Severe Osmotic Stress Research Articles

Myocyte Volume and Function in Response to Osmotic Stress

Hypothermic hyperkalemic cardioplegia results in significant myocyte swelling and impaired contractility. These detrimental effects may be eliminated by the addition of an adenosine triphosphate-sensitive potassium (KATP) channel opener. This study evaluated the hypothesis that a KATP channel opener (diazoxide) would benefit volume homeostasis by limiting volume and subsequent contractility changes during osmotic stress. Isolated rabbit ventricular myocyte volume and contractility were evaluated using video microscopy and field stimulation after exposure to osmotic stress at 37 degrees C. Myocytes were exposed to Tyrode's physiological solution for 20 minutes and test solution for 20 minutes, and then reexposed to Tyrode's for 20 minutes. Test solutions included control Tyrode's (1T) and osmotically altered Tyrode's (2.6T, 0.9T, and 0.6T) solutions with or without the KATP channel opener diazoxide. Severe osmotic stress (2.6T and 0.6T) resulted in significant cell shrinkage and swelling, respectively. This was unchanged by the addition of diazoxide. Mild hyposmotic stress (0.9T) resulted in significant cell swelling that was eliminated by the addition of diazoxide. Cell swelling was associated with reduced contractility. Cell swelling, but not shrinkage, was detrimental to myocyte contractility. Diazoxide eliminated volume change due to mild hyposmotic stress, similar to that previously noted with hyperkalemic cardioplegia, but did not alter volume change secondary to severe osmotic stress.

Debaryomyces hansenii, a highly osmo-tolerant and halo-tolerant yeast, maintains activated Dhog1p in the cytoplasm during its growth under severe osmotic stress

The HOG pathway is an important mitogen-activated protein kinase (MAPK) signal transduction pathway in Saccharomyces cerevisiae that mediates adaptation of cells to hyper-osmotic stress. Activation of this pathway causes rapid but transient, phosphorylation of the MAPK Hog1p. Phosphorylated Hog1p is rapidly transported to the nucleus that results in the transcription of target genes. The HOG pathway appears to be ubiquitous in yeast. Components of HOG pathway have also been identified in Debaryomyces hansenii, a highly osmotolerant and halotolerant yeast. We have studied activation of HOG pathway in D. hansenii under different stress conditions. Our experiments demonstrated that the pathway is activated by high osmolarity, oxidative and UV stress but not by heat stress. We have provided evidence, for the first time, that D. hansenii maintains phosphorylated Dhog1p in the cytoplasm during its growth under severe osmotic stress.

Effects of long-term ionic and osmotic stress conditions on photosynthesis in the cyanobacterium Synechocystis sp. PCC 6803.

Salinity is considered to be one of the most severe problems in worldwide agricultural production, but the published investigations give contradictory results of the effect of ionic and osmotic stresses on photosynthesis. In the present study, long-term effects of both ionic and osmotic stresses, especially on photosynthesis, were investigated using the moderately halotolerant cyanobacterium Synechocystis sp. PCC 6803. Our results show that the PSII activity and the photosynthetic capacity tolerated NaCl but a high concentration of sorbitol completely inhibited both activities. In line with these results, we show that the amount of the D1 protein of PSII was decreased under severe osmotic stress, whereas the levels of PsaA / B and NdhF3 proteins remained unchanged. However, high concentrations of sorbitol stress led to a drastic decrease of both psbA (encoding D1) and psaA (encoding PsaA) transcripts, suggesting that severe osmotic stress may abolish the tight coordination of transcription and translation normally present in bacteria, at least in the case of the psaA gene. Taken together, our results indicate that the osmotic stress component is more detrimental to photosynthesis than the ionic one and, furthermore, under osmotic stress, the D1 protein appears to be the target of this stress treatment.

Roles of compatible osmolytes and heat shock protein 70 in the induction of tolerance to stresses in porcine endothelial cells.

