Alteration of membrane permeability to calcium ions during maturation of bovine spermatozoa.

Bovine spermatozoa, maturing in the caudal epididymis, apparently have a low content of mobilizable Ca²⁺ (6 ± 1 nmoles $\text{Ca}{}^{\mathrm{2+}}\text{10}{}^8$ cells) in situ, but will accumulate added Ca²⁺ when simply diluted into isotonic media. It is suggested that the low concentrations of Ca²⁺ (0.5 ± 0.1 mM) and O₂ present in epididymal fluids prevent the accumulatin of Ca²⁺ by sperm prior to ejaculation. Although the seminal plasma that surrounds ejaculated sperm contains 9 ± 1 mM Ca²⁺, washed ejaculated sperm also have a low Ca²⁺ content (7 ± 1 nmoles $\text{Ca}{}^{\mathrm{2+}}\text{10}{}^8$ cells). Moreover, in contrast to epididymal sperm, washed ejaculated sperm are incapable of accumulating Ca²⁺ supplied exogenously. Evidence is presented that bovine seminal fluids contain a component that is added to the surface membranes of the sperm at ejaculation which prevents or delays the active uptake of Ca²⁺ by these cells.     

Osmotic tolerance limits and membrane permeability characteristics of stallion spermatozoa treated with cholesterol

Stallion spermatozoa exhibit osmotic damage during the cryopreservation process. Recent studies have shown that the addition of cholesterol to spermatozoal membranes increases the cryosurvival of bull, ram and stallion spermatozoa, but the exact mechanism by which added cholesterol improves cryosurvival is not understood. The objectives of this study were to determine if adding cholesterol to stallion sperm membranes alters the osmotic tolerance limits and membrane permeability characteristics of the spermatozoa. In experiment one, stallion spermatozoa were treated with cholesterol-loaded cyclodextrin (CLC), subjected to anisotonic solutions and spermatozoal motility analyzed. The spermatozoa were then returned to isotonic conditions and the percentages of motile spermatozoa again determined. CLC treatment increased the osmotic tolerance limit of stallion spermatozoa in anisotonic solutions and when returned to isotonic conditions. The second and third experiments utilized an electronic particle counter to determine the plasma membrane characteristics of stallion spermatozoa. In experiment two, stallion spermatozoa were determined to behave as linear osmometers. In experiment three, spermatozoa were treated with CLC, incubated with different cryoprotectants (glycerol, ethylene glycol or dimethyl formamide) and their volume excursions measured during cryoprotectant removal at 5° and 22 °C. Stallion spermatozoa were less permeable to the cryoprotectants at 5 °C than 22 °C. Glycerol was the least permeable cryoprotectant in control cells. The addition of CLC’s to spermatozoa increased the permeability of stallion spermatozoa to the cryoprotectants. Therefore, adding cholesterol to spermatozoal membranes reduces the amount of osmotic stress endured by stallion spermatozoa during cryopreservation.     

On the permeability of water molecules across vesicular lipid bilayers

Summary The permeation of water molccules across single-component lecithin or lecithin-cholesterol bilayers is studied by a new technique. The new technique makes use of the different fluorescence quantum yields of appropriate molecules in D₂O and H₂O. Water-soluble indole derivatives which by experimental manipulation reside almost entirely within the aqueous (H₂O) intravesicular compartment thus can monitor D₂O molecules permeating the bilayer by virtue of an increased quantum yield of the fluorescence. In a stopped-flow instrument, a vesicle solution containing the fluorescent chromophore in the intravesicular space is rapidly mixed with the deuterated solvent. The approach to the steady state, where the intra- and extravesicular D₂O and H₂O concentrations are equal, proceeds in a single-exponential manner. Consequently, the exchange relaxation time for the D₂O molecules passing the bilayer can be deduced from the time-dependent increase of the fluorescence intensity. The method and results on lecithin and lecithin-cholesterol bilayer vesicles are discussed. The exchange relaxation times of temperature-dependent studies are interpreted within the framework of the solubility-diffusion theory. Below the crystalline to liquid-crystalline phase transition temperature and for cholesterol-free vesicles, the rate-limiting step for the D₂O permeation is attributed to the intracore diffusion. Above the phase transition and for cholesterol-containing vesicles, the intracore diffusion seems not to be rate-limiting. Deviations from the linearity below the phase transition in the Arrhenius-type presentation of the data are related to changes of the partition coefficient of water between the solvent and the lipid phase at the premelting temperature.     

