Determination of boar spermatozoa water volume and osmotic response

Boar spermatozoa water volume and osmotic response were determined by a shapeindependent method for measuring cellular volume, electron paramagnetic resonance (EPR), employing the spin label, tempone, and the broadening agent, potassium chromium oxalate (CrOx). A water volume of 18.4 +/- 1.6 mum(3) (X +/- SD) was obtained for individual boar spermatozoa at 290 milliosmolar (mOsm) which, after correction for the presence of cytoplasmic droplets, yields a boar sperm water volume of 13.0 to 15.0 mum(3). Assuming 59% of the total cell volume is water, the total cell volume of boar spermatozoa is 22.0 to 25.4 mum(3). In addition, the experiment indicated that the relative water volume versus the reciprocal of the external osmolality (Boyle van't Hoff plot) was linear over the range of 210 to 1500 mOsm of sodium chloride (r(2) = 0.996), supporting the hypothesis that boar spermatozoa act as ideal osmometers. A non-zero y axis intercept of 0.23 from the Boyle van't Hoff plot indicated a 23% spin label accessible, but osmotically inactive water component.     

Successful cryopreservation of mouse oocytes by using low concentrations of trehalose and dimethylsulfoxide

Sugars such as trehalose, sucrose, and glucose are effectively used by a variety of animals (e.g., brine shrimp, tardigrades, some frogs, and insects), as well as by bacteria, yeasts, and plant seeds to survive freezing and extreme drying. The objective of this study was to examine the potential application of sugars to mammalian oocyte cryopreservation. To this end, we used trehalose, a nonreducing disaccharide, and mouse metaphase II oocytes as models. Our experiments show that extracellular trehalose alone affords some protection at high subzero temperatures (e.g., -15 degrees C), which diminishes with further cooling of the oocytes to -30 degrees C and below. When present both intracellularly and extracellularly, trehalose dramatically improves the cryosurvival with increasing extracellular concentrations to 0.5 M, even after cooling to -196 degrees C. Furthermore, the combination of intracellular and extracellular trehalose with small amounts of a conventional penetrating cryoprotectant (i.e., 0.5 M dimethylsulfoxide) provide high survival, fertilization, and embryonic development rates statistically similar to untreated controls. When transferred to foster mothers, cryopreserved oocytes give rise to healthy offspring showing the proof of principle. Our experiments with differential scanning calorimetry indicate that when cooled using the same cryopreservation protocol, the mixture of 0.5 M trehalose and cryopreservation medium undergoes glass transition at high subzero temperatures, which further substantiates the use of sugars as intracellular and extracellular cryoprotectants. Taken together, our results are in agreement with the survival schemes in nature and demonstrate the successful use of sugars in cryopreservation of mammalian oocytes.     

Quantitative microinjection of trehalose into mouse oocytes and zygotes,and its effect on development

Sugars such as trehalose are effectively used by various organisms as protective agents to undergo anhydrobiosis and cryobiosis. The objective of this study was first to establish a method for quantitative delivery of trehalose as a model sugar into oocytes, and then to evaluate its effect on development of mouse zygotes. To this end, a quantitative microinjection technique was developed using volumetric response of microdroplets suspended in dimethylpolysilaxene. To verify accuracy of this technique, both microdroplets and oocytes were microinjected with fluorophore-labeled dextran. Thereafter, injection volumes were calculated from fluorescence intensity, and volumetric responses of both microdroplets and oocytes. Comparison of calculated injection volumes revealed that this technique reflects microinjection into oocytes with pL-accuracy. The next series of experiments focused on toxicity of injection buffers (i.e., 10mM Tris and 15mM Hepes) and trehalose. Microinjection of Hepes and Tris buffer in the presence of 0.1M trehalose resulted in blastocyst rates of 86 and 72%, respectively, without a significant difference when compared to controls (86%). In subsequent experiments, Hepes was used as the injection buffer, and embryonic development of zygotes was studied as a function of intracellular trehalose concentrations. Microinjection of trehalose up to 0.15M resulted in development to blastocyst stage similar to controls (85 and 87%, respectively) while the blastocyst rate was significantly decreased (43%) in the presence of 0.20M intracellular trehalose. When transferred to foster mothers, trehalose-injected zygotes (0.1M) implanted and developed to day 16 fetuses similar to controls, healthy pups were born. The findings of this study suggest that trehalose at effective intracellular concentrations does not impair development of mouse zygotes.     

