Perfect Osmometers Research Articles

A century of exercise physiology: key concepts in muscle cell volume regulation

Skeletal muscle cells can both gain and lose volume during periods of exercise and rest. Muscle cells do not behave as perfect osmometers because the cell volume changes are less than predicted from the change in extracellular osmolality. Therefore, there are mechanisms involved in regulating cell volume, and they are different for regulatory volume decreases and regulatory volume increases. Also, after an initial rapid change in cell volume, there is a gradual and partial recovery of cell volume that is effected by ion and water transport mechanisms. The mechanisms have been studied in non-contracting muscle cells, but remain to be fully elucidated in contracting muscle. Changes in muscle cell volume are known to affect the strength of contractile activity as well as anabolic/catabolic signaling, perhaps indicating that cell volume should be a regulated variable in skeletal muscle cells. Muscles contracting at moderate to high intensity gain intracellular volume because of increased intracellular osmolality. Concurrent increases in interstitial (extracellular) muscle volume occur from an increase in osmotically active molecules and increased vascular filtration pressure. At the same time, non-contracting muscles lose cell volume because of increased extracellular (blood) osmolality. This review provides the physiological foundations and highlights key concepts that underpin our current understanding of volume regulatory processes in skeletal muscle, beginning with consideration of osmosis more than 200years ago and continuing through to the process of regulatory volume decrease and regulatory volume increase.

Modelling the osmotic behaviour of human mesenchymal stem cells

In this work, a novel mathematical model for the description of the osmotic behavior during the cryopreservation of human Mesenchymal Stem Cells (hMSCs) from Umbilical Cord Blood (UCB) is proposed. In cryopreservation, the two-parameter formalism of perfect osmometer behavior is typically adopted and preferred due to its simplicity: cell volume osmotic excursions are described as due only to the passive trans-membrane transport of water and permeant solutes such as cryoprotectant agents (CPAs); intracellular solutes, responsible of the isotonic osmolality, are assumed to be impermeant. The application of the two-parameter model fails to capture the osmotic response of hMSCs whenever a swelling phase is involved, as demonstrated by the authors. To overcome this limitation, the imperfect osmometer behavior of hMSCs is successfully modelled herein by improving the two-parameter formalism through the coupling of osmosis with cell mechanics and cell membrane Surface Area Regulation (SAR): now the transmembrane permeation of solutes (ions/salt) during the swelling phase through the temporary opening of mechanosensitive channels is allowed. This way cells can reach an equilibrium volume different from the initial isotonic one, when isotonic conditions are re-established after contact with impermeant or permeant solutes, such as sucrose or dimethyl-sulfoxide (DMSO), respectively. The sequential best-fit procedure adopted to adjust model parameters is reported herein along with model validation through full predictions.

Applied osmotic loading for promoting development of engineered cartilage

This study investigated the potential use of static osmotic loading as a cartilage tissue engineering strategy for growing clinically relevant grafts from either synovium-derived stem cells (SDSCs) or chondrocytes. Bovine SDSCs and chondrocytes were individually encapsulated in 2% w/v agarose and divided into chondrogenic media of osmolarities 300 (hypotonic), 330 (isotonic), and 400 (hypertonic, physiologic) mOsM for up to 7 weeks. The application of hypertonic media to constructs comprised of SDSCs or chondrocytes led to increased mechanical properties as compared to hypotonic (300mOsM) or isotonic (330mOsM) media (p<0.05). Constant exposure of SDSC-seeded constructs to 400mOsM media from day 0 to day 49 yielded a Young's modulus of 513±89kPa and GAG content of 7.39±0.52%ww on day 49, well within the range of values of native, immature bovine cartilage. Primary chondrocyte-seeded constructs achieved almost as high a Young's modulus, reaching 487±187kPa and 6.77±0.54%ww (GAG) for the 400mOsM condition (day 42). These findings suggest hypertonic loading as a straightforward strategy for 3D cultivation with significant benefits for cartilage tissue engineering strategies. In an effort to understand potential mechanisms responsible for the observed response, cell volume measurements in response to varying osmotic conditions were evaluated in relation to the Boyle–van't Hoff (BVH) law. Results confirmed that chondrocytes behave as perfect osmometers; however SDSCs deviated from the BVH relation.

Do Acartia tonsa (Dana) eggs regulate their volume and osmolality as salinity changes?

