Effect of 6% hydroxyethyl starch 130/0.4 on kidney and haemostatic function in cardiac surgical patients: a randomised controlled trial.

Whether third-generation hydroxyethyl starch solutions provoke kidney injury or haemostatic abnormalities in patients having cardiac surgery remains unclear. We tested the hypotheses that intra-operative administration of a third-generation starch does not worsen postoperative kidney function or haemostasis in cardiac surgical patients compared with human albumin 5%. This triple-blind, non-inferiority, clinical trial randomly allocated patients aged 40-85 who underwent elective aortic valve replacement, with or without coronary artery bypass grafting, to plasma volume replacement with 6% starch 130/0.4 vs. 5% human albumin. Our primary outcome was postoperative urinary neutrophil gelatinase-associated lipocalin concentrations, a sensitive and early marker of postoperative kidney injury. Secondarily, we evaluated urinary interleukin-18; acute kidney injury using creatinine RIFLE criteria, coagulation measures, platelet count and function. Non-inferiority (delta 15%) was assessed with correction for multiple comparisons. We enrolled 141 patients (69 starch, 72 albumin) as planned. Results of the primary analysis demonstrated that postoperative urine neutrophil gelatinase-associated lipocalin (median (IQR [range])) was slightly lower with hydroxyethyl starch (5 (1-68 [0-996]) ng.ml-1 ) vs. albumin (5 (2-74 [0-1604]) ng.ml-1 ), although not non-inferior [ratio of geometric means (95%CI) 0.91 (0.57, 1.44); p = 0.15] due to higher than expected variability. Urine interleukin-18 concentrations were reduced, but interleukin-18 and kidney injury were again not non-inferior. Of 11 individual coagulation measures, platelet count and function, nine were non-inferior to albumin. Two remaining measures, thromboelastographic R value and arachidonic acid-induced platelet aggregation, were clinically similar but with wide confidence intervals. Starch administration during cardiac surgery produced similar observed effects on postoperative kidney function, coagulation, platelet count and platelet function compared with albumin, though greater than expected variability and wide confidence intervals precluded the conclusion of non-inferiority. Long-term mortality and kidney function appeared similar between starch and albumin.

Molecular p-doping in organic liquid crystalline semiconductors: influence of the charge transfer complex on the properties of mesophase and bulk charge transport.

We explore the molecular nature of doping in organic semiconductors (OSCs) by employing a liquid crystalline organic semiconductor based on phenyl naphthalene as a model. The mesophase nature of composites that include a charge transfer complex (CTC) between the OSC (8-PNP-O12) and an electron acceptor (F4TCNQ) has been investigated by means of differential scanning calorimetry, polarized optical microscopy and X-ray scattering. Optical and vibrational spectroscopies allow us to explore the characteristics and the amount of charge transfer in the CTC and expose some properties that appear only in the complexed state. We have found this system to exhibit partial charge transfer, which manifests itself in all the phase states of the host 8-PNP-O12, as well as in solution. Due to the lowering of molecular symmetry as a result of the charge transfer, one of the previously IR-only vibrational bands of the nitrile group is found to be now active in the Raman spectrum. We have also made an attempt to further investigate the influence of dopant introduction on the bulk hole mobility of 8-PNP-O12. It is found that the presence of the CTC promotes the hole transport in the Smectic B mesophase, however it seems to have a somewhat negative influence in the less ordered smectic A mesophase. This work aims to establish the link between the inevitable change of molecular geometry that occurs on charge transfer with the results obtained by spectroscopic techniques and electronic charge carrier mobility measurements.

Prolonged concurrent hypotension and low bispectral index ('double low') are associated with mortality, serious complications, and prolonged hospitalization after cardiac surgery.

BACKGROUND: Low bispectral index (BIS) and low mean arterial pressure (MAP) are associated with worse outcomes after surgery. We tested the hypothesis that a combination of these risk factors, a 'double low', is associated with death and major complications after cardiac surgery. METHODS: We used data from 8239 cardiac surgical patients from two US hospitals. The primary outcomes were 30-day mortality and a composite of in-hospital mortality and morbidity. We examined whether patients who had a case-averaged double low, defined as time-weighted average BIS and MAP (calculated over an entire case) below the sample mean but not in the reference group, had increased risk of the primary outcomes compared with patients whose BIS and/or MAP were at or higher than the sample mean. We also examined whether a prolonged cumulative duration of a concurrent double low (simultaneous low MAP and BIS) increased the risk of the primary outcomes. RESULTS: Case-averaged double low was not associated with increased risk of 30-day mortality {odds ratio [OR] 1.73 [95% confidence interval (CI) 0.94-3.18] vs reference; P =0.01} or the composite of in-hospital mortality and morbidity [OR 1.47 (95% CI 0.98-2.20); P =0.01] after correction for multiple outcomes. A prolonged concurrent double low was associated with 30-day mortality [OR 1.06 (95% CI 1.01-1.11) per 10-min increase; P =0.001] and the composite of in-hospital mortality and morbidity [OR 1.04 (95% CI 1.01-1.07), P =0.004]. CONCLUSIONS: A prolonged concurrent double low, but not a case-averaged double low, was associated with higher morbidity and mortality after cardiac surgery.

