Rapid shape change and release of ninhydrin-positive substances by Leishmania major promastigotes in response to hypo-osmotic stress

Leishmania major promastigotes were grown to late-log phase and washed and resuspended in an isosmotic buffer. When osmolality was suddenly decreased by 50%, the cells rapidly became shorter and increased in width. Cell volume, calculated assuming a prolate-ellipsoidal shape, increased 1.4 times after 1 min. Over the next several minutes, the average length and width returned to control values while the volume returned to baseline, indicating the ability to regulate volume. Concomitantly with the swelling, large amounts of alanine and other ninhydrin-positive substances were released. All of the alanine pool was released within 1 min after reduction of the osmolality by 66%. Cells pre-loaded with [14C]-aminoisobutyric acid also released it very rapidly upon hypo-osmotic stress. Release of ninhydrin-positive substances resulted from decreased osmolality rather than changes in ionic composition. The same results were obtained if osmolality was decreased by reducing only the NaCl content of the buffer instead of diluting it with water, and mannitol could substitute for the NaCl. Promastigotes were able to grow well over several days in media as low as 154 mOsm/kg. The nature of the signalling mechanisms(s) that initiates the rapid shape change and efflux of ninhydrin-positive substances in response to hypo-osmotic stress is at present unknown.     

Effect of hyper-osmotic stress on alanine content of Leishmania major promastigotes

Earlier studies showed that Leishmania major promastigotes are sensitive to osmotic conditions. A reduction in osmolality caused the cells to shorten and to rapidly release most of their large internal pool of alanine. In this study some effects of hyper-osmotic stress were examined. An increase in osmolality of the culture medium from 308 to 625 mOsm/kg caused only a small decrease in growth rate. When cells grown in the usual culture medium (308 mOsm/kg) were washed, resuspended in iso-osmotic buffer, and subjected to acute hyper-osmotic stress by addition of mannitol, the alanine content increased even in the absence of exogenous substrate. Promastigotes, depleted of alanine by a 5-min exposure to hypo-osmotic conditions, also synthesized alanine when resuspended in iso-osmotic buffer. Washed cells resuspended in iso-osmotic buffer consume their internal pool of alanine under aerobic conditions. Rates of consumption decreased on addition of mannitol, becoming zero at about 440 mOsm/kg. At higher osmolalities, alanine synthesis occurred. To estimate whether proteolysis could account for alanine synthesis in the absence of exogenous substrate, cells that had been grown with [1-14C]leucine were washed and resuspended under hypo-, iso-, and hyper-osmotic conditions and the amounts of 14CO2 and 14C-labelled peptides released in 1 h were measured. Little proteolysis occurred under these conditions, but the possibility that proteolysis was the source of the alanine increase, observed in response to hyper-osmotic stress, cannot be ruled out.     

Effect of Hyper-Osmotic Stress On Alanine Content of Leishmania Major Promastigotes

ABSTRACT Earlier studies showed that Leishmania major promastigotes are sensitive to osmotic conditions. A reduction in osmolality caused the cells to shorten and to rapidly release most of their large internal pool of alanine. In this study some effects of hyper-osmotic stress were examined. an increase in osmolality of the culture medium from 308 to 625 mOsm/kg caused only a small decrease in growth rate. When cells grown in the usual culture medium (308 mOsm/kg) were washed, resuspended in iso-osmotic buffer, and subjected to acute hyper-osmotic stress by addition of mannitol, the alanine content increased even in the absence of exogenous substrate. Promastigotes, depleted of alanine by a 5-min exposure to hypo-osmotic conditions, also synthesized alanine when resuspended in iso-osmotic buffer. Washed cells resuspended in iso-osmotic buffer consume their internal pool of alanine under aerobic conditions, Rates of consumption decreased on addition of mannitol, becoming zero at about 440 mOsm/kg. At higher osmolalities, alanine synthesis occurred. to estimate whether proteolysis could account for alanine synthesis in the absence of exogenous substrate, cells that had been grown with [1-¹⁴C]leucine were washed and resuspended under hypo-, iso-, and hyper-osmotic conditions and the amounts of ¹⁴CO₂ and ¹⁴C-labelled peptides released in 1 h were measured. Little proteolysis occurred under these conditions, but the possibility that proteolysis was the source of the alanine increase, observed in response to hyper-osmotic stress, cannot be ruled out.     