Studies of the responses of porcine pulmonary endothelial cells to acute hypertonic stress have been extended by examining the induction and underlying mechanisms of cell tolerance to both osmotic and heat stresses. Preliminary adaptation of these cells to 0.4osmol (kg H(2)O)(-1) rendered them tolerant either to subsequent severe osmotic stress (0.7osmol (kg H(2)O)(-1)) or to subsequent severe heat shock (50 min at 49 degrees C). In contrast, preliminary exposure of the cells to mild heat shock (44 degrees C for 30 min) induced tolerance only to severe heat shock, not to hyperosmotic stress. Induction of tolerance to heat shock by either procedure correlated with the induced expression of heat shock protein 70 (HSP70). Induction of tolerance to hyperosmotic stress, on the other hand, was associated with the cellular accumulation of osmolytes, such as amino acids, betaine and myo-inositol, and did not correlate with the induced expression of HSP70. It also required a reduction in the final change of osmotic pressure applied to the cells, such that maximum cell shrinkage would not be much more than 40%. In general, therefore, HSP70 and compatible osmolytes have distinct roles in cellular adaptation to these stresses.

Impact of osmotic stress on volume regulation, cytoplasmic solute composition and lysine production in Corynebacterium glutamicum MH20-22B

The response of the l-lysine producing Corynebacterium glutamicum strain MH20-22B to osmotic stress was studied in batch cultures. To mimic the conditions during a fermentation process the long term adaptation of cells subjected to a constant osmotic stress between 1.0 and 2.5 osM was investigated. Cytoplasmic water content and volume of C. glutamicum cells were found to depend on growth phase, extent of osmotic stress and availability of betaine. The maximal cytoplasmic volumes, which were highest at maximal growth rate, were linearily related to osmotic stress, whereas in stationary cells no active volume regulation was observed. Under severe osmotic stress proline was the prominent compatible solute in growing cells. Uptake of betaine, if available in the medium, reduced the concentration of proline from 750 to 300 mM, indicating that uptake of compatible solutes is preferred to synthesis. Furthermore, betaine was shown to have a higher efficiency to counteract osmotic stress, since the overall concentration of compatible solutes was lower in the presence of betaine. Under severe osmotic stress, the addition of betaine shifted l-lysine production in MH20-22B to earlier fermentation times and increased both product concentration and yield in these phases, but did not improve the final l-lysine yield.

Multi-barrelled ion-selective microelectrodes as tools for the investigation of volume regulation mechanisms in invertebrate nerve cells under hyperosmotic conditions

Changes in cell volume can be recorded by a potentiometric method based on ion-selective microelectrodes filled with the classical K + exchanger Corning 477317, which is very sensitive to large quaternary ammonium ions (PNAS 82 (1985) 6014; Electrochim. Acta 47 (2001) 309). We investigated the effect of hyperosmotic bathing solutions on cell volume, the intracellular concentrations of Na +, Cl −, and K + ([Na +] i, [Cl −] i, [K +] i) as well as on the membrane potential of leech Retzius neurones by using multi-barrelled ion-selective microelectrodes. We found that an increase in the extracellular NaCl concentration induced a shrinkage of the cells as well as an increase in [Na +] i, [Cl −] i and [K +] i. Provided that the change in osmolality was moderate (≤+40%), cell shrinkage and the changes in [Na +] i, [Cl −] i and [K +] i were proportional to the increase in the extracellular osmolality, i.e. the cells behaved like ideal osmometers. When the bath osmolality was increased more strongly, significant differences from the ideal behaviour were observed. In particular, the changes in [Na +] i and [Cl −] i were larger than expected from the changes in cell volume. The results show that leech neurones raise their intracellular osmolyte content in response to severe osmotic stress, thereby reducing changes in their cellular volume.