Cell membrane water permeability of rabbit cortical collecting duct

Summary The water permeability (P _osm) of the cell membranes of isolated perfused rabbit cortical collecting ducts was measured by quantitative light microscopy. Water permeability of the basolateral membrane, corrected for surface area, was 66 m·sec^–1 for principal cells and 62.3 m·sec^–1 for intercalated cells. Apical membraneP _osm values corrected for surface area, were 19.2 and 25 m·sec^–1 for principal and intercalated cells, respectively, in the absence of antidiuretic hormone (ADH). Principal and intercalated cells both responded to ADH by increasingP _osm of their apical membranes to 92.2 and 86.2 ·sec^–1 respectively. The ratio of the total basolateral cell membrane osmotic water permeability to that of the apical cell membrane was 271 in the absence of ADH and 71 in the presence of the hormone for both cell types. This asymmetry in water permeability is most likely due to the fact that basolateral membrane surface area is at least 7 to 8 times greater than that of the apical membrane. Both cell types exhibited volume regulatory decrease when exposed to dilute serosal bathing solutions. Upon exposure to a hyperosmotic serosal bath (390 mosm), pricipal cells did not volume regulate while two physiologically distinct groups of intercalated cells were observed. One group of intercalated cells failed to volume regulate; the second group showed almost complete volume regulatory increase behavior.     

Structural determinants of water permeability through the lipid membrane

Despite intense study over many years, the mechanisms by which water and small nonelectrolytes cross lipid bilayers remain unclear. While prior studies of permeability through membranes have focused on solute characteristics, such as size, polarity, and partition coefficient in hydrophobic solvent, we focus here on water permeability in seven single component bilayers composed of different lipids, five with phosphatidylcholine headgroups and different chain lengths and unsaturation, one with a phosphatidylserine headgroup, and one with a phosphatidylethanolamine headgroup. We find that water permeability correlates most strongly with the area/lipid and is poorly correlated with bilayer thickness and other previously determined structural and mechanical properties of these single component bilayers. These results suggest a new model for permeability that is developed in the accompanying theoretical paper in which the area occupied by the lipid is the major determinant and the hydrocarbon thickness is a secondary determinant. Cholesterol was also incorporated into DOPC bilayers and X-ray diffuse scattering was used to determine quantitative structure with the result that the area occupied by DOPC in the membrane decreases while bilayer thickness increases in a correlated way because lipid volume does not change. The water permeability decreases with added cholesterol and it correlates in a different way from pure lipids with area per lipid, bilayer thickness, and also with area compressibility.     

The water permeability of basement membrane under increasing pressure: evidence for a new theory of permeability

The water permeability and physical characteristics of the basement membrane (lens capsule) of the crystalline lens of the adult rat have been investigated. The hydraulic conductivity of the basement membrane at low pressure is 50 +/- 9.5 X 10(-12) m s-1 Pa-1 and at high pressure 17 +/- 7.5 X 10(-12) m s-1 Pa-1. This decrease in permeability occurs despite a 75% increase in area of the membrane and a 65% reduction in its thickness. Conventional theories of a membrane possessing pores or of a fibre matrix of filaments of a constant diameter fail to explain the decreasing permeability of the membrane with increasing hydrostatic pressure. The present data suggest that the structure of the membrane is changed by pressure and the coiled filaments of which it is composed are extended as stress is applied to the membrane. If allowance is made both for thinning and for compaction of the membrane and the extension of its area the permeability of the membrane can be predicted satisfactorily at varying pressures. Thus the hydraulic conductivity of basement membrane at a given pressure can be adequately described by the product of a constant and a dimensionless 'deformation coefficient'. This deformation coefficient is equal to the square of the product of the thickness ratio and elongation ratio of the membrane at the given filtration pressure.     