Subitaneous eggs from an euryhaline calanoid copepod Acartia tonsa were challenged by changes in salinity within the range from full strength salinity, down to zero and up to >70psu. Egg volume changed immediately, increasing from 2.8×10(5)μm(3) at full strength salinity (35psu) to 3.8×10(5)μm(3) at 0psu and back to its initial volume when gradually being returned to full strength salinity. Egg osmolality followed the molality of the surrounding water when challenged within a salinity range from 2 to 50psu. Egg respiration was not affected when eggs kept at 35psu was exposed to low salinity (2psu). These results suggest that eggs are unable to regulate their volume or osmolality when challenged with changes in salinity. Gradual changes in salinity from 35 to 2psu and back did not harm the eggs (embryos), since the hatching success remained unaffected by such changes in salinity. In contrast, extreme hyper-saline conditions (76psu) made the eggs implode and killed the embryo. We propose that the embryo is protected from salinity stress by its plasma membrane and that water exchange driven by osmosis is restricted to the perivitelline space of the egg, which acts as a perfect osmometer in the salinity range of 5-35psu. We hypothesize further that the embryo is able to keep its volume and osmolality constant due to the impermeability of the inner plasma membrane of the egg or by a combination of osmoregulation and reduced permeability of the inner plasma membrane.

Water flux through human aquaporin 1: inhibition by intracellular furosemide and maximal response with high osmotic gradients

This work studies water permeability properties of human aquaporin 1 (hAQP1) expressed in Xenopus laevis oocyte membranes, applying a technique where cellular content is replaced with a known medium, with the possibility of measuring intracellular pressure. Consequences on water transport-produced by well-known anisotonic gradients and by the intracellular effect of probable aquaporin inhibitors-were tested. In this way, the specific intracellular inhibition of hAQP1 by the diuretic drug furosemide was demonstrated. In addition, experiments imposing anisotonic mannitol gradients with a constant ionic strength showed that the relationship between water flux and the applied mannitol gradient deflects from a perfect osmometer response when the gradient is higher than 150 mosmol kg (W) (-1) . These results would indicate that the passage of water molecules through hAQP1 may have a maximum rate. As a whole, this work demonstrates the technical advantage of controlling both intracellular pressure and medium composition in order to study biophysical properties of hAQP1, and contributes information on water channel behavior under osmotic challenges and the discovery of new inhibitors.

Root pressure and a solute reflection coefficient close to unity exclude a purely apoplastic pathway of radial water transport in barley (Hordeum vulgare)

*Aquaporins can contribute to the control of root water uptake, provided that at least one membrane is crossed between the root medium and the xylem and that water does not move only along the apoplast. In the present study we have critically assessed the possibility of such a purely apoplastic pathway. *A range of methods were used to analyse root water flow (root pressure probe, root exudation, vacuum perfusion and cell pressure probe) and associated driving forces (gradients in hydrostatic pressure and osmolality). These methods were complemented by theoretical approaches in which we predicted, for a particular main pathway of water movement, values for root pressure and osmolality and compared these with measured values. *The mere existence of root pressure excludes the possibility of a purely apoplastic pathway of radial water uptake. A membrane(s) must be crossed. Equilibrium measurements of gradients in hydrostatic and osmotic pressure between the root medium and the xylem point to a root reflection coefficient for solutes close to 1.0. *The generally accepted composite model of water transport across roots should be revised. Barley (Hordeum vulgare) roots behave as perfect osmometers. Root reflection coefficients significantly smaller than unity may be experimental artefacts.

A Volume Regulatory Response Can Be Triggered by Nucleosides in Human Erythrocytes, a Perfect Osmometer No Longer