Branchial carbonic anhydrase activity and ninhydrin positive substances in the Pacific white shrimp, Litopenaeus vannamei, acclimated to low and high salinities

The Pacific white shrimp, Litopenaeus vannamei, acclimated to 30 ppt salinity, was transferred to either low (15 and 5 ppt), or high (45 ppt) salinity for 7 days. Hemolymph osmolality, branchial carbonic anhydrase activity, and total ninhydrin-positive substances (TNPS) in abdominal muscle were then measured for each condition. Hemolymph osmotic concentration was regulated slightly below ambient water osmolality in shrimp acclimated to 30 ppt. At 15 and 5 ppt, shrimp were strong hyper-osmotic regulators, maintaining hemolymph osmolality between 200 and 400 mOsm above ambient. Shrimp acclimated to 30 ppt and transferred to 45 ppt salinity were strong hypo-osmotic and hypo-ionic regulators, maintaining hemolymph osmolality over 400 mOsm below ambient. Branchial carbonic anhydrase (CA) activity was low (approximately 100 micromol CO(2) mg protein(-1) min(-1)) and uniform across all 8 gills in shrimp acclimated to 30 ppt, but CA activity increased in all gills after exposure to both low and high salinities. Anterior gills had the largest increases in CA activity, and levels of increase were approximately the same for low and high salinity exposure. Branchial CA induction appears to be functionally important in both hyper- and hypo-osmotic regulations of hemolymph osmotic concentrations. Abdominal muscle TNPS made up between 19 and 38% of the total intracellular osmotic concentration in shrimp acclimated to 5, 15, and 30 ppt. TNPS levels did not change across this salinity range, over which hemolymph osmotic concentrations were tightly regulated. At 45 ppt, hemolymph osmolality increased, and muscle TNPS also increased, presumably to counteract intracellular water loss and restore cell volume. L. vannamei appears to employ mechanisms of both extracellular osmoregulation and intracellular volume regulation as the basis of its euryhalinity.     

Effect of osmolality on 86Rb+ uptake and release by Leishmania donovani.

Promastigotes from late-log phase cultures of Leishmania donovani were washed and resuspended in Hanks' Balanced Salt Solution without glucose or phenyl red but with 20 mM (N-[2-hydroxyethyl] piperazine-N'-[2-ethanesulfonic acid]) (HEPES) (HBSS-, 305 mOsm/kg). They were then added to a solution containing 86Rb such that the final osmolality and ionic composition was as desired. Samples were taken at known times and the amount of intracellular 86Rb was measured. Similarly, experiments were performed in which 86Rb was added to the cultures about 18 hr before collection, and the amount of 86Rb released from the washed cells was measured. Under iso-osmotic conditions only about 1.3% of the intracellular 86Rb was released in 900 sec. This increased about 4-fold if the osmolality was reduced from 305-153 mOsm/kg. This is much slower than the very rapid release of alanine in response to hypo-osmotic stress, indicating that alanine release is not via a non-specific pore. Reducing the temperature from 26 degrees C to 3-4 degrees C completely inhibits 86Rb release under iso-osmotic conditions and largely inhibits it under hypo-osmotic conditions. The rate of 86Rb release was not sensitive to K+ concentration and was not altered if chloride was replaced by sulfamate. Ouabain had no effect on either 86Rb uptake or release, but carbonylcyanide P-trifluoromethoxyphenylhydrazone (FCCP) reduced the rate of 86Rb release and, after about a 300 sec exposure, completely inhibited 86Rb uptake. Amiloride partially inhibited 86Rb release, but had no effect on uptake. A decrease in pH from 7.1-5.9 had little effect on 86Rb release under iso-osmotic conditions and slightly increased the rate of release under hypo-osmotic conditions, but it decreased the rate of uptake under both iso-osmotic and hypo-osmotic conditions. Cells taken from 3-day stationary phase cultures released 86Rb more slowly under iso-osmotic conditions than cells from late log phase cultures, but were more responsive to hypo-osmotic stress than were log phase cells. These data appear to rule out an [Na-K-Cl] transporter or a [K-Cl] cotransporter as the means of K+ release, but are consistent with the possibility that a K+/H+ exchanger is present. The possibility that other carrier systems may be present is also discussed.     

