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.     

The Saccharomyces cerevisiae ARO1 gene. An example of the co-ordinate regulation of five enzymes on a single biosynthetic pathway

The ARO1 gene of Saccharomyces cerevisiae encodes the arom multifunctional enzyme. Specific inhibitors of amino acid biosynthesis have been used to obtain evidence that expression of a cloned ARO1 gene is regulated in response to amino acid limitation. Northern blot analysis and sequence studies indicate that ARO1 is regulated by the well characterised S. cerevisiae 'general control' mechanism. This provides a very economical means of simultaneously tailoring the synthesis of five shikimate pathway enzymes to the needs of the cell.     

Effect of streptolysin S on liposomes Influence of membrane lipid composition on toxin action

The effect of the bacterial cytolytic toxin, streptolysin S, on liposomes composed of various phospholipids was investigated. Large unilamellar vesicles containing [¹⁴C]sucrose were prepared by reverse-phase evaporation, and membrane damage produced by the toxin was measured by following the release of labeled marker. The net charge of the liposomes had little or no effect on their susceptibility to steptolysin S and the toxin was about equally effective on liposomes composed of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and phosphatidylglycerol. Experiments with liposomes composed of synthetic phospholipids showed that the ability of the toxin to produce membrane damage depended on the degree of unsaturation of the fatty acyl chains. The order of sensitivity was C18 : 2 phosphatidylcholine > C18 : 1 phosphatidylcholine > C18 : 0 phosphatidylcholine = C16 : 0 phosphatidylcholine. Liposomes containing the latter two phospholipids were virtually unaffected by streptolysin S, and experiments with C18 : 0 phosphatidylcholine suggested that toxin activity does not bind to liposomes composed of phospholipids with saturated fatty acyl chains. The inclusion of 40 mol% cholesterol in C16 : 0 phosphatidylcholine and C18 : 0 phosphatidylcholine liposomes made these vesicles sensitive to streptolysin S. Egg phosphatidylcholine liposomes, which were unaffected at 0°C and 4°C became susceptible to the toxin at these temperatures when cholesterol was included. Liposomes composed of C14 : 0 phosphatidylcholine were unaffected by streptolysin S at temperatures below the chain-melting transition temperature (23°C) of this phospholipid, but became increasingly susceptible above this temperature. The results suggest that the fluidity of the phospholipid hydrocarbon chains in the membrane is important in streptolysin S action.