Osmotic tolerance and membrane permeability characteristics of rhesus monkey (Macaca mulatta) spermatozoa

Biophysical characteristics of the plasma membrane, such as osmotic sensitivity and water and cryoprotectant permeability are important determinants of the function of spermatozoa after cryopreservation. A series of experiments was conducted with rhesus macaque spermatozoa at 23 degrees C to determine their: (1) cell volume and osmotically inactive fraction of the cell volume; (2) permeability coefficients for water and the cryoprotectants dimethyl sulfoxide, glycerol, propylene glycol, and ethylene glycol; (3) tolerance to anisosmotic conditions; and (4) motility after a one step addition and removal of the four cryoprotectants. An electronic particle counter and computer aided semen analysis were used to determine the cell volume and permeability coefficients, and motility, respectively. Rhesus spermatozoa isosmotic cell volume was 27.7+/-3.0 microm3 (mean+/-SEM) with an osmotically inactive cell fraction of 51%. Hydraulic conductivity in the presence of dimethyl sulfoxide, glycerol, propylene glycol, and ethylene glycol was 1.09+/-0.30, 0.912+/-0.27, 1.53+/-0.53, and 1.94+/-0.47 microm/min/atm, respectively. Cryoprotectant permeability was 1.39+/-0.31, 2.21+/-0.32, 3.38+/-0.63, and 6.07+/-1.1 (x10(-3)cm/min), respectively. Rhesus sperm tolerated all hyposmotic exposures. However, greater than 70% motility loss was observed after exposure to solutions of 600 mOsm and higher. A one step addition and removal of all four cryoprotectants did not cause significant motility loss. These data suggest that rhesus sperm are tolerant to hyposmotic conditions, and ethylene glycol may be the most appropriate cryoprotectant for rhesus sperm cryopreservation, as it has the highest permeability coefficient of the tested cryoprotectants.     

Nature of virally mediated changes in membrane permeability to small molecules.

1. The changes in membrane permeability to small molecules caused by Sendai virus [Pasternak & Micklem (1973) J. Membr. Biol. 14, 293-303] have been further characterized. The uptake of substances that are concentrated within cells is inhibited. Choline and 2-deoxyglucose, which become phosphorylated, and aminoisobutyrate and glycine, which are driven by a Na+-linked mechanism, are examples. The uptake of each compound under conditons where its diffusion across the plasma membrane is rate-limiting is stimulated by virus. Choline, 2-deoxyglucose and amino acids at high concentration, amino acids in Na+-free medium, and most substances at low temperature, are examples. It is concluded that virally mediated decrease of uptake is due to one of two causes. Substances that are accumulated by phosphorylation are not retained because of leakage of the phosphorylated metabolites out of cells. Substances that are accumulated by linkage to a Na+ gradient are no longer accumulated because of collapse of the gradient resulting from an increased permeability to Nat 2. Increased permeability to K+ and Na+ results in (a) membrane depolarization and (b) cell swelling. The latter event leads to haemolysis (for erythrocytes) and can lead to giant-cell (polykaryon) formation (for several cell types). 3. Recovery of cells can be temporarily achieved by the addition of Ca2+; permanent recovery requires incubation for some hours at 37 degrees C. 4. The possible significance of virally mediated permeability changes, with regard to clinical situations and to cell biology, is discussed.     