Hemolymph levels of methyl farnesoate increase in response to osmotic stress in the green crab, Carcinus maenas

The salinity of estuarine environments can vary widely, exposing resident organisms to considerable osmotic stress. The green crab Carcinus maenas is well known for its ability to osmoregulate in response to such stress. Therefore, we tested the relationship between osmoregulation and hemolymph levels of methyl farnesoate (MF), a compound previously shown to rise in response to various types of environmental stresses. When crabs were transferred from 100% seawater to dilute (hypo-osmotic) seawater, hemolymph osmolality dropped rapidly, reaching an acclimation level 48 h after transfer. Hemolymph levels of MF also rose in these animals after a delay of 6 h, and reached a maximum level at 48 h. MF levels remained elevated as long as the crabs were maintained in dilute seawater, and quickly returned to basal levels when the animals were returned to full strength seawater. In most (but not all) animals, MF levels were elevated when hemolymph osmolality fell below the isosmotic point (approx. 800 mOsm/kg). These data suggest that MF may have a role in osmoregulation by this species. In addition, the elevation of MF by hypo-osmotic seawater suggests an experimental strategy for manipulating MF levels in crustaceans.     

Effects of Osmotic Pressure on the Oxidative Metabolism of Leishmania major Promastigotes

Leishmania major promastigotes were washed and resuspended in an iso-osmotic buffer. The rate of oxidation of ¹⁴C-labeled substrates was then measured as a function of osmolality. An acute decrease in osmolality (achieved by adding H₂O to the cell suspension) caused an increase in the rates of ¹⁴CO₂ production from [6-¹⁴C]glucose and, to a lesser extent, from [1, (3)-¹⁴C]glycerol. An acute increase in osmolality (achieved by adding NaCl, KCl, or mannitol) strongly inhibited the rates of ¹⁴CO₂ production from [1-: ¹⁴C]alanine, [1-¹⁴C]glutamate, and [1, (3)-¹⁴C]glycerol. The rates of ¹⁴CO₂ formation from [1-¹⁴C]laurate, [1-¹⁴C]acetate, and [2-¹⁴C]glucose (all of which form [1-¹⁴C]acetyl CoA prior to oxidation) were also inhibited, but less strongly, by increasing osmolality. These data suggest that with increasing osmolality there is an inhibition of mitochondrial oxidative capacity, which could facilitate the increase in alanine pool size that occurs in response to hyper-osmotic stress. Similarly, an increase in oxidative capacity would help prevent a rebuild up of the alanine pool after its rapid loss to the medium in response to hypo-osmotic stress.     

Effects of osmotic pressure on the oxidative metabolism of Leishmania major promastigotes.

Leishmania major promastigotes were washed and resuspended in an iso-osmotic buffer. The rate of oxidation of 14C-labeled substrates was then measured as a function of osmolality. An acute decrease in osmolality (achieved by adding H2O to the cell suspension) caused an increase in the rates of 14CO2 production from [6-14C]glucose and, to a lesser extent, from [1,(3)-14C]glycerol. An acute increase in osmolality (achieved by adding NaCl, KCl, or mannitol) strongly inhibited the rates of 14CO2 production from [1-14C]alanine,[1-14C]glutamate, and [1,(3)-14C]glycerol. The rates of 14CO2 formation from [1-14C]laurate,[1-14C]acetate, and [2-14C]glucose (all of which form [1-14C]acetyl CoA prior to oxidation) were also inhibited, but less strongly, by increasing osmolality. These data suggest that with increasing osmolality there is an inhibition of mitochondrial oxidative capacity, which could facilitate the increase in alanine pool size that occurs in response to hyper-osmotic stress. Similarly, an increase in oxidative capacity would help prevent a rebuild up of the alanine pool after its rapid loss to the medium in response to hypo-osmotic stress.     

