Daniel V. Lynch

Lipid composition and thermotropic phase behavior of boar, bull, stallion, and rooster sperm membranes

Composition and thermotropic phase behavior of sperm membrane lipids from species ranging in sensitivity to cold shock were determined. Lipids from whole sperm and sperm plasma membrane were fractionated into neutral lipid, glycolipid, and phospholipid fractions. Compositional analyses were completed for free sterols, phospholipids and phospholipid-bound fatty acids. Phase transition temperatures were determined for phospholipid and glycolipid fractions using differential scanning calorimetry. Cholesterol was the major sterol in sperm lipids of all species. Cholesterol to phospholipid molar ratios were 0.26, 0.30, 0.36, and 0.45 for sperm plasma membrane of the boar, rooster, stallion, and bull, respectively. Choline and ethanolamine phosphoglycerides and sphingomyelin were the major phospholipid classes in sperm and their proportions differed across species. Phospholipid-bound fatty acyl compositions of choline and ethanolamine phosphoglycerides were characterized by a high proportion of docosapentanoyl and docosahexanoyl groups in mammalian sperm and shorter, more saturated groups in rooster sperm. Glycolipids represented less than 10% of total polar lipids for all species. Thin-layer chromatographic analysis indicated that the major glycolipid component of rooster sperm was different from that of mammalian sperm. Peak phase transition temperatures (Tm) for sperm membrane phospholipids were 24.0, 25.4, 20.7 and 24.5, for the boar, stallion, and rooster, respectively. Corresponding Tms for glycolipids were 36.2, 42.8, and 33.4 with no exotherm for rooster sperm glycolipids. These results demonstrate a difference in both composition and thermotropic phase behavior of glycolipids between rooster and mammalian sperm which may be related to the greater tolerance of rooster sperm to rapid cooling.

Freeze/thaw-induced destabilization of the plasma membrane and the effects of cold acclimation.

Disruption of the plasma membrane is a primary cause of freezing injury. In this review, the mechanisms of injury resulting from freeze-induced cell dehydration are presented, including destabilization of the plasma membrane resulting from (a) freeze/thaw-induced osmotic excursions and (b) lyotropic phase transitions in the plasma membrane lipids. Cold acclimation dramatically alters the behavior of the plasma membrane during a freeze/thaw cycle—increasing the tolerance to osmotic excursions and decreasing the propensity for dehydration-induced lamellar to hexagonal-II phase transitions. Evidence for a casual relationship between the increased cryostability of the plasma membrane and alterations in the lipid composition is reviewed.

Lyotropic phase behavior of unsaturated phosphatidylcholine species: relevance to the mechanism of plasma membrane destabilization and freezing injury

Abstract The phase behavior and hydration characteristics of phosphatidylcholine (PC) species having one or two unsaturated acyl chains (i.e., 1-palmitoyl-2-oleoyl-, 1-palmitoyl-2-linoleoyl-, dilinoleoyl-, and dilinolenoylphosphatidylcholine) were characterized. Using differential scanning calorimetry, it was determined that the phase transition temperatures (Tm values) of these PC species increased by 30–40 C° over the range of water contents between 20 wt% and 5 wt%. To determine the relevance of dehydration-induced phase transitions to membrane destabilization during freezing, the hydration characteristics of PC species were investigated by constructing desorption isotherms. For all the species, the water content decreased from an average 17 wt% to 4 wt% water over the range of water potentials from −16 MPa to −160 MPa. The results of these and related studies of mixtures of 1-palmitoyl-2-linoleoyl-PC and dilinoleoyl-PC indicate that under the conditions of temperature and dehydration encountered during freezing and which result in membrane destabilization, unsaturated species of PC would be expected to remain in the liquid-crystalline phase.

Characterization of intracellular ice formation in Drosophila melanogaster embryos

Cryomicroscopy and differential scanning calorimetry (DSC) were used to characterize the incidence of intracellular ice formation (IIF) in 12- to 13-hr-old embryos of Drosophila melanogaster (Oregon-R strain P2) as influenced by the state of the eggcase (untreated, dechorionated, or permeabilized), the composition of the suspending medium (with and without cryoprotectants), and the cooling rate. Untreated eggs underwent IIF over a very narrow temperature range when cooled at 4 or 16 degrees C/min with a median temperature of intracellular ice formation (TIIF50) of -28 degrees C. The freezable water volume of untreated eggs was approximately 5.4 nl as determined by DSC. IIF in dechorionated eggs occurred over a much broader temperature range (-13 to -31 degrees C), but the incidence of IIF increased sharply below -24 degrees C, and the cumulative incidence of IIF at -24 degrees C decreased with cooling rate. In permeabilized eggs without cryoprotectants (CPAs), IIF occurred at much warmer temperatures and over a much wider temperature range than in untreated eggs, and the TIIF50 was cooling rate dependent. At low cooling rates (1 to 2 degrees C/min), TIIF50 increased with cooling rate; at intermediate cooling rates (2 to 16 degrees C/min), TIIF50 decreased with cooling rate. The total incidence of IIF in permeabilized eggs was 54% at 1 degree C/min, and volumetric contraction almost always occurred during cooling. Decreasing the cooling rate to 0.5 degree C/min reduced the incidence of IIF to 43%. At a cooling rate of 4 degrees C/min, ethylene glycol reduced the TIIF50 by about 12 degrees C for each unit increase in molarity of CPA (up to 2.0 M) in the suspending medium. The TIIF50 was cooling rate dependent when embryos were preequilibrated with 1.0 M propylene glycol or ethylene glycol, but was not so in 1.0 M DMSO. For embryos equilibrated in 1.5 M ethylene glycol and then held at -5 degrees C for 1 min before further cooling at 1 degree C/min, the incidence of IIF was decreased to 31%. Increasing the duration of the isothermal hold to 10 min reduced the incidence of IIF to 22% and reduced the volume of freezable water in embryos when intracellular ice formation occurred. If the isothermal hold temperature was -7.5 or -10 degrees C, a 10- to 30-min holding time was required to achieve a comparable reduction in the incidence of IIF.