Determination of cells' water membrane permeability: unexpected high osmotic permeability of Saccharomyces cerevisiae

Water permeability (Lp), calculated from the volume variations of cells subjected to an osmotic shock, is classically used to characterize cell membrane properties. In this work, we have shown the importance of the kind of mixing reactor used to measure the Lp parameter. A mathematical model including the mixing time constant has been proposed allowing an accurate Lp estimation even though the mixing time constant is higher than the cell time constant obtained in response to a perfect shock. The estimated Lp values of human leukemia K562 cells were found to be the same whatever the mixing time constant. The Lp value of Saccharomyces cerevisiae could not be exactly estimated. However, S. cerevisiae has unexpectedly high water permeability, implying that this yeast may contain water channels in the membrane. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 62-70, 1997.     

Plant plasma membrane water permeability and slow freezing injury

Abstract Evaluation of existing experimental evidence supports the hypothesis that, under natural or slow rates of freezing, plasma membrane permeability does not significantly limit water efflux from plant cells. Water efflux at slow freezing rates is controlled largely by the rate of heat removal from the plant cells. These conclusions are in direct contrast to the recent views of Levitt (1978).     

The water permeability of the monoolein/triolein bilayer membrane

The water permeability of the lipid bilayer can be used as a probe of membrane structure. A simple model of the bilayer, the liquid hydrocarbon model, views the membrane as a thin slice of bulk hydrocarbon liquid. A previous study (Petersen, D. (1980) Biochim. Biophys. Acta 600, 666–677) showed that this model does not accurately predict the water permeability of the monoolein/n-hexadecane bilayer: the measured activation energy for water permeation is 50% above the predicted value. From this it was inferred that the hydrocarbon chains in the lipid bilayer are more ordered than in the bulk hydrocarbon liquid. The present study tests the liquid hydrocarbon model for the monoolein/triolein bilayer, which has been shown to contain very little triolein in the plane of the membrane (Waldbillig, R.C. and Szabo, G. (1979) Biochim. Biophys. Acta 557, 295–305). Measurements of the water permeability coefficient of the bilayer are compared with predictions of the liquid hydrocarbon model based on measurements of the water permeability coefficient of bulk 8-heptadecene. The predicted and measured values agree quite closely over the temperature range studied (15–35°C): the predicted activation energy is 11.1±0.2 kcal/mol, whereas the measured activation energy for the bilayer is 9.8±0.7 kcal/mol. This close agreement is in contrast with the monoolein/n-hexadecane results and suggests that, insofar as water permeation is concerned, the liquid hydrocarbon model quite closely represents the monoolein/triolein bilayer.     

A note on the units of membrane permeability to water

In the usual experimental technique, the membrane permeability to water in the units of cm/sec is not measured directly. The quantity which is usually obtained may be considered to be a measure of a coefficient of penetrability having the dimensions time/length. The purpose of this paper is to show that by introducing the bulk modulus of water, an expression is obtained from which it is possible to calculate the water permeability in the units of cm/sec from the values in conventional units.     

Regulation of luminal membrane water permeability by water flow in toad urinary bladder

Intramembranous particle aggregates (presumed sites for water flow) which appear in the luminal membrane consequent to ADH treatment are derived from cytoplasmic membrane structures (now termed "aggrephores") which fuse with the luminal membrane. We have previously shown that bladders stimulated in the absence of an osmotic gradient have about twice as many aggregates and about three times as many sites of aggrephore fusion as bladders stimulated with ADH in the presence of a 175 milliosmolal gradient. The present studies show that the frequency of fused aggrephores and luminal membrane aggregates can be modified as a consequence of alterations in transmembrane water flow initiated by changing the transbladder osmotic gradient during hormone stimulation. Bladders treated with ADH for 1 hr without a gradient and then for 1 hr with a gradient had approximately 1/3 as many aggregates and fusion sites as paired bladders treated for 2 hr without a gradient. Conversely, bladders treated with ADH for 1 hr with a gradient and then for 1 hr without a gradient had approximately 2x as many aggregates and fusion sites as bladders treated for 2 hr with a gradient. In other experiments we demonstrate that the time course of hormone washout is greatly accelerated if carried out in the presence of an osmotic gradient. In paired bladders that were first stimulated with ADH for 30 min in the absence of a gradient, aggregates and fusion sites as well as osmotic water permeability determined in fixed bladders, persisted at near maximum levels for 15 min of washout in the absence of a gradient.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro maturation of the potential for movement of carp spermatozoa