Does proline accumulated in leaves of water deficit stressed barley plants confine cell membrane injuries? II. Proline accumulation during hardening and its involvement in reducing membrane injuries in leaves subjected to severe osmotic stress

The purpose of this research was to examine whether proline accumulation in leaves of barley under conditions of mild water deficit (PEG — 0.75 MPa imposed on roots) may modify membrane injuries caused by subsequent severe osmotic stress (PEG — 1.6 MPa imposed on leaves). Six-day-old seedlings of four barley genotypes were used in the experiments. Substantial and different proline accumulation was found in the leaves of mild water deficit-stressed plants of the most investigated genotypes. This stress factor caused rather a small decrease in RWC and did not lead to membrane injuries. Severe osmotic stress imposed on leaves caused considerable membrane injuries in all the genotypes investigated. Leaves of plants pre-stressed with mild water deficit and then subjected to severe osmotic stress exhibited about a 50% lower membrane injury than those of not pre-stressed plants. A possible role of proline accumulated in the leaves of pre-stressed plants in the process of alleviating cell membrane injuries in the leaves subsequently exposed to severe water deficit is discussed.

The effect of water stress on the activities of key regulatory enzymes of the sucrose to starch pathway in wheat

Developmental changes in the starch and sucrose content of grains andthe activities of enzymes of starch synthesis in wheat were studied under waterstress conditions. Water stress caused a marked reduction in the sucrose andstarch content of the grains. Sucrose synthase (SS) and UDP-glucosepyrophosphorylase (UDP-Gppase), showed higher catalytic activity and moreresistance to water stress compared with amyloplastic enzymes. ADP-glucosepyrophosphorylase (ADP-Gppase) activity was reduced to a low level under bothin situ and osmotic stress conditions in which grainsfailed to accumulate dry matter in vivo. Granule-boundstarch synthase (GBSS) also responded rapidly to in situwater stress treatments as did ADP-Gppase. Reduction in GBSS activity at thetime of growth cessation in situ was less than that ofADP-Gppase and the enzyme did not respond to severe osmotic stress. Solublestarch synthase (SSS) was the enzyme most sensitive to water stress in that itresponded earlier, and to a greater extent, than the other enzymes. However,under severe dehydration conditions, leading to cessation of growth, thedeclinein SSS activity was less than that for ADP-Gppase. SSS showed the lowestin vitro activity followed by GBSS. These results suggestthat SSS is the site of response to water stress by which the rate of graingrowth can be affected, whereas growth cessation is due mainly to theinactivation of ADP-Gppase.

Yeast Cdc42 GTPase and Ste20 PAK-like kinase regulate Sho1-dependent activation of the Hog1 MAPK pathway.

The adaptive response to hyperosmotic stress in yeast, termed the high osmolarity glycerol (HOG) response, is mediated by two independent upstream pathways that converge on the Pbs2 MAP kinase kinase (MAPKK), leading to the activation of the Hog1 MAP kinase. One branch is dependent on the Sho1 transmembrane protein, whose primary role was found to be the binding and translocation of the Pbs2 MAPKK to the plasma membrane, and specifically to sites of polarized growth. The yeast PAK homolog Ste20 is essential for the Sho1-dependent activation of the Hog1 MAP kinase in response to severe osmotic stress. This function of Ste20 in the HOG pathway requires binding of the small GTPase Cdc42. Overexpression of Cdc42 partially complements the osmosensitivity of ste20Delta mutants, perhaps by activating another PAK-like kinase, while a dominant-negative Cdc42 mutant inhibited signaling through the SHO1 branch of the HOG pathway. Since activated Cdc42 translocates Ste20 to sites of polarized growth, the upstream and downstream elements of the HOG pathway are brought together through the membrane targeting function of Sho1 and Cdc42.

Response of Saccharomyces cerevisiae to severe osmotic stress: evidence for a novel activation mechanism of the HOG MAP kinase pathway.