Stoichiometry determination for carbon monoxide binding to Rhodospirillum molischianum cytochrome c'

The stoichiometry of CO ligation to the dimer heme protein Rhodospirillum molischianum cytochrome c' is determined. We have recently measured the enthalpy change of CO ligation to this molecule by the van't Hoff method and found the value of -10.7 +/- 1.2 kcal/mol CO (aqueous) (Doyle, M. L., Weber, P. C., and Gill, S. J. (1985) Biochemistry 24, 1987-1991). In the present paper the enthalpy change of CO ligation, measured directly by titration calorimetry, is found to be -9.5 +/- 0.2 kcal/mol heme. Since the van't Hoff method gives the heat value in units/mole of CO and the calorimetric method gives the heat value in units/mole of heme, the stoichiometry of the reaction is given by the ratio of the two values and found to be 0.9 +/- 0.1, or within experimental error, one CO molecule bound per heme.     

The effects of osmotic stress on the viscoelastic and physical properties of articular chondrocytes.

The metabolic activity of chondrocytes in articular cartilage is influenced by alterations in the osmotic environment of the tissue, which occur secondary to mechanical compression. The mechanism by which osmotic stress modulates cell physiology is not fully understood and may involve changes in the physical properties of the membrane or the cytoskeleton. The goal of this study was to determine the effect of the osmotic environment on the mechanical and physical properties of chondrocytes. In isoosmotic medium, chondrocytes exhibited a spherical shape with numerous membrane ruffles. Normalized cell volume was found to be linearly related to the reciprocal of the extracellular osmolality (Boyle van't Hoff relationship) with an osmotically active intracellular water fraction of 61%. In deionized water, chondrocytes swelled monotonically until lysis at a mean apparent membrane area 234 +/- 49% of the initial area. Biomechanically, chondrocytes exhibited viscoelastic solid behavior. The instantaneous and equilibrium elastic moduli and the apparent viscosity of the cell were significantly decreased by hypoosmotic stress, but were unchanged by hyperosmotic stress. Changes in the viscoelastic properties were paralleled by the rapid dissociation and remodeling of cortical actin in response to hypoosmotic stress. These findings indicate that the physicochemical environment has a strong influence on the viscoelastic and physical properties of the chondrocyte, potentially through alterations in the actin cytoskeleton.     

Osmotically inactive volume, hydraulic conductivity, and permeability to dimethyl sulphoxide of human mature oocytes

Controlled ovarian stimulation during an in vitro fertilization cycle usually produces large numbers of oocytes and, consequently, it is likely that more embryos will be generated than can be transferred in a given cycle. It is desirable to freeze-bank surplus oocytes before insemination to avoid the ethical and legal complications of disposing of or storing embryos. Although many attempts have been made to cryopreserve human oocytes, to date, post-thaw survival has been poor, and viable pregnancies after in vitro fertilization have been rare. A possible explanation for the lack of success is that the freezing methods have been adapted from animal studies but have not been optimized for the human oocyte. In this study, video microscopy was used to determine the volumetric responses of mature human oocytes to changes in osmolarity during preparation for freezing. A Boyle van't Hoff plot of data collected in static experiments with fresh human oocytes gave a value of 0.19 +/- 0.01 (mean +/- SEM) for the osmotically inactive volume. Dynamic measurements during exposure to dimethyl sulphoxide at room temperature (22 degrees C) were analysed by a two-parameter transport model and produced values of 1.30 x 10(-6) cm atm-1 s-1 for the hydraulic conductivity of the plasma membrane and 3.15 x 10(-5) cm s-1 for dimethyl sulphoxide permeability (chi-squared = 0.43, df = 20) of fresh human oocytes. Oocytes that had failed to fertilize had a slightly lower hydraulic conductivity and dimethyl sulphoxide permeability and, after exposure to 1.5 mol dimethyl sulphoxide l-1, these cells appeared to become permeable to normally impermeable solutes. These permeability properties have been used to design a protocol for the addition and removal of dimethyl sulphoxide to control the magnitude of volumetric changes.     

Energetics of thrombin-fibrinogen interaction.