Human erythrocytes have been regarded as perfect osmometers, which swell or shrink as dictated by their osmotic environment. In contrast, in most other cells, swelling elicits a regulatory volume decrease (RVD) modulated by the activation of purinic and pyrimidinic receptors (P receptors). For human erythrocytes this modulation has not been tested, and we thus investigated whether P receptor activation can induce RVD in these cells. Further, because ectonucleotidases may scavenge ATP or ADP or act as a source for extracellular adenosine and therefore modulate P receptor activation and RVD, we also determined their activity in intact erythrocytes. We found relatively low ectoATPase but significant ectoADPase and ectoAMPase activities. When erythrocytes were exposed to hypotonic medium alone, they swelled as expected for an osmometric response and showed no RVD. Activation of P2 receptors by exogenous ATP or ADP did not trigger RVD, whereas P1 agonists adenosine and adenosine-5'-N-ethylcarboxamide induced significant RVD. The effect of adenosine-5'-N-ethylcarboxamide was dose-dependent (maximal RVD of 27%; apparent K((1/2)) of 1.6 +/- 1.7 microM). The RVD induced by adenosine was blocked 80% with the non-selective P1 antagonist 8-(p-sulfophenyl theophylline) or the P1-A(2B) inhibitor MRS1754, but not by inhibitors of P1 subtypes A(1), A(2A), and A(3). In addition, forskolin (an inducer of intracellular cAMP formation) could mimic the effect of adenosine, supporting the idea of P1-A(2B) receptor activation. In conclusion, we report a novel P1-A(2B) receptor-mediated RVD activation in mature human erythrocytes and thus indicate that these long held perfect osmometers are not so perfect after all.

Sodium-dependent activity of aquaporin-1 in rat glioma cells: a new mechanism of cell volume regulation

Cell volume controls many functions and is itself regulated. To study cell volume regulations, the mean volume of C6-BU-1 rat glioma cells was electronically measured under isotonic and anisotonic conditions. Two isotonic solutions were used containing either normal (solution 1) or low (solution 2) NaCl. Anisotonicity was induced by changing NaCl or sucrose concentrations in solutions 1 and 2, respectively. The cells behaved like perfect osmometers when the tonicity was increased. In contrast, just after hypotonic challenges, the cell volume was smaller than that predicted by a perfect osmometer. This deviation reveals a new mechanism, which we call the volume increase limitation (VIL). When hypotonicity was induced by decreasing NaCl, a classical slow regulatory volume decrease (RVD) was also observed in addition to VIL. The cells expressed aquaporin-1 sensitive to HgCl(2) and decreased by siRNA, which both reduced fast volume changes. In this study, we show that: (1) RVD is proportional to the change in external Cl(-) concentration and is inhibited by Cl(-) channel and K(+)-Cl(-) cotransporter blockers; (2) cell swelling due to the influx of H(2)O through aquaporins shows rectification with decreasing osmolarity and is sensitive to the internal Na(+) concentration; (3) VIL is linearly related with hypotonicity and is abolished in solutions 1 and 2 by the Na(+) ionophore monensin and in solution 1 by the Na(+)-K(+) ATPase inhibitor ouabain. These results suggest that VIL is triggered by the decrease in internal Na(+) caused by hyponatrema and cell swelling. In addition to RVD, VIL should protect cells during hyposmotic stress.

Conduction velocities in amphibian skeletal muscle fibres exposed to hyperosmotic extracellular solutions

Early quantitative analyses of conduction velocities in unmyelinated nerve studied in a constantly iso-osmotic volume conductor were extended to an analysis of the effects of varying extracellular osmolarities on conduction velocities of surface membrane action potentials in Rana esculenta skeletal muscle fibres. Previous papers had reported that skeletal muscle fibres exposed to a wide range of extracellular sucrose concentrations resemble perfect osmometers with increased extracellular osmolarity proportionally decreasing fibre volume and therefore diminishing fibre radius, a. However, classical electrolyte theory (Robinson and Stokes 1959, Electrolyte solutions 2nd edn. Butterworth & Co. pp 41–42) would then predict that the consequent increases in intracellular ionic strength would correspondingly decrease sarcoplasmic resistivity, Ri. An extension of the original cable analysis then demonstrated that the latter would precisely offset its expected effect of alterations in a on the fibre axial resistance, ri, and leave action potential conduction velocity constant. In contrast, other reports (Hodgkin and Nakajima J Physiol 221:105–120, 1972) had suggested that Riincreased with extracellular osmolarity, owing to alterations in cytosolic viscosity. This led to a prediction of a decreased conduction velocity. These opposing hypotheses were then tested in muscle fibres subject to just-suprathreshold stimulation at a Vaseline seal at one end and measuring action potentials and their first order derivatives, dV/dt, using 5–20 MΩ, 3 M KCl glass microelectrodes at defined distances away from the stimulus sites. Exposures to hyperosmotic, sucrose-containing, Ringer solutions then reversibly reduced both conduction velocity and maximum values of dV/dt. This was compatible with an increase in Ri in the event that conduction depended upon a discharge of membrane capacitance by propagating local circuit currents through initially passive electrical elements. Conduction velocity then showed graded decreases with increasing extracellular osmolarity from 250–750 mOsm. Action potential waveforms through these osmolarity changes remained similar, including both early surface and the late after-depolarisation events reflecting transverse tubular activation. Quantitative comparisons of reduced-χ 2 values derived from a comparison of these results and the differing predictions from the two hypotheses strongly favoured the hypothesis in which Riincreased rather than decreased with hyperosmolarity.Electronic supplementary materialThe online version of this article (doi:10.1007/s10974-007-9115-8) contains supplementary material, which is available to authorized users.