A two-step method for permeabilization of Drosophila eggs☆

As a first step in developing a procedure for the cryopreservation of Drosophila melanogaster embryos, we have established a method for permeabilization of the eggcase and have initiated studies of the hydraulic conductivity of permeabilized embryos and the permeation of selected cryoprotective agents. The eggcase of D. melanogaster embryos has a wax layer that precludes any flux of water. A two-step procedure employing organic solvents was developed to effect removal of the wax layer with minimal deleterious effects on the embryos. Dechorionated embryos (Oregon-R strain P2, 12 to 13 hr old) were rinsed sequentially in isopropanol and hexane. After removal of solvent, embryos were held in a modified cell culture medium for further manipulation. This procedure routinely yielded 80 to 95% of the eggs permeabilized (as determined by osmotic contraction in 1 M sucrose) and 75 to 90% survival (incidence of hatching). Hydraulic conductivity of permeabilized embryos and permeation of cryoprotectants were determined using a microdiffusion chamber and computerized video microscopy. Regression analysis of the volumetric data from individual embryos yielded the Boyle-vant Hoff function FVeq = 0.124 (osm-1) + 0.541 with the standard deviations of slope and intercept (Vb) being 0.010 and 0.040, respectively. Permeabilized embryos exhibited ideal osmotic behavior over the range of 0.265 to 2.00 osm. The mean hydraulic conductivity coefficient (Lp) was 0.722 +/- 0.366 micron/(min.atm) at 20 degrees C, based on observations of contraction following a step change in concentration of Ringers solution.(ABSTRACT TRUNCATED AT 250 WORDS)

Cryobiology of Drosophila Melanogaster Embryos

The common fruit fly Drosophila melanogaster is the subject of investigation in many diverse areas of biology. It has been studied intensively by geneticists, developmental and molecular biologists, neurobiologists, population and evolutionary biologists, entomologists, and chronobiologists. Currently, interest in D. melanogaster is most intense among molecular biologists, but studies of D. melanogaster have a long and distinguished history, dating back to Thomas Hunt Morgan in the first decade of this century. As a result of both past and present activity, there is an enormous number of D. melanogaster genetic stocks. In 1985 it was estimated that the number of different stocks was in excess of 30,000 and was rapidly increasing because of the increased number of investigators studying Drosophila, the increased number of large scale mutant screens, and the generation of new stocks by DNA transformation. Since then, the number of mutant stocks is even greater, especially since so many germ line transformants have been obtained; for example, in Drosophila Information Service (June 1988), some 1350 entries were recorded in the “clone list.” Many of these clones have been reinserted in several different places in the germ line via P-element mediated transformation. We estimate that over 50,000 different genetic lines of D. melanogaster are now maintained in national and international stock centers and in the laboratories of individual investigators.

Plasma Membrane Lipid Alterations Following Cold Acclimation: Possible Relevance to Freeze Tolerance

The plasma membrane plays a central role in cellular behavior during a freeze/thaw cycle and lysis or alterations in its semipermeable characteristics is a primary cause of freezing injury1. In protoplasts isolated from nonacclimated (NA) rye leaves, injury over the range of 0° to –5°C is a consequence of freeze-induced osmotic contraction resulting in endocytotic vesiculation of the plasma membrane - with sufficiently large area contractions being irreversible. As a result, lysis of the protoplasts occurs during osmotic expansion following thawing of the suspending medium. Alternatively, cooling to temperatures below -5°C results in destabilization of the plasma membrane so that the protoplasts are osmotically unresponsive during thawing of the suspending medium. This form of injury is associated with several changes in the ultrastructure of the plasma membrane, including the formation of lateral phase separations, aparticulate lamellae, and hexagonalII configurations2. Cold acclimation dramatically alters the behavior of the plasma membrane during freeze-induced osmotic contraction and dehydration. In protoplasts from acclimated (ACC) rye leaves, osmotic contraction results in the formation of exocytotic extrusions of the plasma membrane; and at severe levels of dehydration, lateral phase separations, aparticulate lamellae, or HII configurations are not observed.