The kinetic mechanism of thrombin-fibrinogen interaction has been elucidated by steady-state measurements of synthetic substrate hydrolysis by human alpha-thrombin in the presence of human fibrinogen used as a competitive inhibitor and sucrose used as a viscogenic agent. Sucrose greatly affects the FKm for thrombin-fibrinogen interaction, without altering the intrinsic properties of the system. Under conditions of pH 7.5 and 0.1 M NaCl, fibrinogen behaves like a sticky substrate for thrombin, with acylation being comparable to dissociation in the temperature range 20-37 degrees C. In the same temperature range, deacylation is much faster than acylation. The van't Hoff enthalpy of binding for thrombin-fibrinogen interaction is -24 +/- 3 kcal/mol and the entropy is -55 +/- 11 cal mol-1 deg-1. A chemical compensation effect is present in the binding of fibrinogen and synthetic amide substrates to thrombin, with the delta H and delta G values being linked through a linear relationship.     

Osmotic properties of the rabbit corneal endothelium and their relevance to cryopreservation

The process of cryopreservation subjects cells to gross changes in the composition of the solution that surrounds them, changes that cause the cells first to shrink and then to swell by an osmotic mechanism. Empirical methods have been developed that permit many cells to survive freezing and thawing, but the cornea, which is crucially dependent upon the function of its endothelial monolayer, has proved quite refractory. In this paper we explore the osmotic response of the corneal endothelium of the rabbit to solutions ranging in osmolality from 0.25 to 8.6 X isotonic. Boyle van't Hoff behavior was observed between 0.43 and 8.6 X isotonic, and there was an apparent nonosmotic volume of 33.6%. However, ultrastructural damage was observed at the limits of this range, and it appeared that the tolerated range was 0.64-4.4 X isotonic. We show the extent to which dimethyl sulfoxide (Me2SO) would be expected to moderate changes in volume during freezing and suggest that its initial concentration should be at least 2M to prevent excessive shrinkage. We also show that cell swelling during removal of Me2SO is especially likely to be hazardous.     

Studies on membrane permeability of zebrafish (Danio rerio) oocytes in the presence of different cryoprotectants

Investigation into fish oocyte membrane permeability is essential for developing successful protocols for their cryopreservation. The aim of the present work was to study the permeability of the zebrafish (Danio rerio) oocyte membrane to water and cryoprotectants before cryopreservation protocol design. The study was conducted on stage III and stage V zebrafish oocytes. Volumetric changes of stage III oocytes in different concentrations of sucrose were measured after 20 min exposure at 22 degrees C and the osmotically inactive volume of the oocytes (Vb) was determined using the Boyle-van't Hoff relationship. Volumetric changes of oocytes during exposure to different cryoprotectant solutions were also measured. Oocytes were exposed to 2 M dimethyl sulphoxide (DMSO), propylene glycol (PG), and methanol for 40 min at 22 degrees C. Stage III oocytes were also exposed to 2 M DMSO at 0 degrees C. Oocyte images were captured on an Olympus BX51 cryomicroscope using Linkham software for image recording. Scion Image was used for image analysis and diameter measurement. The experimental data were fitted to a two-parameter model using Berkeley Madonna 8.0.1 software. Hydraulic conductivity (L(p)) and solute (cryoprotectant) permeability (Ps) were estimated using the model. The osmotically inactive volume of stage III zebrafish oocytes was found to be 69.5%. The mean values+/-SE of Lp were found to be 0.169+/-0.02 and 0.196+/-0.01 microm/min/atm in the presence of DMSO and PG, respectively, at 22 degrees C, assuming an internal isosmotic value for the oocyte of 272 mOsm. The Ps values were 0.000948+/-0.00015 and 0.000933+/-0.00005 cm/min for DMSO and PG, respectively. It was also shown that the membrane permeability of stage III oocytes decreased significantly with temperature. No significant changes in cell volume during methanol treatment were observed. Fish oocyte membrane permeability parameters are reported here for the first time. The Lp and Ps values obtained for stage III zebrafish oocytes are generally lower than those obtained from successfully cryopreserved mammalian oocytes and higher than those obtained with fish embryos and sea urchin eggs. It was not possible to estimate membrane permeability parameters for stage V oocytes using the methods employed in this study because stage V oocytes experienced the separation of outer oolemma membrane from inner vitelline during exposure to cryoprotectants.     