Enhanced Binding of Sperm With Superior Volume Regulation to Oviductal Epithelium

The plasma membrane is a key organelle with respect to sperm fertilizing ability. A sensitive way of testing plasma membrane functionality is to examine the sperm ability to moderate its swelling in response to hypo-osmotic stress (volume regulatory ability) using an electronic cell counter to assess cell volume changes. In this study of frozen-thawed bull sperm, we examined the relationship among sperm-oviductal epithelium binding capacity, osmotically induced swelling response, volume regulatory ability, and standard spermatologic parameters. Sperm cell volume distributions were measured under iso-osmotic conditions and after hypo-osmotic stress. The relative volume shift was calculated by comparing modal values of the cell volume distributions during transition from iso-osmotic to hypo-osmotic conditions. Significant correlations were found between volumetric parameters and sperm-oviduct binding capacity. Both the relative volume shift and regulative volume decrease correlated positively and significantly with the sperm-oviduct binding capacity. No significant correlations were found between sperm volumetric parameters and any of the standard sperm parameters with the exception of forward motility of Percoll-washed sperm. However, the use of multiple regression models improved the prediction level for binding capacity when motility parameters were combined with membrane integrity and volumetric parameters (R2 = .84). Spermatozoa of bulls with high nonreturn rates responded to hypotonicity as "perfect osmometers." Subfertile bulls had lower binding indices and deficiencies in volume recovery after hypotonic challenge, indicating that intact volume regulatory ability is a necessary prerequisite for binding to oviductal epithelium and is related to fertility. Volumetric parameters therefore could be used as tools in semen evaluation programs.

Membrane potentials in Rana temporaria muscle fibres in strongly hypertonic solutions.

Conventional microelectrode methods were used to measure variations in resting membrane potentials, E(m), of intact amphibian skeletal muscle fibres over a wide range of increased extracellular tonicities produced by inclusion of varying extracellular concentrations of sucrose. Moderate increases in extracellular tonicity to up to 2.6x normal (2.6tau) under Cl(-) free conditions produced negative shifts in E(m) that followed expectations for the K(+) Nernst equation (E(K)) applied to a perfect osmometer containing a conserved intracellular K(+) content despite any accompanying cell volume change. In contrast, E(m) remained stable in fibres studied in otherwise similar Cl(-) containing solutions, consistent with E(m) stabilization despite negative shifts in E(K) through inward cation-Cl(-) co-transport activity. Short exposures to higher tonicities (>3tau) similarly produced negative shifts in E(m) in Cl(-) free but not Cl(-) containing solutions. However, prolonged exposures to solutions of >3tau caused gradual net positive changes in E (m) in both Cl(-) containing and Cl(-) free solutions suggesting that these changes were independent of cation-Cl(-) transport. Indeed, there was no evidence of cation-Cl(-) co-transport activity in strongly hypertonic solutions despite its predicted energetic favourability, suggesting its possible regulation by E (m) in muscle. Additional findings implicated a failure to maintain greatly increased transmembrane [K(+)] gradients in these E(m) changes. Thus: (1) halving or doubling [K(+)](e) produced negative or positive shifts in E(m), respectively in isotonic or moderately hypertonic (<2.7tau), but not strongly hypertonic (>3tau) solutions; (2) subsequent restoration of isotonic extracellular conditions produced further positive changes in E(m) consistent with a dilution of the depleted [K(+)](i) by fibres regaining their original resting volumes; (3) quantitative modelling similarly predicted a gradual net efflux of K(+) as the balance between active and passive [K(+)] fluxes altered due to increased transmembrane [K(+)] gradients in hypertonic and low [K(+)](e) solutions. However, the observed positive changes in E(m) in the most strongly hypertonic solutions eventually exceeded these predictions suggesting additional limitations on Na(+)/K(+)-ATPase activity in strongly hypertonic solutions.