Effects of Pregnancy on the Contents of Water, Taurine, and Total Amino Nitrogen in Rat Cerebral Cortex

Plasma osmolality and the levels of water, taurine, and total amino nitrogen (detected as ninhydrin-positive substances) have been measured in the cerebral cortices of nonpregnant and 19-day pregnant Wistar rats. Plasma osmolality fell by 11 mosmol/kg during pregnancy. Brain water content remained unaltered, but levels of taurine and ninhydrin-positive substances fell by 48.5 and 21.9%, respectively. It is suggested that one way in which brain cells are prevented from swelling during the mild hypoosmolality of pregnancy is through loss of cellular amino nitrogen, particularly taurine.     

Accumulation of amino acids in muscle of perfused rat heart. Effect of insulin

1. Rat heart perfused with Krebs-Henseleit bicarbonate buffer released material containing ninhydrin-positive nitrogen, but the amount was less than that reported to be released by diaphragm; glucose, but not insulin, decreased the release of ninhydrin-positive nitrogen and increased the concentration of the same material in the intracellular water of heart. 2. When heart was perfused with a mixture of amino acids and glucose, there was actually a net uptake, and an increase in intracellular concentration, of ninhydrin-positive nitrogen. Changes in the concentration of ninhydrin-positive nitrogen did not accurately reflect changes in concentration of amino acids. 3. The effect of insulin on the actual concentration of individual amino acids in heart muscle was examined by perfusing the heart with a mixture of amino acids and other ninhydrin-positive substances in the same concentration as they are found in plasma. 4. The effect of insulin on the concentrations of amino acids in the medium and in the intracellular water of the heart was determined after perfusion for different periods of time. No clear or meaningful effect of insulin was observed, despite the fact that insulin significantly increased the accumulation, in each of the same hearts, of radioactivity from amino[(14)C]isobutyric acid.     

Volume regulation mechanisms in Rana castebeiana cardiac tissue under hyperosmotic stress

Volume changes of cardiac tissue under hyperosmotic stress in Rana catesbeiana were characterized by the identification of the osmolytes involved and the possible regulatory processes activated by both abrupt and gradual changes in media osmolality (from 220 to 280mosmol/kg H(2)O). Slices of R. catesbeiana cardiac tissue were subjected to hyperosmotic shock, and total tissue Na(+), K(+), Cl(-) and ninhydrin-positive substances were measured. Volume changes were also induced in the presence of transport inhibitors to identify osmolyte pathways. The results show a maximum volume loss to 90.86+/-0.73% of the original volume (measured as 9% decrease in wet weight) during abrupt hyperosmotic shock. However, during a gradual osmotic challenge the volume was never significantly different from that of the control. During both types of hyperosmotic shock, we observed an increase in Na(+) but no significant change in Cl(-) contents. Additionally, we found no change in ninhydrin-positive substances during any osmotic challenge. Pharmacological analyses suggest the involvement of the Na(+)/H(+) exchanger, and perhaps the HCO(3)(-)/Cl(-) exchanger. There is indirect evidence for decrease in Na(+)/K(+)-ATPase activity. The Na(+) fluxes seem to result from Mg(2+) signaling, as saline rich in Mg(2+) enhances the regulatory volume increase, followed by a higher intracellular Na(+) content. The volume maintenance mechanisms activated during the gradual osmotic change are similar to that activated by abrupt osmotic shock.     