Carp semen obtained from isolated fish after hormonal stimulation was highly variable in terms of volume of semen, osmotic pressure of the seminal plasma, and sperm capacity to move. Moreover, this last parameter was unstable when the spermatozoa were kept within the seminal plasma, and the present work was designed to investigate and possibly correct this phenomenon. Sperm potential movement was the major parameter studied and was measured by the percentage of motile cells in a final 3.000-fold dilution in a medium of low osmotic pressure in which sperm movement is known to occur (Morisawa and Suzuki, Science 210:1145-1147, 1980). This was completed with occasional measurements of flagellar beat frequencies and demembranation-reactivation of axonemal movement. The results showed that sperm potential movement was preserved upon dilution of the semen into cold 200 mM KCl medium and that semen of initially "poor" quality or spermatozoa that had lost their capacity to move during storage in the semen recovered gradually their potential movement during incubation at 2 degrees C in the same medium. The K+ dependence for both the conservation and the regeneration of sperm capacity to move showed a minimal requirement of 50 mM KCl in media of high osmotic pressure. Na+ ions had similar properties but not divalent cations. The K+ activation was not pH dependent between pH 9.03 and 6.04. Whatever the functional state of live spermatozoa, demembranation-reactivation occurred in ATP-Mg2+. It is concluded that, with dilution of the semen in appropriate conditions, carp spermatozoa retain or acquire potential movement and therefore are a lower vertebrate spermatozoa model available year-round. In addition, obtaining potentially nonmotile sperm and reversion in vitro might be useful to study the control of in vitro maturation.     

High membrane permeability for melatonin

The pineal gland, an endocrine organ in the brain, synthesizes and secretes the circulating night hormone melatonin throughout the night. The literature states that this hormone is secreted by simple diffusion across the pinealocyte plasma membrane, but a direct quantitative measurement of membrane permeability has not been made. Experiments were designed to compare the cell membrane permeability to three indoleamines: melatonin and its precursors N-acetylserotonin (NAS) and serotonin (5-HT). The three experimental approaches were (1) to measure the concentration of effluxing indoleamines amperometrically in the bath while cells were being dialyzed internally by a patch pipette, (2) to measure the rise of intracellular indoleamine fluorescence as the compound was perfused in the bath, and (3) to measure the rate of quenching of intracellular fura-2 dye fluorescence as indoleamines were perfused in the bath. These measures showed that permeabilities of melatonin and NAS are high (both are uncharged molecules), whereas that for 5-HT (mostly charged) is much lower. Comparisons were made with predictions of solubility-diffusion theory and compounds of known permeability, and a diffusion model was made to simulate all of the measurements. In short, extracellular melatonin equilibrates with the cytoplasm in 3.5 s, has a membrane permeability of ∼1.7 µm/s, and could not be retained in secretory vesicles. Thus, it and NAS will be “secreted” from pineal cells by membrane diffusion. Circumstances are suggested when 5-HT and possibly catecholamines may also appear in the extracellular space passively by membrane diffusion.     