Cannabis or alcohol first? Differences by ethnicity and in risk for rapid progression to cannabis-related problems in women.

BACKGROUND: Initiation of cannabis use typically follows alcohol use, but the reverse order does occur and is more common for African-Americans (AAs) than European-Americans (EAs). The aim of this study was to test for differences in the order of initiation of cannabis and alcohol use between AA and EA women and to determine whether order and ethnicity contribute independently to risk for rapid progression to cannabis-related problems. Method Data were drawn from structured psychiatric interviews of 4102 women (mean age = 21.6 years), 3787 from an all-female twin study and 315 from a high-risk family study; 18.1% self-identified as AA, 81.9% as EA. Ethnicity and order of initiation of cannabis and alcohol use were modeled as predictors of transition time from first use to onset of cannabis use disorder symptom(s) using Cox proportional hazards regression analyses. RESULTS: AA women were nearly three times as likely as EA women to initiate cannabis use before alcohol use. Using cannabis before alcohol [hazard ratio (HR) 1.44, 95% confidence interval (CI) 1.08-1.93] and AA ethnicity (HR 1.59, 95% CI 1.13-2.24) were both associated with rapid progression from first use to cannabis symptom onset even after accounting for age at initiation and psychiatric risk factors. CONCLUSIONS: The findings indicate that AA women are at greater risk for rapid development of cannabis-related problems than EA women and that this risk is even higher when cannabis use is initiated before alcohol use. Prevention programs should be tailored to the various patterns of cannabis use and relative contributions of risk factors to the development of cannabis-related problems in different ethnic groups.

Common genetic and environmental contributions to post-traumatic stress disorder and alcohol dependence in young women.

BACKGROUND: The few genetically informative studies to examine post-traumatic stress disorder (PTSD) and alcohol dependence (AD), all of which are based on a male veteran sample, suggest that the co-morbidity between PTSD and AD may be attributable in part to overlapping genetic influences, but this issue has yet to be addressed in females.MethodData were derived from an all-female twin sample (n=3768) ranging in age from 18 to 29 years. A trivariate genetic model that included trauma exposure as a separate phenotype was fitted to estimate genetic and environmental contributions to PTSD and the degree to which they overlap with those that contribute to AD, after accounting for potential confounding effects of heritable influences on trauma exposure. RESULTS: Additive genetic influences (A) accounted for 72% of the variance in PTSD; individual-specific environmental (E) factors accounted for the remainder. An AE model also provided the best fit for AD, for which heritability was estimated to be 71%. The genetic correlation between PTSD and AD was 0.54. CONCLUSIONS: The heritability estimate for PTSD in our sample is higher than estimates reported in earlier studies based almost exclusively on an all-male sample in which combat exposure was the precipitating traumatic event. However, our findings are consistent with the absence of evidence for shared environmental influences on PTSD and, most importantly, the substantial overlap in genetic influences on PTSD and AD reported in these investigations. Additional research addressing potential distinctions by gender in the relative contributions of genetic and environmental influences on PTSD is merited.

Algorithms for sequence analysis via mutagenesis.

MOTIVATION: Despite many successes of conventional DNA sequencing methods, some DNAs remain difficult or impossible to sequence. Unsequenceable regions occur in the genomes of many biologically important organisms, including the human genome. Such regions range in length from tens to millions of bases, and may contain valuable information such as the sequences of important genes. The authors have recently developed a technique that renders a wide range of problematic DNAs amenable to sequencing. The technique is known as sequence analysis via mutagenesis (SAM). This paper presents a number of algorithms for analysing and interpreting data generated by this technique. RESULTS: The essential idea of SAM is to infer the target sequence using the sequences of mutants derived from the target. We describe three algorithms used in this process. The first algorithm predicts the number of mutants that will be required to infer the target sequence with a desired level of accuracy. The second algorithm infers the target sequence itself, using the mutant sequences. The third algorithm assigns quality values to each inferred base. The algorithms are illustrated using mutant sequences generated in the laboratory.