The behavior of the human erythrocyte as an imperfect osmometer: A hypothesis

The human erythrocyte does not behave as a perfect osmometer that is its volume does not change as predicted with the change of the tonicity of the medium, as if there was a fraction of the cell water not participating in the osmotic exchange. A mechanism of control of the erythrocyte shape has been previously proposed in which Band 3 (AE1), the protein anion exchanger of Cl − and HCO 3 −, plays a central role. Specifically, decrease and increase of the ratio of its outward-facing conformation and inward-facing conformation (Band 3 o /Band 3 i ) contract and relax the membrane skeleton, thus favoring echinocytosis and stomatocytosis, respectively. The equilibrium Band 3 o /Band 3 i ratio is determined by the Donnan equilibrium ratio of anions and protons, increasing with it ( r = Cl i - / Cl o - = HCO 3 i - / HCO 3 o - = H o + / H i + ). The Donnan ratio is influenced by the erythrocyte transport and metabolic activities. The volume change of the human erythrocyte alters the skeleton conformation as it is accompanied by a change of the membrane curvature. Thus, the mechanism could be a hypothesis for explaining the behavior of the human erythrocyte as an imperfect osmometer since the Donnan ratio controls the Band 3 o /Band 3 i ratio which controls the volume by a control of the degree of contraction or relaxation of the skeleton. Predictions made by the hypothesis on the Ponder's coefficient R′ values in the presence of sucrose or Band 3 substrates slowly transported as well as on the participation of Band 3 in the osmotic hemolysis appear to be corroborated by previous observations. If the hypothesis was valid, it would follow that there is a pressure gradient across the erythrocyte membrane. The equilibrium volume is antagonistically determined by the Donnan ratio per se and Band 3. Band 3, rather than the ratio of surface-to-volume, primarily controls the osmotic hemolysis.

Regulatory and necrotic volume increase in boar spermatozoa

Spermatozoa of many species initially respond to hypotonicity as perfect osmometers. Thereafter they undergo a regulatory process resulting in a decrease in cell volume, similar to that reported for somatic cells. Regulatory volume increase (RVI), a complementary process which is assumed to occur following initial shrinkage of sperm volume after exposure to a hypertonic medium, has not yet been described in detail for spermatozoa. In this study, we investigated whether spermatozoa are able to regulate their volume after hypertonic stress and whether this ability is maintained in preserved sperm. Cell volume changes were recorded using electronic cell sizing. Sperm response to the ion channels blockers quinidine, tamoxifen, and dydeoxyforskolin, and to protein kinase/phosphatase inhibitors lavendustin, staurosporine, and vanadate was studied to investigate possible mechanisms of RVI. Annexin V staining was used in combination with propidium iodide to determine whether hypertonic stress may induce apoptosis. Overall protein tyrosine phosphorylation under hypertonic conditions was measured via flow cytometry using antiphosphotyrosine antibody. Spermatozoa exposed to hypertonic stress initially responded with an abundant subpopulation according to the perfect osmometer model and recovered their volume from this shrinkage after 20 min. RVI was inhibited by quinidine and tamoxifen, which indicates the involvement of the important cellular ions sodium and chloride in this process. Volume regulatory ability was essentially maintained during storage of liquid semen. However, the response of the sperm population was heterogeneous. A second population raised, containing spermatozoa with larger volumes, which demonstrated irregularities in the volume response with respect to osmotic challenge, ion channel blockers, and storage. Under hypertonic conditions, both protein kinase inhibitors (PKI) led to increased isotonic volumes and to elevated initial relative volumes and subsequent volume decrease. RVI was inhibited by the vanadate. Hypertonic stress did not result in an increase in early apoptotic cells, but produced a shift toward late necrotic cells. Substitution of sodium and chloride by choline and sulfate resulted in decreased isotonic volume of sperm treated with lavendustin. Tyrosine phosphorylation levels were reduced after 20 min under hypertonic conditions. It was concluded that RVI is regulated via a protein tyrosine kinase-dependent pathway, and that dephosphorylation occurs when volume regulation is required. The necrotic volume increase (NVI) is associated with the accumulation of sodium and chloride following uncontrolled opening of the channels. The ability to regulate volume after exposure to hypertonic conditions is important for sperm functionality and can have practical applications in spermatological diagnostics and cryopreservation.