Hepatocyte membrane water permeability measured by silicone layer filtering centrifugation

We previously found that hepatocytes are able to control their osmotic membrane water permeability (P(f)) by regulating the number of surface aquaporin water channels. Hepatocyte P(f) has been assessed by phase-contrast microscopy and cell image analysis, an established but relatively laborious procedure. We report here an alternative method to assess hepatocyte P(f) based on a single silicone layer filtering centrifugation system. Isolated rat hepatocytes were incubated in hypotonic or isotonic buffers containing (3)H(2)O as a tracer and, then, were filtered by rapid centrifugation through a silicone layer down to a lysis layer. Osmotically driven radioactivity (i.e., (3)H(2)O) within hepatocytes was calculated as the difference between the dpm in lysis media measured under hypotonic and isotonic conditions. The P(f) calculated from the initial slope of the radioactivity-versus-time curve was 18 microm/s at 4 degrees C. Hepatocytes treated with dibutyryl cyclic AMP, to increase P(f) through the plasma membrane insertion of aquaporins, showed an increased P(f) value of 37 microm/s. The aquaporin blocker dimethyl sulfoxide selectively prevented the agonist-induced hepatocyte P(f). These data are in good agreement with the corresponding values determined by quantitative phase-contrast microscopy; thus, the method developed allows the rapid and reliable measurement of hepatocyte P(f).     

Lack of influence of phytochrome on membrane permeability to tritiated water

The water permeability of tissues was investigated by measuring the efflux of ³HHO from previously loaded (in darkness) etiolated bean buds (Phaseolus vulgaris L. var. Red Kidney), pea epicotyl segments (Pisum sativum L. var. Alaska), and oat coleoptile segments (Avena sativa L. var. Garry). Red light, far red light, or darkness was applied at the time of transfer of tissue from labeled to unlabeled medium. There were no effects of light on half-time for efflux or on the maximum level of radioactivity in the medium. Based on these criteria, phytochrome exerts no apparent control over water permeability.     

Dynamic changes in the osmotic water permeability of protoplast plasma membrane

The osmotic water permeability coefficient (P(f)) of plasma membrane of maize (Zea mays) Black Mexican Sweet protoplasts changed dynamically during a hypoosmotic challenge, as revealed using a model-based computational approach. The best-fitting model had three free parameters: initial P(f), P(f) rate-of-change (slope(P(f))), and a delay, which were hypothesized to reflect changes in the number and/or activity of aquaporins in the plasma membrane. Remarkably, the swelling response was delayed 2 to 11 s after start of the noninstantaneous (but accounted for) bath flush. The P(f) during the delay was < or =1 microm s(-1). During the swelling period following the delay, P(f) changed dynamically: within the first 15 s P(f) either (1) increased gradually to approximately 8 microm s(-1) (in the majority population of low-initial-P(f) cells) or (2) increased abruptly to 10 to 20 microm s(-1) and then decreased gradually to 3 to 6 microm s(-1) (in the minority population of high-initial-P(f) cells). We affirmed the validity of our computational approach by the ability to reproduce previously reported initial P(f) values (including the absence of delay) in control experiments on Xenopus oocytes expressing the maize aquaporin ZmPIP2;5. Although mercury did not affect the P(f) in swelling Black Mexican Sweet cells, phloretin, another aquaporin inhibitor, inhibited swelling in a predicted manner, prolonging the delay and slowing P(f) increase, thereby confirming the hypothesis that P(f) dynamics, delay included, reflected the varying activity of aquaporins.     

The possible influence of osmotic poration on cell membrane water permeability

It has been hypothesized that pores in the plasma membrane form under conditions of rapid water efflux, allowing extracellular ice to grow into the cytoplasm under conditions of rapid freezing. When cells with intracellular ice are thawed slowly, the transmembrane ice crystal expands through recrystallization causing the cell to lyse. One of the implications of this hypothesis is that osmotic pores will provide an alternative route for water movement under conditions of osmotically induced flow. We show that the plasma membrane water permeability of a fibroblast cell changes as a function of the osmotic pressure gradient that is used to drive water movement. It is further shown that cell volume is more important than the magnitude of water flux in causing this departure from a uniform water permeability. We suggest that these data provide evidence of a transient route for water movement across cell membranes.