Properties of Hemoglobin Solutions in Red Cells

The present studies are concerned with a detailed examination of the apparent anomalous osmotic behavior of human red cells. Red cell water has been shown to behave simultaneously as solvent water for nonelectrolytes and nonsolvent water, in part, for electrolytes. The nonsolvent properties are based upon assumptions inherent in the conventional van't Hoff equation. However, calculations according to the van't Hoff equation give osmotic volumes considerably in excess of total cell water when the pH is lowered beyond the isoelectric point for hemoglobin; hence the van't Hoff equation is inapplicable for the measurement of the solvent properties of the red cell. Furthermore, in vitro measurements of osmotic and other properties of 3.7 millimolal solutions of hemoglobin have failed to reveal the presence of any salt exclusion. A new hypothesis has been developed from thermodynamic principles alone, which predicts that, at constant pH, the net charge on the hemoglobin molecule decreases with increased hemoglobin concentration. The existence of such cooperative interaction may be inferred from the effect of pH on the changes in hemoglobin net charge as the spacing between the molecules decreases. The resultant movement of counterions across the cell membrane causes the apparent anomalous osmotic behavior. Quantitative agreement has been found between the anion shift predicted by the equation and that observed in response to osmotic gradients. The proposed mechanism appears to be operative in a variety of tissues and could provide an electrical transducer for osmotic signals.     

Enhanced trehalose production improves growth of Escherichia coli under osmotic stress

The biosynthesis of trehalose has been previously shown to serve as an important osmoprotectant and stress protectant in Escherichia coli. Our results indicate that overproduction of trehalose (integrated lacI-Ptac-otsBA) above the level produced by the native regulatory system can be used to increase the growth of E. coli in M9-2% glucose medium at 37 degrees C to 41 degrees C and to increase growth at 37 degrees C in the presence of a variety of osmotic-stress agents (hexose sugars, inorganic salts, and pyruvate). Smaller improvements were noted with xylose and some fermentation products (ethanol and pyruvate). Based on these results, overproduction of trehalose may be a useful trait to include in biocatalysts engineered for commodity chemicals.     

Increase in the stability and helical content of estrogen receptor alpha in the presence of the estrogen response element: analysis by circular dichroism spectroscopy

Ligand-dependent stabilization of the estrogen receptor (ER) is often postulated, with limited support from experimental data. We studied the thermal unfolding of recombinant ERalpha by circular dichroism (CD) spectroscopy. The T(M) of unfolding of ERalpha was 38 +/- 2.4 degrees C, and the van't Hoff enthalpy of unfolding was 31.7 +/- 3.4 kcal/mol in the absence of ligands. Addition of estradiol (E(2)) increased the T(M) to 43.6 +/- 2.3 degrees C, while addition of E(2) and an oligonucleotide harboring the estrogen response element (ERE) increased the T(M) to 47.9 +/- 1.6 degrees C. Addition of the antiestrogen 4-hydroxytamoxifen (HT) alone did not increase the T(M); however, a combination of HT and the ERE increased the T(M) to 48.9 +/- 1.0 degrees C. The ERE alone increased the T(M) to 46.1 +/- 0.9 degrees C. Addition of E(2) alone had no effect on the apparent enthalpy of unfolding; however, the ERE alone increased the apparent enthalpy from 31.7 to 36.1 kcal/mol. ERalpha samples containing the ERE also exhibited an increase in the negative ellipticity at 208 and 222 nm, relative to that of ligand-free ERalpha, suggesting a stabilization of the alpha-helix. CD data analysis further showed that the presence of the ERE caused a large increase in alpha-helical content of ERalpha in both the presence and absence of the ligands. This increase in alpha-helical content of ERalpha was not observed in the presence of a nonspecific oligonucleotide. These results show that the ERE can increase the thermal stability of ERalpha, enhance its alpha-helical content, and facilitate the cooperativity of the folding transition.     

Heat capacity microcalorimetry of the in vitro reconstitution of calf brain microtubules.