The HOG/p38 MAP kinase route is an important stress-activated signal transduction pathway that is well conserved among eukaryotes. Here, we describe a novel mechanism of activation of the HOG pathway in budding yeast. This mechanism operates upon severe osmostress conditions (1.4 M NaCl) and is independent of the Sln1p and Sho1p osmosensors. The alternative input feeds into the HOG pathway MAPKK Pbs2p and requires activation of Pbs2p by phosphorylation. We show that, upon severe osmotic shock, Hog1p nuclear accumulation and phosphorylation is delayed compared with mild stress. Moreover, both events lost their transient pattern, presumably because of the absence of negative feedback mediated by Ptp2p tyrosine phosphatase, which we found to be localized in the nucleus. Under severe osmotic stress conditions, the delayed nuclear accumulation correlates with a delay in stress-responsive gene expression. Severe osmoshock leads to a situation in which active and nuclear-localized Hog1p is transiently unable to induce transcription of osmotic stress-responsive genes. It also appeared from our studies that the Sho1p osmosensor is less active under severe osmotic stress conditions, whereas the Sln1p/Ypd1p/Ssk1p sensor and signal transducer functions normally under these circumstances.

Behaviour of Sugar-beet Plants (Beta vulgaris L. ssp. vulgaris var. altissima [Doell]) under Conditions of Changing Water Supply: Abscisic Acid as Indicator

Moderate drought stress increases the root-to-shoot ratio in sugar-beet plants. The question arises whether abscisic acid occurs in these plants and whether it plays a role in their adaptation to limited water supply. Thus, after identification by GC/MS, the content of free ABA and its alkaline hydrolysable conjugates was determined by capillary GLC in sugar-beet plants of pot and field experiments with differentiated water supply. It was shown that leaves had a higher content of free ABA than tap roots. Within the leaf apparatus the young organs contained more ABA than the fully developed leaves. Moderate drought stress increased both free and alkaline hydrolysable ABA contents in leaves of intact plants, whereas the ABA content of tap roots was not demonstrably influenced. In contrast, severe and rapid osmotic stress treatment of detached leaves by dipping the petioles in mannitol solutions increased the free ABA content at the expense of the hydrolysable conjugates. This suggests an ABA release by the latter. Therefore, sugar-beet plants seem to have two mechanisms for increasing the free ABA content under drought stress conditions: the gradual synthesis of ABA including its conjugates and the release of free ABA from ABA conjugates.

Role of trehalose in survival of Saccharomyces cerevisiae under osmotic stress.

Trehalose is an enigmatic compound that accumulates in Saccharomyces cerevisiae and has been implicated in survival under various stress conditions by acting as membrane protectant, as a supplementary compatible solute or as a reserve carbohydrate that may be mobilized during stress. In this study, specific mutants in trehalose metabolism were used to evaluate whether trehalose contributes to survival under severe osmotic stress and generates the compatible solute glycerol under moderate osmotic stress. The survival under severe osmotic stress (0.866 aw' NaCl or sorbitol) of mutants was compared to that of the wild-type strain when cultivated to either the mid-exponential or the stationary growth phase on glucose, galactose or ethanol. Stationary-phase cells survived better than exponential-phase cells. The death rates of ethanol-grown cells were lower than those of galactose-grown cells, which in turn survived better than glucose-grown cells. There was a strong relationship between intracellular trehalose levels and resistance to osmotic stress. The mutant strains unable to produce trehalose (tps1 delta tps2 delta and tps1 delta hxk2 delta) were more sensitive to severe osmotic stress (0.866 aw) than the isogenic wild-type strain, confirming a role for trehalose in survival. Hyperaccumulation of trehalose found in the nth1 delta and the nth1 delta gpd1 delta mutant strains, however, did not improve survival rates compared to the wild-type strain. When wild-type, nth1 delta and nth1 delta gpd1 delta cells were exposed to moderate osmotic stress (0.98 and 0.97 aw' NaCl), which permits growth, glycerol production did not appear to be related to the intracellular trehalose levels although glycerol levels increased more rapidly in nth1 delta cells than in wild-type cells during the initial response to osmotic stress. These data indicate that trehalose does not act as a reserve compound for glycerol synthesis under these conditions. No evidence was found for solutes other than glycerol and trehalose being significant for the survival of or growth by S. cerevisiae under osmotic stress conditions.