Cell composition as assessed from osmolality and concentrations of sodium, potassium and chloride: total contributions of other substances to osmolality and charge balance

From published data for various tissues on intracellular Na, K and Cl were calculated the net anionic charge on all other substances present and also the total contribution of these to osmolality, assuming osmotic equilibrium with extracellular fluid. These parameters were compared for muscle and nerve of animals differing widely in osmolality and also for other mammalian cells. Cell volume regulation in some euryhaline species was considered; it is only partly due to ninhydrin-positive materials. In sheep mammary glands K seems to be sequestered with lactose and anion. Changes in mammalian muscle due to adrenalectomy, hypophysectomy, K deficiency, myotonia, acid-base imbalance and treatment with deoxycorticosterone or insulin were discussed.     

Sensing of osmotic pressure changes in tomato cells

Cells of tomato (Lycopersicon esculentum) growing in suspension gradually depleted their culture medium and caused a steady decrease in its osmolality. When confronted with a sudden change in medium osmolality (a hypo-osmotic or hyperosmotic shock), respectively, these cells responded with volume changes and stress symptoms such as rapid extracellular alkalinization, efflux of K(+)-ions, and induction of 1-aminocyclopropane-1-carboxylate synthase acid, the key enzyme of ethylene biosynthesis. This array of stress symptoms is well known from cultured plant cells treated with microbial elicitors. Compared with elicitor treatment, induction of responses by hyperosmotic shock was slow and occurred only after increases of approximately 200,000 Pa in osmotic pressure. In contrast, hypo-osmotic shock induced responses without measurable lag and faster than elicitor treatments. Measurable medium alkalinization was induced when medium osmolality was reduced by as little as approximately 10 mosmol, a change corresponding to only approximately 0.2 bar in osmotic pressure. Like treatment with elicitors, hypo-osmotic shock induced specific changes in protein phosphorylations as demonstrated by in vivo labeling with [(33)P]orthophosphate. Exposure of cells to consecutive up- and down-shifts in medium osmolality showed that sensing of osmotic changes occurred within seconds, whereas adaptation to new osmotic conditions proceeded over hours. In conclusion, suspension-cultured plant cells display rapid, easily measurable macroscopic responses to osmotic shock and provide an interesting model system to study osmoregulation, a key process in plant growth and development.     

Osmoregulation and muscle water control of the Amazon fish Oscar Astronotus ocellatus (Cichlidae) when facing salinity stress

Specimens of Oscar Astronotus ocellatus from a fish farm were abruptly submitted to salt stress of 14 ppt and 20 ppt, for 3 and 8 h to determine their plasma osmolality. Muscle wet body mass change in vitro was analyzed from control freshwater animals. Fish in 14 ppt presented no osmolality distress even after 8 h. In 20 ppt, a slight increase (10%) in plasma osmolality was observed for both times of exposure when compared to control fish. Muscle slices submitted in vitro to hyper-osmotic saline displayed decreased body mass after 75 min, and slices submitted to hypo-osmotic saline displayed increased body mass after 45 min when compared to control (isosmotic saline). These results reinforce A. ocellatus’s euryhalinity. The fish were able to regulate its internal medium and tolerate 14 ppt, but presented an intense osmotic challenge and low muscle hydration control when facing salinities of 20 ppt.     

Osmotic and Metabolic-Induced Changes in Light Scattering of Leishmania donovani as Measured by Flow Cytometry

Leishmania donovani promastigotes were collected from cultures in log and stationary phases of growth and resuspended in Hank's Balanced Salt Solution containing 1 mM sodium acetate. Changes in the forward and side scattering of the cells were measured by flow cytometry in response to acute changes in osmolality and to the addition of several different substrates. Forward and side scattering of cells from log phase cultures decreased when the osmolality was decreased by the addition of H₂O and increased when the osmolality was increased by the addition of NaCl. Cells from stationary phase cultures had about the same forward scatter as cells from log phase cultures, but almost a four-fold lower side scatter, and their side scatter values did not change significantly in response to a reduction in osmolality. Microscopic observation showed that both log and stationary cells got longer and thinner, on average, in response to hyperosmolality. The light scattering properties of log (but not of stationary) cells changed in a reproducible manner when substrates were added to the buffer. The ratio of forward to side scatter increased in the following order: controls in balanced salt solution >aspartate >glutamate, glucose or 2-deoxyglucose >alanine >proline. Thus the light scattering properties of L. donovani promastigotes change with culture age, in response to changes in osmolality, and, in log phase cells, in response to the presence of several substrates.     