The self-assembly of calf brain tubulin, purified by the modified Weisenberg procedure, was examined in an adiabatic differential heat capacity microcalorimeter. Tubulin solutions at concentrations between 6 and 17 mg/mL were heated from 8 to 40 degrees C at heating rates between 0.1 and 1.0 deg/min in a pH 7.0 phosphate buffer containing 1 X 10(-3) M GTP, 1.6 X 10(-2) M MgCl2, and 3.4 M glycerol. The heat capacity change, deltaCp of the microtubule growth reaction was found to be -1600 +/- 500 cal/(deg mol) per 110 000 molecular weight tubulin dimer incorporated into microtubules, in agreement with the reported van't Hoff deltaCp value of -1500 cal/(deg mol) [Lee, J.C., & Timasheff, S.N. (1977) Biochemistry 16, 1754-1765]. The assembly reaction is characterized by a complex heat uptake pattern comprising both endothermic and exothermic processes.     

Thermodynamic analysis of protein unfolding in aqueous solutions as a multisite reaction of protein with water and solute molecules

Thermal unfolding of ribonuclease A, lysozyme, and chymotrypsinogen A was analyzed as a multisite reaction of a protein molecule with water and solute molecules. The protein unfolding process in various solutions of sugars and denaturants was described well by the van't Hoff equation. The reciprocal form of the Wyman-Tanford equation, which describes the unfolded-to-folded protein ratio as a function of water activity, was successfully applied to obtain a good linear relationship. From this analysis, the role of water activity on protein stability was clearly explained and the contributions of hydration and solute binding to protein molecule were separately discussed in protein unfolding. General solution for the free energy of protein stability was obtained as a simple function of solute concentration.     

A non-ideal replacement for the Boyle van't Hoff equation

A non-ideal osmotic equilibrium equation is proposed as a replacement for the Boyle van’t Hoff equation to describe the equilibrium volume of a living cell as a function of external osmolality. Contrary to common understanding, the Boyle van’t Hoff equation is only thermodynamically correct for ideal, dilute solutions. However, the Boyle van’t Hoff equation is commonly used to determine the osmotically inactive fraction of the cell. This involves extrapolating to infinite osmolality, which violates the ideal, dilute solution constraint. It has been noted that the osmotically inactive fractions obtained from the Boyle van’t Hoff equation for human erythrocytes are markedly larger than measured values of the dry volume fraction of the cell. Using the new osmotic equilibrium equation to analyze experimental osmotic equilibrium data reduces the inferred osmotically inactive fraction of human erythrocytes by approximately 20%.     

Platelet cryopreservation using a trehalose and phosphate formulation

Long-term storage of platelets is infeasible due to platelet activation at low temperatures. In an effort to address this problem, we evaluated the effectiveness of a formulation combining trehalose and phosphate in protecting platelet structure and function following cryopreservation. An annexin V binding assay was used to quantify the efficacy of the trehalose and phosphate formulation in suppressing platelet activation during cryopreservation. Of the platelets cryopreserved with the trehalose plus phosphate formulation, 23% +/- 1.2% were nonactivated, compared with 9.8% +/- 0.26% nonactivated following cryopreservation with only trehalose. The presence of both trehalose and phosphate in the cryopreservation medium is critical for cell survival and preincubation in trehalose plus phosphate solutions further enhances viability. The effectiveness of trehalose plus phosphate in preserving platelets in a nonactivated state is comparable to 6% dimethyl sulfoxide (Me(2)SO). Measurements of platelet metabolic activity using an alamarBlue assay also established that trehalose plus phosphate is superior to trehalose alone. Finally, platelets protected by the trehalose plus phosphate formulation exhibit similar aggregation response upon thrombin addition as fresh platelets, but an increase of cytosolic calcium concentration upon thrombin addition was not observed in the cryopreserved platelets. These results suggest that trehalose and phosphate protect several aspects of platelet structure and function during cryopreservation, including an intact plasma membrane, metabolic activity, and aggregation in response to thrombin, but not intracellular calcium release in response to thrombin.     