Unlocking hidden genomic sequence.

Despite the success of conventional Sanger sequencing, significant regions of many genomes still present major obstacles to sequencing. Here we propose a novel approach with the potential to alleviate a wide range of sequencing difficulties. The technique involves extracting target DNA sequence from variants generated by introduction of random mutations. The introduction of mutations does not destroy original sequence information, but distributes it amongst multiple variants. Some of these variants lack problematic features of the target and are more amenable to conventional sequencing. The technique has been successfully demonstrated with mutation levels up to an average 18% base substitution and has been used to read previously intractable poly(A), AT-rich and GC-rich motifs.

Proteomic analysis of chronic lymphocytic leukemia subtypes with mutated or unmutated Ig V(H) genes.

Chronic lymphocytic leukemia (CLL) is a common hematopoietic malignant disease with variable outcome. CLL has been divided into distinct groups based on whether somatic hypermutation has occurred in the variable region of the immunoglobulin heavy-chain locus or alternatively if the cells express higher levels of the CD38 protein. We have analyzed the proteome of 12 cases of CLL (six mutated (M-CLL) and six unmutated (UM-CLL) immunoglobulin heavy-chain loci; seven CD38-negative and five CD38-positive) using two-dimensional electrophoresis and mass spectrometry. Statistical evaluation using principal component analysis indicated significant differences in patterns of protein expression between the cases with and without somatic mutation. Specific proteins indicated by principal component analysis as varying between the prognostic groups were characterized using mass spectrometry. The levels of F-actin-capping protein beta subunit, 14-3-3 beta protein, and laminin-binding protein precursor were significantly increased in M-CLL relative to UM-CLL. In addition, primary sequence data from tandem mass spectrometry showed that nucleophosmin was present as several protein spots in M-CLL but was not detected in UM-CLL samples, suggesting that several post-translationally modified forms of nucleophosmin vary between these two sample groups. No specific differences were found between CD38-positive and -negative patient samples using the same approach. The results presented show that proteomic analysis can complement other approaches in identifying proteins that may have potential value in the biological and diagnostic distinction between important clinical subtypes of CLL.

In vitro maintenance of boar sperm viability by a soluble fraction obtained from oviductal apical plasma membrane preparations.

Oviductal apical plasma membrane fractions have been successfully used to provide an in vitro model to study the role of direct membrane contact in sperm-oviduct interactions. Apical plasma membrane preparations from pig oviductal tissues show a dose-response in their ability to maintain boar sperm viability in vitro. Membrane preparations obtained from other tissues (lung and duodenum) are incapable of maintaining boar sperm viability to the same extent as oviductal tissue. The present study examined the validity of two hypotheses that arise from current knowledge of sperm-oviduct interactions, namely, that (i) apical plasma membranes prepared from ampullar regions of the oviduct are less effective than those from isthmus regions, and (ii) sperm survival is more effective in apical plasma membrane preparations derived from follicular phase oviducts than those derived from luteal phase oviducts. Both hypotheses were proved false. The nature of the active component(s) in the oviductal apical plasma membrane fractions was further investigated. Heat treatment (100 degrees C for 20 min) diminished the capacity of membranes to support boar sperm viability. Furthermore, a soluble salt-extracted fraction obtained from oviductal apical plasma membrane preparations was biologically active and supported boar sperm viability in vitro. This may indicate that the active factor(s) responsible for the maintenance of boar sperm viability is not an integral part of oviductal membranes and is peripherally bound to these membranes.

A simulated annealing algorithm for finding consensus sequences.

MOTIVATION: A consensus sequence for a family of related sequences is, as the name suggests, a sequence that captures the features common to most members of the family. Consensus sequences are important in various DNA sequencing applications and are a convenient way to characterize a family of molecules. RESULTS: This paper describes a new algorithm for finding a consensus sequence, using the popular optimization method known as simulated annealing. Unlike the conventional approach of finding a consensus sequence by first forming a multiple sequence alignment, this algorithm searches for a sequence that minimises the sum of pairwise distances to each of the input sequences. The resulting consensus sequence can then be used to induce a multiple sequence alignment. The time required by the algorithm scales linearly with the number of input sequences and quadratically with the length of the consensus sequence. We present results demonstrating the high quality of the consensus sequences and alignments produced by the new algorithm. For comparison, we also present similar results obtained using ClustalW. The new algorithm outperforms ClustalW in many cases.