Regulation of compatible solute accumulation in bacteria

In their natural habitats, microorganisms are often exposed to osmolality changes in the environment. The osmotic stress must be sensed and converted into an activity change of specific enzymes and transport proteins and/or it must trigger their synthesis such that the osmotic imbalance can be rapidly restored. On the basis of the available literature, we conclude that representative Gram-negative and Gram-positive bacteria use different strategies to respond to osmotic stress. The main focus of this paper is on the initial response of bacteria to hyper- and hypo-osmotic conditions, and in particular the osmosensing devices that allow the cell to rapidly activate and/or to synthesize the transport systems necessary for uptake and excretion of compatible solutes. The experimental data allow us to discriminate the transport systems by the physicochemical parameter that is sensed, which can be a change in external osmotic pressure, turgor pressure, membrane strain, internal osmolality and/or concentration of specific signal mmicule. We also evaluate the mmicular basis for osmosensing by reviewing the unique structural features of known osmoregulated transport systems.     

Cell swelling activates stress-activated protein kinases, p38 MAP kinase and JNK, in renal epithelial A6 cells

Osmotic shock is well recognized as one of the factors activating stress-activated protein kinases (SAPKs), p38 MAP kinase and c-Jun N-terminal kinases (JNKs). In renal epithelial A6 cells, hypo-osmotic shock transiently activated SAPKs with maximal activation at 5 min. A6 cells showed a regulatory volume decrease (RVD) after swelling when the cells were exposed to a hypo-osmotic solution. In contrast, activation of SAPKs was maintained over 90 min after hypo-osmotic shock in the presence of 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB, a Cl(-) channel blocker), which completely blocked the RVD and kept the cells continuously swelling. Exposure of the cells to a high K(+) iso-osmotic solution containing nystatin, which induces continuous cell swelling, also continuously activated SAPKs. Furthermore, membrane deformation induced by chlorpromazine activated SAPKs. These results suggest that changes in membrane tension by cell swelling or chlorpromazine, but not osmolality, are important steps for activation of SAPKs in A6 cells.     

INFLUENCE OF GLUTARALDEHYDE AND/OR OSMIUM TETROXIDE ON CELL VOLUME, ION CONTENT, MECHANICAL STABILITY, AND MEMBRANE PERMEABILITY OF EHRLICH ASCITES TUMOR CELLS

Effects of fixation with glutaraldehyde (GA), glutaraldehyde-osmium tetroxide (GA-OsO₄), and osmium tetroxide (OsO₄) on ion and ATP content, cell volume, vital dye staining, and stability to mechanical and thermal stress were studied in Ehrlich ascites tumor cells (EATC). Among variables investigated were fixation time, fixative concentration, temperature, osmolality of the fixative agent and buffer, total osmolality of the fixative solution, osmolality of the postfixation buffer, and time of postfixation treatment in buffer (Sutherland, R. M., et al. 1967. J. Cell Physiol. 69:185.). Rapid loss of potassium, exchangeable magnesium, and ATP, and increase of vital dye uptake and electrical conductivity occurred with all fixatives studied. These changes were virtually immediate with GA-OsO₄ or OsO₄ but slower with GA (in the latter case they were dependent on fixative temperature and concentration) (Foot, N. C. 1950. In McClung's Handbook of Microscopical Technique. 3rd edition. 564.). Total fixative osmolality had a marked effect on cell volume with OsO₄ but little or no effect with GA or GA-OsO₄. Osmolality of the buffer had a marked effect on cell volume with OsO₄, whereas with GA or GA-OsO₄ it was only significant at very hypotonic buffer osmolalities. Concentration of GA had no effect on cell volume. Osmolality of the postfixation buffer had little effect on cell volume, and duration of fixation or postfixation treatment had no effect with all fixatives. Freezing and thawing or centrifugal stress (up to 100,000 g) had little or no effect on cell volume after all fixatives studied. Mechanical stress obtained by sonication showed that OsO₄ alone produced poor stabilization and that GA fixation alone produced the greatest stabilization. The results indicate that rapid membrane permeability changes of EATC follow fixative action. The results are consistent with known greater stabilizing effects of GA on model protein systems since cells were also rendered relatively stable to osmotic stress during fixation, an effect not noted with OsO₄. After fixation with GA and/or OsO₄ cells were stable to osmotic, thermal, or mechanical stress; this is inconsistent with several earlier reports that GA-fixed cells retain their osmotic properties.     

