06 and 7.48 Kcal/mol for glycerol and EG permeability, respectively. These membrane characteristics were used to calculate volume flux during addition and removal of cryoprotectant agents as well as during cooling and warming. In addition, the potential for intracellular ice formation during cooling and warming was calculated.

Transport parameters in the human red cell membrane: solute-membrane interactions of hydrophilic alcohols and their effect on permeation

A systematic study has been made of the three coefficients that describe the human red cell membrane transport of a series of short straight-chain hydrophilic alcohols: the permeability coefficient, ω i , the reflection coefficient, σ i , and the hydraulic conductivity, L p . Ethylene glycol transport is saturable with K m = 220 ± 50 mM ; there is a second, low-affinity, ethylene glycol site which inhibits water transport ( K l = 570 ± 140 mM , max. inhib. = 90 ± 10% ). σ eth gly = 0.71 ± 0.04 which is significantly less than 1 ( n = 6, P < 0.001), as are σ i for six other alcohols ( n = 23), thus providing strong thermodynamic evidence that water and these alcohols cross the red cell membrane, at least in part, in an aqueous channel. The solute/membrane frictional coefficient, f sm , for all seven alcohols has been determined and found to decrease monotonically as membrane permeability increases. The red cell membrane has been perturbed by treatments with phenylglyoxal and Bs 3 (bis(succinimidyl suberate)); these treatments are accompanied by correlated modulation of both ethylene glycol and urea permeability. In one set of experiments in control cells, urea permeability is correlated with water permeability; and, in another set, ethylene glycol permeability is correlated with water permeability. All of these observations support the proposiion that the urea class of solutes, the ethylene glycol class of solutes and water all cross the membrane through the same aqueous pore. A schematic model of the red cell pore, consistent with the experimental observations, is presented.

Urea and ethylene glycol-facilitated transport systems in the human red cell membrane. Saturation, competition, and asymmetry.

The equilibrium exchange of [14C]urea and ethylene glycol was measured using a new type of fast flow system. Approximately equal volumes of saline and air were mixed to form a segmented fluid stream into which 14C-loaded red cells are injected. The stream flows through three filter chambers which allow sampling of the 14C in the extracellular fluid at three time points. The chambers are designed so that they do not disrupt the segmented bubble pattern. The alternating air and saline segments prevent laminar dispersion in the flowing stream and ensure good mixing at the injection and sampling sites. The equilibrium exchange of both urea and ethylene glycol showed saturation kinetics. The maximum permeability (Po) measured in the limit of zero solute concentration is 1.6 X 10(-3) cm/s for urea and 4.8 X 10(-4) cm/s for ethylene glycol (T = 23 degrees C). The apparent dissociation constant (Km) was 218 mM for urea and 175 mM for ethylene glycol. The Po for thiourea is 2.3 X 10(-6) cm/s and the Km is 19 mM. Urea and thiourea inhibit the transport of each other and the inhibition constant (KI) is approximately equal to the Km for both compounds. 53 other analogues of urea were screened for their inhibition of urea or thiourea transport. Several analogues [e.g., 1-(3,4-dichloro-phenyl)-2-thiourea] had a KI in the range of 0.03 mM. The affinity of the inhibitor increased as it was made more hydrophobic. The urea analogues did not significantly inhibit the ethylene glycol or osmotic permeability. Glycerol inhibited ethylene glycol permeability with a KI of 1,200 mM.

Nonelectrolyte Permeability, Sodium Influx, Electrical Potentials, and Axolemma Ultrastructure in Squid Axons of Various Diameters

The penetration of (14)C-labeled ethylene glycol, erythritol, mannitol, and sucrose was measured in giant axons of various diameters isolated from the hindmost stellar nerves of Doryteuthis plei squid. Axon diameter depends mainly on the age of the squid. The influx of (22)Na, some electrical properties, and the ultrastructure of the axolemma were also studied. The results confirm our previous observation that in medium sized axons of D. plei stimulation causes an increase in the permeability to the penetration of erythritol, mannitol, and sucrose. They also demonstrate that the magnitude of the increase in the penetration of these probing molecules diminishes progressively as the axon diameter increases. The diminution in permeability may be due to a reduction in size of the pathways used by nonelectrolytes to enter the axon. No effect of stimulation on the ethylene glycol permeability is observed. The sodium influx and electrical properties are independent of axon size. The ultrastructural study shows that the axolemma thickness increases with axon diameter. The present experiments indicate that the nonelectrolyte permeability of stimulated axons depends on nerve fiber properties related to axon diameter and on the size of the hydrophilic nonelectrolyte probe.

Temperature Coefficients of Hemolysis of a Few Types of Nucleated Erythrocytes.

Jacobs et al. have presented a series of data giving temperature coefficients for the hemolysis of various types of mammalian erythrocytes in non-electrolytes. These data have recently been utilized in an attempt to relate species differences in permeability to possible chemical differences in the cell membrane (Dziemian and Ballentine) Because of their possible use in future studies, and for comparative purposes, the data in Table I are presented giving temperature coefficients of hemolysis of nucleated erythrocytes of several species. The technic employed in these experiments was essentially that used by Jacobs et al. In general, the temperature coefficients of hemolysis for these nucleated erythrocytes are of the same order of magnitude as those obtained using mammalian red cells. These data are too fragmentary to justify a detailed analysis. However, certain comparisons can well be made with data described by Jacobs and Glassman. These authors point out that in fishes (elasmobranchs and teleosts) the permeability to ethylene glycol is high, in birds the permeability to ethylene glycol and glycerol is very great and nearly equal, and much less to urea, except in the duck and chicken, and permeability to urea is great in reptiles. The present data are in complete agreement. Ethylene glycol permeability is high in the four fishes studied, urea and glycerol permeability is high in the chicken, and the turtle exhibits a very high permeability to urea.