Sperm-oviduct interaction: induction of capacitation and preferential binding of uncapacitated spermatozoa to oviductal epithelial cells in porcine species.

After mating, inseminated spermatozoa are transported to the oviduct. They attach to and interact with oviductal epithelial cells (OEC). To investigate sperm-OEC interactions, we used chlortetracycline to study the capacitation status of boar spermatozoa in coculture with homologous OEC and cells of nonreproductive origin (LLC-PK1, porcine kidney epithelial cell line). Boar spermatozoa were cocultured with OEC and LLC-PK1 cells for 15, 60, 120, or 240 min. The proportion of capacitated spermatozoa in coculture with the isthmic and ampullar cells increased significantly (p < 0.05) during incubation. However, most spermatozoa in coculture with LLC-PK1 cells or blank (medium only) remained uncapacitated. In addition, preferential binding of uncapacitated, capacitated, or acrosome-reacted boar spermatozoa to OEC and the other cell type was investigated. Our approach was to vary the proportions of uncapacitated, capacitated, or acrosome-reacted boar spermatozoa in suspension using long preincubation and lysophosphatidylcholine treatment of semen prior to a very short incubation with OEC or LLC-PK1 cells. The results showed that the majority of spermatozoa that were bound to OEC or LLC-PK1 cells were uncapacitated and that a significant relationship existed between the relative proportion of uncapacitated spermatozoa in the control samples and those bound to LLC-PK1 cells (r2 = 0.43, p < 0.005). However, there was no correlation between the proportion of uncapacitated spermatozoa in the control samples and the proportion of those bound to isthmic or ampullar cells. In conclusion, the results clearly demonstrated the specific nature of the sperm-OEC interaction in the porcine species. This interaction is initiated by uncapacitated spermatozoa binding to OEC and is continued by the induction of capacitation in cocultured spermatozoa.

Adenosine analogues with specificity for A2 receptors bind to mouse spermatozoa and stimulate adenylate cyclase activity in uncapacitated suspensions.

Adenosine and its analogues, known to stimulate adenylate cyclase activity in somatic cells via A2 receptors, can accelerate capacitation in mouse spermatozoa and thereby enhance fertilizing ability in vitro. Indirect evidence has suggested that adenosine can modulate mouse sperm adenylate cyclase, implicating this enzyme and cAMP in the observed functional responses. In the present study we provide evidence that [3H]5'-N-ethylcarboxamidoadenosine (NECA), an adenosine analogue with specificity for stimulatory A2 adenosine receptors, can bind to mouse spermatozoa. This binding can be displaced by both unlabelled NECA and 2-chloroadenosine, another A2 receptor agonist, but not by cyclopentyladenosine, an inhibitory A1 receptor agonist, suggesting that the NECA binding is specific for A2 receptors. The presence of S-(p-nitrobenzyl)-6-thioinosine, an adenosine transport inhibitor, did not affect binding, indicating an external site for interaction with sperm cells. Saturable specific binding of [3H]NECA to mouse spermatozoa incubated at 37 degrees C was observed, with a Bmax of 5.17 pmol mg-1 protein and a Kd value of 930 nmol l-1. Binding data were consistent with the presence of a single major class of receptor. In addition to demonstrable binding of [3H]NECA, both NECA and 2-chloroadenosine significantly stimulated adenylate cyclase activity in a concentration-dependent manner, with NECA being effective at a lower concentration. Furthermore, the hydrolysis-resistant GTP analogue Gpp(NH)p, alone and in the presence of either NECA or 2-chloroadenosine, also significantly stimulated enzyme activity. In somatic cells, expression of responses to adenosine usually requires GTP and G proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Cyclic AMP-dependent phosphorylation of epididymal mouse sperm proteins during capacitation in vitro: identification of an M(r) 95,000 phosphotyrosine-containing protein.