Evidence against Volume Regulation by Cortical Brain Cells during Acute Osmotic Stress

The cell bodies of neurons and glia examined in culture respond to severe osmotic stress (100 to 200 mOsm) by passive volume change that is followed within several minutes by volume regulation, even in the face of maintained osmotic change. However, in clinical situations, the brain does not experience such precipitous and severe changes in brain hydration. In this study we examined if there is evidence from the hippocampal slice preparation supporting the type of volume regulation observed in cultured brain cells. Within the CA1 region we imaged changes in light transmittance (LT), recorded the evoked field potential, and monitored tissue resistance (all measures of cell volume change) during the first hour of osmotic stress to search for evidence of volume regulation. During superfusion of hypo-osmotic aCSF (−40 mOsm), LT increased 24 to 28% in the dendritic regions of CA1 neurons. The LT reached a plateau which was maintained throughout a 45-min application interval, more than enough time to reveal a regulatory volume decrease. Upon return to control saline, LT immediately returned to baseline and settled there. Hypo-osmolality reversibly increased the relative tissue resistance (RREL) measured across the CA1 region with a time course identical to the increase in LT. Conversely, hyperosmotic aCSF (mannitol, +40 mOsm)decreasedbothRRELby 8% and LT by 15.5% with no indication of a regulatory volume increase. The CA1 cell body layer showed only slight hypo-osmotic swelling whereas exposure to the glutamate agonist quinolinic acid caused pronounced swelling in this region. Even when osmolality was decreased by 120 mOsm for 20 min, dendritic regions responded passively with no regulatory volume decrease. However, when aCSF Cl−was substituted, the CA1 dendritic regions displayed immediate swelling followed by a dramatic volume reduction under normosmotic conditions, indicating that such behavior can be evoked by extreme aCSF dilution. We conclude that in the brain slice preparation, the cortical cells do not exhibit classic volume regulation in response to sudden physiological changes in osmolality. Moreover it is the dendritic region, not the cell body region, that displays dynamic volume change during osmotic challenge.

Abscisic acid changes in osmotic stressed leaves of alfalfa genotypes varying in drought tolerance

Endogenous ABA has been measured in detached alfalfa leaves during severe osmotic stress (40 % PEG) and recovery. Genotypes differing in their osmotic tolerance, including regenerants obtained after selection in vitro, have been compared. A relation between the capability to withstand the osmotic shock, expressed as a recovery potential, and the maintenance of high ABA levels under stress has been found.

Salinity tolerance of the larvae and first juveniles of a semiterrestrial grapsid crab, Armases miersii (Rathbun)

The tropical crab Armases miersii breeds in supratidal rock pools, where great salinity variations occur. In laboratory experiments, all larval stages (zoea I–III, megalopa) and the first juvenile were studied as to their tolerance of acute salinity changes (5–55%., intervals of 10%.) after preceding adaptation to ambient sea water (32–33%.). Our observations suggest that some osmoregulatory capability exists from hatching. Survival was frequently higher and development faster at 15–25%. than in sea water (35%.), possibly indicating a phylogenetic adaptation of A. miersii to brackish water. Extremely low and high salinities (5, 45–55%.) caused prolonged development durations and increased mortality rates. Tolerance of very low salinity, but not that of hypersaline conditions tended to increase during development. This suggests that late stages attain an increasing capability for hyper-osmoregulation in dilute media, but probably remain poor regulators in concentrated media. The megalopa and crab I were, in some experiments, less affected by chronic than by acute exposure to unfavourable salinities, suggesting that non-genetic resistance adaptation (acclimatization) had taken place during earlier development. During continual exposure to extreme salinities, however, effects of severe osmotic stress outweighed those of acclimatization. The range which allowed for successful development through metamorphosis widened with consecutively later change of salinity: 15–35%. (exposure from hatching), 15–45%. (from zoea II), 5–45%. (from zoea III), 5–55%. (from megalopa or crab I). It is concluded that tolerance of variable, predominantly low salinities in larval and early juvenile A. miersii has evolved as an adaptation to breeding in a physically unstable environment, where rapidly decreasing or continually low salinities are more likely to occur than hypersaline conditions. The degree of salinity tolerance in its early life-cycle stages shows that this species has reached, within the Grapsidae, a phylogenetically intermediate stage of adaptation to conditions in freshwater and terrestrial environments.