Stabilization of bovine intestine alkaline phosphatase by sugars

Bovine intestine alkaline phosphatase (BIALP) is widely used as a signaling enzyme in sensitive assays such as enzyme immunoassay. In this study, we evaluated the effects of sugars on the kinetic stability of BIALP in the hydrolysis of p-nitrophenylphosphate (pNPP). The temperatures reducing initial activity by 50% in a 30-min incubation, T(50), of BIALP with 1.0 M disaccharide (sucrose and trehalose) or 2.0 M monosaccharide (glucose and fructose) were 55.0-55.5 °C, 4.7-5.2 °C higher than without sugar (50.3±0.1 °C). The T(50) of BIALP increased to 58.4±0.3 °C when the trehalose concentration was from 1.0 to 1.5 M, but did not change when the glucose concentration was from 2.0 to 3.0 M. Thermodynamic analysis revealed that the stabilization of BIALP by sugars was driven by the increase in the enthalpy change of activation for thermal inactivation of BIALP. No sugars affected the k(cat) of BIALP in the hydrolysis of pNPP. These results suggest that not only trehalose, which is considered the most effective stabilizer of enzymes, but also sucrose, glucose, and fructose can be used as stabilizers of BIALP.     

The peculiar nature of the guanidine hydrochloride-induced two-state denaturation of staphylococcal nuclease: a calorimetric study.

This work determines the ratio of DeltaH(vH) /DeltaH(cal) for staphylococcal nuclease (SN) denaturation in guanidine hydrochloride (GdnHCl) to test whether GdnHCl-induced denaturation is two-state. Heats of mixing of SN as a function of [GdnHCl] were determined at pH 7.0 and 25 degrees C. The resulting plot of DeltaH(mix) vs [GdnHCl] exhibits a sigmoid shaped curve with linear pre- and post-denaturational base lines. Extending the pre- and post-denaturational lines to zero [GdnHCl] gives a calorimetric DeltaH (DeltaH(cal)) of 24.1 +/- 1.0 kcal/mol, for SN denaturation in the limit of zero GdnHCl concentration. Guanidine hydrochloride-induced denaturation Gibbs energy changes in the limit of zero denaturant concentration (DeltaG degrees (N)(-)(D)) at pH 7. 0 were determined for SN from fluorescence measurements at fixed temperatures over the range from 15 to 35 degrees C. Analysis of the resulting temperature-dependent DeltaG degrees (N)(-)(D) data defines a van't Hoff denaturation enthalpy change (DeltaH(vH)) of 26. 4 +/- 2.8 kcal/mol. The model-dependent van't Hoff DeltaH(vH) divided by the model-independent DeltaH(cal) gives a ratio of 1.1 +/- 0.1 for DeltaH(vH)/DeltaH(cal), a result that rules out the presence of thermodynamically important intermediate states in the GdnHCl-induced denaturation of SN. The likelihood that GdnHCl-induced SN denaturation involves a special type of two-state denaturation, known as a variable two-state process, is discussed in terms of the thermodynamic implications of the process.     

Gustatory perception and metabolic utilization of sugars by<Emphasis Type="Italic"> Myrmica rubra</Emphasis> ant workers

The suitability of various nectar and honeydew sugars as a food source for the polyphagous ant species M. rubra (L.) was studied. The sugars used included monosaccharides (fructose, glucose, galactose, mannose, rhamnose), disaccharides (sucrose, maltose, trehalose, melibiose, lactose) and trisaccharides (melizitose, raffinose, erlose). Single-sugar solutions were tested on ant workers in a long-term laboratory bioassay in which acceptance of the solutions and ant survival were recorded. The acceptance of the sugars was confirmed in a second bioassay in which feeding time was established. Enzymatic hydrolysis of sucrose, maltose and melibiose was investigated through HPLC analyses of workers fed these disaccharides. Sugar acceptance and feeding time were related to ant survival. Considering the monosaccharide units of which the sugars are composed, fructose seems especially suitable as a short-term energy source, while glucose appears to be used both directly and for storage. The presence of a galactose unit appears to reduce sugar suitability. It is suggested that the workers possess invertase and maltase and to a lesser degree also galactosidase. The gustatory perception is correlated with the profitability of sugars in further metabolic processes.     

Significant discrepancies between van't Hoff and calorimetric enthalpies. II.

Isothermal calorimetric titration of 18-crown-6 ether with BaCl2 in pure aqueous solution over the temperature range 7-40 degrees C gives precise binding constants and enthalpy changes. Nonlinear least-squares fitting of the binding constants to the integrated van't Hoff equation, including a temperature-independent change in heat capacity, leads to van't Hoff enthalpies that differ significantly from the observed calorimetric enthalpies. This perplexing discrepancy appears at present to be very widely occurring.