The role of osmotic resistance on equine spermatozoal function

Cryopreservation requires exposure of sperm to extreme variations in temperature and osmolality. The goal of this experiment was to determine the osmotic tolerance levels of equine sperm by analyzing motility, viability, mitochondrial membrane potential (MMP), and mean cell volume (MCV). Spermatozoa were incubated at 22 degrees C for 10 min in isosmolal TALP (300 mOsm/kg), or a range of anisosmolal TALP solutions (75-900 mOsm/kg), for initial analysis, and then returned to isosmolal conditions for 10 min for further analysis. Total sperm motility was lower (P < 0.05) in anisosmolal conditions compared to sperm motility in control medium. When cells were returned to isosmolal conditions, only sperm previously incubated in 450 mOsm/kg TALP were able to recover to control levels of motility. Sperm viability and MMP were lower (P < 0.05) when exposed to hypotonic solutions in comparison to control solutions. Sperm suspensions that were returned to isosmolal conditions from 75, 150, and 900 mOsm/kg had lower (P < 0.05) percentages of viable sperm than control suspensions (300 mOsm/kg). MMP was lower (P < 0.05) in cells previously incubated in 75 and 900 mOsm/kg when returned to isosmolal, as compared to control cells. MCV differed (P < 0.05) from control cell volume in all anisosmolal solutions. Cells in all treatments were able to recover initial volume when returned to isosmolal medium. Although most spermatozoa are able to recover initial volume after osmotic stress, irreversible damage to cell membranes may render some sperm incapable of fertilizing an oocyte following cryopreservation.     

The effect of aldehyde fixation on selected substrates for energy metabolism and amino acids in mouse brain.

The effect of aldehyde fixation on concentrations of low molecular weight constituents was determined by comparing amounts of selected intermediates in brains of mice exposed to aldehyde fixative solutions with those perfused with phosphate buffer solution alone. Aldehyde perfusion resulted in excellent preservation of cerebral cortex ultrastructure in the presence of dramatic declines in adenosine triphosphate, phosphocreatine, glucose and glucose-6-phosphate that occureed before exposure of the tissue to aldehyde fixatives. Decreases in hexose were accompanied by approximately a 4-fold increase in lactate and a 2-fold increase in pyruvate. Glycogen levels decreased by about 60% during the initial operative procedure but remained constant after aldehyde fixation. Glycogen content declined approximately 90% in tissues that were not treated with aldehyde. Concentrations of aspartate and glutamate changed only slightly during the initial period (1-5 min) and remained constant for at least 90 min in cerebral cortices fixed with aldehydes. Alanine levels increased in both fixed and unfixed tissue; however, this increase was much smaller in tissues exposed promptly to aldehydes. Total ninhydrin-positive material in perchloric acid extracts of brain decreased in mice exposed to aldehyde solutions but increased in tissues that were not. These results indicated that several amino acids may be measured reliably in tissues preserved for light and electron microscopy. In addition, determination of glutamate: alanine ratios in tissues perfused with aldehydes may provide an indication of the timing of fixation.