Relative contribution of chloride channels and transporters to regulatory volume decrease in human glioma cells

Primary brain tumors (gliomas) often present with peritumoral edema. Their ability to thrive in this osmotically altered environment prompted us to examine volume regulation in human glioma cells, specifically the relative contribution of Cl(-) channels and transporters to this process. After a hyposmotic challenge, cultured astrocytes, D54-MG glioma cells, and glioma cells from human patient biopsies exhibited a regulatory volume decrease (RVD). Although astrocytes were not able to completely reestablish their original prechallenge volumes, glioma cells exhibited complete volume recovery, sometimes recovering to a volume smaller than their original volumes (V(Post-RVD) < V(baseline)). In glioma cells, RVD was largely inhibited by treatment with a combination of Cl(-) channel inhibitors, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and Cd(2+) (V(Post-RVD) > 1.4*V(baseline)). Volume regulation was also attenuated to a lesser degree by the addition of R-(+)-[(2-n-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5-yl)oxy]acetic acid (DIOA), a known K(+)-Cl(-) cotransporter (KCC) inhibitor. To dissect the relative contribution of channels vs. transporters in RVD, we took advantage of the comparatively high temperature dependence of transport processes vs. channel-mediated diffusion. Cooling D54-MG glioma cells to 15 degrees C resulted in a loss of DIOA-sensitive volume regulation. Moreover, at 15 degrees C, the channel blockers NPPB + Cd(2+) completely inhibited RVD and cells behaved like perfect osmometers. The calculated osmolyte flux during RVD under these experimental conditions suggests that the relative contribution of Cl(-) channels vs. transporters to this process is approximately 60-70% and approximately 30-40%, respectively. Finally, we identified several candidate proteins that may be involved in RVD, including the Cl(-) channels ClC-2, ClC-3, ClC-5, ClC-6, and ClC-7 and the transporters KCC1 and KCC3a.

On water availability in saline soils

We present a formulation for the effect of the osmotic pressure of the soil solution on the availability of soil water for plant uptake in the extreme case that the reflection coefficient of root-cell walls is always unity. We also present a new equation to fit the water retention curve, which allows for an inflection point and is solidly anchored at both the wet end (saturated water content) and the dry end (water content at 150 m head, the permanent wilting point). By differentiating the fitting-equation one finds the differential water capacity, which is subsequently multiplied by a weighting function to account for the impediment caused by soluble salts. The weighted differential water capacity is then integrated over the entire range of the matric head from zero to infinity. This produces the integral water capacity and constitutes the total amount of water the soil can hold and release to a hypothetical plant that behaves like a perfect osmometer. We illustrate the approach using data found in the literature for a wide range of soil textures. In this paper the lower boundary of water availability in the presence of soluble salts is defined and calculated as would be registered by a perfect osmometer (reflection coefficient of unity). The upper boundary of water availability is found by setting the weighting coefficient at unity at all times, which implies a reflection coefficient of zero, and in turn that the salts in the soil solution have no influence on the availability of water (as would be registered by a tensiometer). The upper and the lower boundaries constitute the envelope within which the actual availability of water to real plants occurs, and implies a variable reflection coefficient plus the occurrence of active plant osmo-regulation. This establishes a framework within which water availability to real plants experiencing real osmo-matric conditions can be evaluated.