Osmoregulation of fission yeast: cloning of two distinct genes encoding glycerol-3-phosphate dehydrogenase, one of which is responsible for osmotolerance for growth.

Many types of microorganisms, including both prokaryotes and eukaryotes, have developed mechanisms to adapt to severe osmotic stress. In this study, we isolated multicopy suppressor genes for a Schizosaccharomyces pombe mutant, which exhibited the clear phenotype of being osmosensitive for growth (Osms) on agar plates containing high concentrations of either non-ionic or ionic osmotic solutes. Two genes were thus identified, and each was suggested to encode an NADH-dependent glycerol-3-phosphate dehydrogenase (GPD), which is required for glycerol synthesis. The nucleotide sequences, determined for these genes (named gpd1+ and gpd2+, respectively), revealed that S. pombe has two distinct GPD isozymes. They are only 60% identical to each other in their amino acid sequences. One such isozyme, GPD1, was shown to be directly involved in osmoregulation, based on the following observations. (i) Expression of gpd1+ was regulated at the mRNA level in response to osmotic upshift. (ii) It was demonstrated that wild-type cells markedly accumulated internal glycerol under high-osmolarity growth conditions. (iii) delta gpd1 mutants, however, failed to do so even in a high-osmolarity medium, and thus exhibited an Osms phenotype. On the other hand, the gpd2+ gene was constitutively expressed at a particular low level, regardless of the osmolarity of the medium.

Induced Morphological Changes in Synthetic Giant Vesicles: Growth, Fusion, Undulation, Excretion, Wounding, and Healing

Giant unilamellar vesicles, prepared by hydrating a synthetic lipid, are visible under phase-contrast microscopy. Additives injected into or onto the vesicles induce various morphological changes that have been recorded photographically. For example, KI/I 2 creates a large, slowly-healing hole in the vesicle surface to form a solvent-tilled nanocup. Sodium cholate (a bile salt) also injures the vesicle surface, but the defect is smaller and heals rapidly. Sodium acetate induces vesicle fusion, a process explainable by the Svetina-Zeks mechanism. Poly(vinyl alcohol) causes filament-connected vesicles (but not isolated vesicles) to fuse. This observation leads to the speculation that many fusion experiments with submicroscopic vesicles might also reflect, unknowingly, the presence ofintervesicular filaments. Severe osmotic stress, as provided by 0.1 M NaBr, forces the vesicles to undulate vigorously. Finally, injection of a fluorescent dye into the vesicles allows, via fluorescent microscopy, the detection of outward diffusion by the dye. Giant vesicles provide a particularly valuable membrane model because, unlike submicroscopic vesicles on which the bulk of bilayer research has thus far been focused, the results are not affected by an unnatural membrane curvature. Moreover, morphological changes can be monitored as a function of membrane composition and experimental conditions without relying on indirect spectroscopic methods.

Growth and solute adjustment of calli of Populus clones cultured on nutrient medium containing polyethylene glycol