Cyclic AMP-dependent changes in phosphorylation of epididymal mouse sperm suspensions were examined in media designed to manipulate capacitation and the expression of parameters associated with full fertilizing ability, i.e. hyperactivated motility and the acrosome reaction. After initial assessment of cAMP-dependent protein kinase activity in frozen-thawed and lyophilized sperm suspensions using exogenous substrate, phosphorylation of endogenous sperm phosphoproteins was examined using sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by autoradiography or immunoblotting. Numerous phosphoproteins were detected in both incapacitated and capacitated suspensions, the majority of which were probably concerned with motility; full expression of fertilizing ability appeared to involve an increase in the amount of endogenous phosphorylation as deduced from the decreased amount of 32P incorporation in these suspensions. The addition of the cAMP-dependent protein kinase inhibitors, H8 and PKI (6-22) amide, demonstrated that most of the phosphoproteins detected were phosphorylated in a cAMP-dependent manner. Of particular interest was a phosphoprotein with an M(r) of about 95,000 which was consistently observed in capacitated suspensions. Evidence suggests that this may be phosphorylated on tyrosine residues, since the inclusion of orthovanadate, a phosphoryltyrosine phosphatase inhibitor, altered phosphorylation of this protein. Furthermore, immunodetection using the antiphosphotyrosine antibody, PY-20, identified five proteins with approximate M(r) 116,000, 105,000, 95,000, 86,000, and 76,000, and possibly a sixth at 54,000. The 95,000 protein was consistently diminished in ionophore-treated spermatozoa, indicating that the protein was located in the acrosomal cap region. These results suggest that the protein may be the same phosphotyrosine-containing protein as that described by Leyton and Saling (1989) which has been proposed to play a role in acrosomal exocytosis.

Predictive water loss curves for ram spermatozoa during cryopreservation: comparison with experimental observations.

Calculated curves predicting intracellular water loss during cryopreservation at different cooling rates were calculated from published equations. To compute these curves, basic cell parameters specific to ram spermatozoa were measured, i.e., the total surface area (139 microns2), the hydraulic conductivity (0.222 micron3/micron2.atm-1.min-1), and its temperature dependence (0.045/degree C). Cell surface area was derived from measurements of physical dimensions. Hydraulic conductivity was calculated from measurements of the critical medium hypotonicity on exposure of sperm to various hypotonic solutions and the time taken for membrane rupture in sperm exposed to distilled water (spermolysis time). The temperature dependence of the water permeability was derived from measurements of spermolysis time at various temperatures above zero. Several discrepancies were noted between the resulting calculated curves and experimental observations made on the effects of cooling rate on sperm cell survival. These could be due to errors in the estimates of the basic parameters, or to false assumptions in the basic equations used to compute the curves, e.g., the validity of the Boyle-van't Hoff relationship. Nevertheless, this study represents a first attempt to predict intracellular water loss from ram sperm during cooling and may provide a novel approach for the interpretation of the many empirical studies carried out to investigate optimal conditions for the cryopreservation of sperm.

Interspecies differences in stereoselective protein binding and clearance of MK-571.

(+)-3-(((3-(2-(7-chloro-2-quinolinyl)ethenyl)phenyl)((3-(dimethylamino)- 3-oxopropyl)thio)methyl)thio)propanoic acid (MK-571), is a potent and specific antagonist of leukotriene D4 action in vitro and in vivo. The compound, which is being developed for the treatment of asthma, contains a chiral center at the methine carbon of the dithio side chain and exists in two forms. The binding of MK-571 enantiomers to plasma protein was extensive (greater than 99.5%), stereoselective, and species dependent. The R-(-)-enantiomer was bound to rat plasma to a greater extent than the S-(+)-enantiomer, while in dog and monkey plasma the reverse was the case. The elimination clearance of the enantiomers was inversely related to the stereoselective plasma protein binding, that with the greater unbound fraction being cleared more rapidly. Thus, the pharmacologically more active S-(+)-enantiomer was cleared 3.7 times more rapidly than its antipode in rats following iv administration of the racemate (10 mg/kg), whereas in dogs and monkeys the R-(-)-enantiomer was cleared more rapidly. Kinetic analysis of the data revealed that the intrinsic clearances of the unbound enantiomers were similar within species, suggesting that stereoselectivity in elimination is not attributable to differences in metabolism and biliary excretion. Bioavailabilities of the S-(+)- and R-(-)-enantiomers in the rat were similar (75% and 71%, respectively) suggesting that MK-571 was not stereoselectively absorbed in that species.

The physiological disposition of aerosolized 4-[(3-(4-acetyl-3-hydroxy-2-propylphenoxy)propyl)sulfonyl]-gamma -oxobenzenebutanoate (L-648,051) in rats.