111VOLUMETRIC CHANGES OF BOVINE OOCYTES RESULTING FROM DEHYDRATION IN DISACCHARIDE SOLUTIONS

Vitrification of mammalian oocytes employs hypertonic solutions of cryoprotectant additives (CPAs), causing a rapid loss of water from the cell. This severe dehydration can result in osmotic shock. Oocytes are known to be altered by abrupt changes in osmotic pressure (1987 Cryobiology 24, 387–402), which may result in ultrastructural changes. Therefore, the objective of this study was to examine the effects of severe dehydration of mature bovine oocytes on the cytoskeletal arrangement of microtubules and actin filaments as well as the metaphase spindle prior to vitrification. In Experiment I, oocytes (n=30) obtained from Ovagenix (San Angelo, TX, USA) were sequentially exposed (5 or 6/treatment group) to increasing hypertonic solutions of two saccharides (0.15, 0.3, 0.5, 0.65, 0.99M sucrose and 0.125, 1.25, 0.35, 0.55, 0.65M trehalose) prepared in TCM-199 or M2 medium. Control oocytes (n=22) were subjected to isotonic control solutions (TCM-199 and M2 alone). Relative average volumes of oocytes were determined as previously described and then Boyle vant Hoff plots were constructed. In this study, bovine oocytes behaved similarly when exposed to increasing osmolalities of sucrose and trehalose in both media. The oocytes exhibited a linear decrease in relative volume as a function of 1/(osmotic pressure)to a minimum of approximately 32% of the isotonic volume for 0.99M sucrose in M2 medium (r2=0.9785) (Graphpad 2.04, GraphPad Software, San Diego, CA, USA). In the second experiment, partially denuded bovine oocytes (n=106) were sequentially dehydrated to determine the effect of dehydration on cytoskeletal structures and the meiotic spindle. Oocytes were suspended in solutions of 0.99M sucrose or 0.65M trehalose in either TCM-199 or M2 medium (concentration of sucrose and trehalose where maximum dehydration occurred in the first experiment). Correspondingly, similar oocytes (n=30) were allotted to either TCM-199 or M2 medium control groups. Oocytes were subsequently fixed, immunolabelled for tubulin (rat anti-α-yeast tubulin and anti-rat FITC) and actin (rhodamine phalloidin), stained for DNA (Hoechst 33342), and then mounted for analysis. Cytoskeletal arrangement and DNA localization were visualized using fluorescence and laser-scanning microscopy. Microtubules in control oocytes were intimately associated with the meiotic spindle, which appeared as a symmetrical, barrel-shaped structure with anastral poles. Intact spindles were observed in 63% of the control oocytes compared with 0% in all treatment groups, with the exception of 3% of the oocytes suspended in 0.99M sucrose in TCM-199. Co-localization of actin with tubulin was observed in all oocytes with intact spindles. In conclusion, bovine oocytes respond as perfect osmometers when suspended in hypertonic solutions of saccharides. Oocytes exposed to such solutions exhibited extensive disruption of their meiotic spindles, which would undoubtedly result in reduced fertilization and abnormal development. Further study is needed to determine whether the effects of dehydration can be reversed by sequential rehydration.

The osmotic sensitivity of isolated and in situ bovine articular chondrocytes

Articular chondrocytes experience changes to matrix hydration during both physiological (static load) and pathophysiological (osteoarthrosis, OA) conditions. Such changes should alter chondrocytes' volume, which has been shown to modify matrix metabolism. However, the osmometric behaviour of chondrocytes is not well understood. Here, using confocal laser scanning microscopy (CLSM), we have investigated the `passive' osmotic responses of fluorescent-labelled chondrocytes within, and isolated from, the matrix. The volume-regulatory pathways normally activated by cell shrinkage/swelling, were blocked by bumetanide/REV5901, respectively. Chondrocytes in situ were broadly grouped into superficial (SZ), mid (MZ) and deep (DZ) zones, and there was a significant increase in resting cell volume with depth into the cartilage. Variation in medium osmolarity (range 0–530 mOsm; corresponding to an extracellular osmolarity of ∼150 to ∼600 mOsm) caused a rapid and sustained change to in situ MZ chondrocytes' volume. Over the range 180–380 mOsm, the change to in situ or isolated chondrocytes' volume was similar. For MZ chondrocytes, ideal osmometric (Boyle–van't Hoff) behaviour was apparent over the extracellular osmolarity range of ∼250 to ∼600 mOsm. Chondrocytes within the SZ appeared to be more sensitive to reduced osmolarity, swelling more for a given reduction in osmolarity, than MZ or DZ chondrocytes. These data show that over wide variations in osmolarity, articular chondrocytes in situ were osmotically sensitive, and for MZ chondrocytes behaved as perfect osmometers with the extracellular matrix (ECM) not restraining cell volume changes. Changes to matrix hydration may therefore alter passive chondrocytes' volume and unless compensated by volume-regulatory pathways, could lead to changes in cell volume, and hence matrix metabolism.