To determine whether in vitro assays can be used as a surrogate for whole plants in the assessment of drought tolerance, six clones in a pedigree consisting of black cottonwood (Populustrichocarpa Torr. & Gray, ♂) and eastern cottonwood (Populusdeltoides Bartr., ♀) and four hybrid progeny were regenerated in tissue culture and subjected to 0–20% polyethylene glycol (PEG) mol. wt. 8000 in the nutrient medium, providing osmotic potentials that ranged from –0.40 to –1.29 MPa. Drought tolerance was assessed by callus growth and solute accumulation under various stress conditions. Among the cultures grown in the light (16-h photoperiod), clones with the highest relative growth rate of callus dry weight after 18 d in the nutrient medium without PEG included hybrids 246 ♂, 242 ♀, and the eastern cottonwood ♀ parent; the same clones that tended to be the most drought tolerant in previous whole-plant studies. However, there were few clonal differences in relative growth rate across PEG concentrations or among light or dark environments, and any differences were not related to solute accumulation. Polyethylene glycol (20%) provided a severe osmotic stress that dehydrated callus cultures, thereby suppressing relative growth rate on a fresh-weight basis, but not on a dry-weight basis. Inorganic cations and free amino acids constituted the bulk of the solutes in calli, whereas concentrations of soluble carbohydrates were low. Therefore, results obtained from callus cultures growing under artificially induced drought regimes did not parallel those obtained from water stress studies on whole plants.

Andrology: Prevention of osmotic injury to human spermatozoa during addition and removal of glycerol*

Use of a cryoprotective agent is indispensable to prevent injury to human spermatozoa during the cryopreservation process. However, addition of cryoprotective agents to spermatozoa before cooling and their removal after warming may create severe osmotic stress for the cells, resulting in injury. The objective of this study was to test the hypothesis that the degree (or magnitude) of human sperm volume excursion can be used as an independent indicator to evaluate and predict possible osmotic injury to spermatozoa during the addition and removal of cryoprotective agents. Glycerol was used as a model cryoprotective agent in the present study. To test this hypothesis, first the tolerance limits of spermatozoa to swelling in hypo-osmotic solutions (iso-osmotic medium diluted with water) and to shrinkage in hyperosmotic solutions (iso-osmotic medium with sucrose) were determined. Sperm plasma membrane integrity was measured by fluorescent staining, and sperm motility was assessed by computer-assisted semen analysis before, during and after the anisosomotic exposure. The result indicate firstly that motility was much more sensitive to anisosmotic conditions than membrane integrity, and secondly that motility was substantially more sensitive to hypotonic than to hypertonic conditions. Based on the experimental data, osmotic injury as a function of sperm volume excursion (swelling or shrinking) was determined. The second step, using these sperm volume excursion limits and previously measured glycerol and water permeability coefficients of human spermatozoa, was to predict, by computer simulation, the cell osmotic injury caused by different procedures for the addition and removal of glycerol. The predicted sperm injury was confirmed by experiment. Based on this study, an analytical methodology has been developed for predicting optimal protocols to reduce osmotic injury associated with the addition and removal of hypertonic concentrations of glycerol in human spermatozoa.

The HOG pathway controls osmotic regulation of transcription via the stress response element (STRE) of the Saccharomyces cerevisiae CTT1 gene.

The HOG signal pathway of the yeast Saccharomyces cerevisiae is defined by the PBS2 and HOG1 genes encoding members of the MAP kinase kinase and of the MAP kinase family, respectively. Mutations in this pathway (deletions of PBS2 or HOG1, or point mutations in HOG1) almost completely abolish the induction of transcription by osmotic stress that is mediated by stress response elements (STREs). We have demonstrated previously that STREs also mediate induction of transcription by heat shock, nitrogen starvation and oxidative stress. This study shows that they are also activated by low external pH, sorbate, benzoate or ethanol stress. Induction by these other stress signals appears to be HOG pathway independent. HOG1-dependent osmotic induction of transcription of the CTT1 gene encoding the cytosolic catalase T occurs in the presence of a protein synthesis inhibitor and can be detected rapidly after an increase of tyrosine phosphorylation of Hog1p triggered by high osmolarity. Consistent with a role of STREs in the induction of stress resistance, a number of other stress protein genes (e.g. HSP104) are regulated like CTT1. Furthermore, catalase T was shown to be important for viability under severe osmotic stress, and heat shock was demonstrated to provide cross-protection against osmotic stress.