14C-labeled 4-[(3-(4-acetyl-3-hydroxy-2-propylphenoxy)propyl)-sulfonyl]-gamma- oxobenzenebutanoate (L-648,051) was suspended in Freon under pressure and injected into rat lungs via a tracheal cannula. The particle size of the drug was 1 to 5 microns and the mean dose was approximately 0.2 mg/kg. Levels of radioactivity in the lung/trachea declined in a monoexponential manner. Absorption, estimated from radioactivity remaining in the lung and trachea, was 73% in 1 hr and 95% in 4 hr. L-648,051 and its pharmacologically active metabolite L-657,098 (formed by ketoreduction of the butanoic acid moiety of L-648,051) accounted for 96% of the radioactivity in the lung at 10 min after the dose and 91% after 60 min. The lung:plasma concentration ratio of active drug (L-648,051 plus L-657,098) was at least 176:1 at 10 min and 17:1 at 60 min (compared with 1:1 after 2 mg/kg iv) suggesting that aerosol administration of L-648,051 in humans may result in an ideal therapeutic ratio, with high levels of pharmacologically active ingredient in the lung and low levels in the plasma.

Disposition and metabolism of sodium 4-[(3-(4-acetyl-3-hydroxy-2-propylphenoxy)propyl)sulfonyl]-gamma -oxobenzenebutanoate in rats and dogs.

The disposition of sodium 4-[(3-(4-acetyl-3-hydroxy-2-propylphenoxy)propyl)sulfonyl]-gamma-o xo benzenebutanoate (L-648,051) was determined in rats and dogs. L-648,051 is a potent receptor antagonist for leukotriene D4 and is potentially useful in the treatment of asthma and other allergic disorders. After a dosage of 10 mg/kg iv, L-648,051 declined rapidly with a half-life of approximately 2 min in rat and dog plasma. Although the compound was well absorbed, it exhibited poor bioavailability due to efficient first-pass metabolism. In rats receiving 25, 50, and 150 mg/kg po, bioavailabilities were 0.5, 4.8, and 38.7%, respectively. In dogs, bioavailability of 10 and 50 mg/kg po was 0 and 23%, respectively. Two metabolites were identified, 4-[(3-(4-acetyl-3-hydroxy-2-propylphenoxy)propyl)sulfonyl-gamma- hydroxybenzenebutanoic acid (metabolite I), formed by ketoreduction, and 4-[(3-(4-acetyl-3-hydroxy-2-propylphenoxy)propyl)sulfonyl] benzeneacetic acid (metabolite II) formed by catabolic oxidation of the butanoic acid moiety of L-648,051. Ketoreduction resulted in the production of a chiral center and two enantiomers of metabolite I. In vitro studies suggest that rat erythrocytes formed the (+)-enantiomer exclusively. When L-648,051 was administered orally or iv to rats, both the (+)- and (-)-enantiomers were observed in the plasma. The data suggest that either two L-648,051 ketoreductases were present or that inversion of the hydroxyl stereocenter of metabolite I occurred.

Effect of salt concentration and unfrozen water fraction on the viability of slowly frozen ram spermatozoa.

Ram spermatozoa were subjected to a slow rate of freezing (1 degree C/min) in various glycerol-NaCl-water solutions of known composition such that the molal concentration of NaCl (ms) and the unfrozen fraction of water (U) could be calculated at subzero temperatures from the relevant phase diagram. Sperm motility was reduced as ms increased and U correspondingly decreased with temperature. However, by freezing spermatozoa in solutions of differing initial tonicities, but with a constant weight ratio of glycerol: salt, to various subzero temperatures, the effects of ms could be separated from those of U. Motility was found to decrease dramatically at values of U less than 0.07 regardless of ms but, at higher values of U, maximum motility was dependent on the final salt concentration in that fraction, being reduced as the osmolality increased. Sperm cell concentration had no apparent effect on the influence of ms or U on viability in the range studied (3-12 x 10(8) spermatozoa/ml). In order to account for these observations, the effects of osmotic stress on spermatozoa were investigated. When subjected to sudden changes in osmolality of the suspending medium by increasing NaCl or sucrose concentration at room temperature, spermatozoa showed a decreased motility with increasing osmolality. Since no improvement in motility was found on returning the cells to isosmolar conditions cell damage appeared to be irreversible. Furthermore, when placed in solutions of increasing hypotonicity the number of swollen spermatozoa with looped tails increased with increasing hypotonicity. Since the drop in motility seen at low values of U corresponded to those spermatozoa exposed to a hypotonic starting solution, it is suggested that a hypotonic stress followed by a hypertonic stress during freezing and thawing may account for the profound loss of motility in these samples, while a hypertonic stress may account for the strong effect of ms seen at higher values of U.