Osmotic Behavior of in Vitro Produced Bovine Blastocysts in Cryoprotectant Solutions as a Potential Predictive Test of Survival

The osmotic behavior of bovine blastocysts produced in vitro was filmed during exposure to and dilution of cryoprotectant solutions used for vitrification. The relationship between the changes in the diameter of embryos and their subsequent survival was assessed. Embryos collected on Day 6 and Day 7 postinsemination were exposed to 10% glycerol (GLY) for 5 min, 10% GLY + 20% ethylene glycol (EG) for 5 min, and 25% Gly + 25% EG for 30 s, before dilution in 0.85 M galactose and finally in embryo transfer freezing medium (ETF). Embryos that had a higher probability of survival behaved as perfect osmometers, shrinking, reexpanding, or swelling according to an identical pattern, whereas embryos that deviated from this standard usually did not survive. The initial embryo diameter, duration of shrinkage and expansion in 10% glycerol, duration of reexpansion in ETF, and final embryo diameter were clearly predictive of the ability to hatch after culture in vitro. On a given day postinsemination, larger blastocysts were more likely than smaller blastocysts to survive and hatch after exposure to cryoprotectants with or without vitrification.

Heterogeneous osmotic behaviour in boar sperm populations and its relevance for detection of changes in plasma membrane.

The spermatozoa of most mammals behave as 'perfect osmometers'. The volume response to osmolality obeys the Boyle-Van't Hoff relationship (i.e. volume changes are determined by the osmotically active fraction of the cell volume (solids and water)). Most evaluations of osmotic sperm cell behaviour have been based on the mean volume of the cell population. In the present study, both mean and modal volumes of samples of sperm were evaluated. Both mean and modal volumes responded to environmental osmolality via the Boyle-Van't Hoff relationship; however, the modal volume showed a more sensitive response than the mean volume. This was confirmed for both ejaculated and epididymal spermatozoa. After incubation under capacitating conditions, the difference in modal and mean volume response of ejaculated sperm was considerably diminished and, in epididymal sperm, completely abolished. The sperm osmotic behaviour was still consistent with the Boyle-Van't Hoff equation, but the apparent osmotically inactive modal cell volume decreased after exposure to capacitating conditions in both ejaculated and epididymal sperm samples. The changes in epididymal sperm were more intensive. Due to its enhanced sensitivity to environmental osmolality and incubation under capacitating conditions, the modal volume could be used as a parameter for evaluating sperm population response, such as for detecting environmentally or cryopreservation-induced membrane changes.

Evidence for several mechanisms of volume regulation in neuroblastoma×glioma hybrid NG108-15 cells

Volumes of neuroblastoma×glioma hybrid NG 108-15 cells were electronically measured in order to characterize the mechanisms involved in volume regulation in isosmotic and anisosmotic conditions. The cells behave as perfect osmometers when tonicity was changed at constant chloride concentration by adding sucrose or replacing NaCl with CaCl 2 or MgCl 2. In contrast, the cell volume was poorly dependent on tonicity when the Cl − concentration was changed by adding NaCl or H 2O. Cell shrinkage was induced by cell stirring or after a hypotonicity-induced swelling. These volume decreases were abolished by caffeine but not by ryanodine or EGTA. Shrinkage was also induced by the Ca 2+ ionophore ionomycin. The ionomycin-induced volume decrease was abolished by EGTA. Cell swelling induced an outwardly rectifying Cl − current which was blocked by 5-nitro-2-(3-phenylpropylamino)benzoic acid, 4,4′-diisothiocyanatostilbene-2,2′-disulphonic acid and dihydroindenyl-oxy-alkanoic acid. When the tonicity was reduced at constant Cl − concentration by replacing NaCl with CaCl 2 or MgCl 2, the volume increased and then slowly decreased towards its control value. This regulatory volume decrease was blocked by 5-nitro-2-(3-phenylpropylamino)benzoic acid, 4,4′-diisothiocyanatostilbene-2,2′-disulphonic acid and dihydroindenyl-oxy-alkanoic acid. Long-term (hours–days) cell shrinkage was induced by a reduction of the culture medium osmolarity. Long-term cell swelling was induced by an increase of the culture medium osmolarity. These volume changes were abolished by the protein translation inhibitor cycloheximide. The results suggest that NG 108-15 cell volume is regulated by at least four interacting mechanisms controlled, respectively, by intracellular Ca 2+, extracellular NaCl, cell volume and intracellular ionic strength. The speculative nature of ionic systems responsible for these volume regulating mechanisms is discussed.