Phospholipid composition and phospholipid asymmetry of ram spermatozoa plasma membranes

The phospholipid composition of ram spermatozoa plasma membranes has been investigated. An exclusively high participation of the choline- and ethanolamine-plasmalogens in the phosphatidylcholine and phosphatidylethanolamine fractions has been established. Phosphatidylcholine of ram spermatozoa plasma membranes contains a great amount of polyunsaturated fatty acids. The phospholipid distribution in spermatozoa plasma membrane was investigated. It was established that the choline containing phospholipids are situated mainly in the outer membrane lipid monolayer, whereas diphosphatidylglycerol and phosphatidylserine are localized predominantly in the inner monolayer. The rest of the phospholipids are evenly distributed among the two monolayers. Ram spermal plasma membranes exhibit high phospholipase A2 activity.     

Effect of lactoferrin on ram sperm motility after cryopreservation

ObjectiveThe objective of this study was to analyse the differentially abundant proteins caused by freeze–thawing of ram sperm and explore candidate proteins of interest for their ability to improve ram sperm cryopreservation outcomes in vitro.MethodsSperm were from three mature Dorper. Fresh and frozen sperm proteins were extracted, and the differentially abundant proteins were analysed by mass spectrometry. Among these proteins, lactoferrin (LTF) was selected to be added before cryopreservation. Next, sperm samples were diluted in Tris extender, with the addition of 0, 10, 100, 500, and 1,000 μg/mL of LTF. After thawing, sperm quality was evaluated by motility, plasma membrane integrity, mitochondrial activity and reactive oxygen species (ROS).ResultsCryopreservation significantly altered the abundance of 40 proteins; the abundance of 16 proteins was increased, while that of 24 proteins was decreased. Next, LTF was added to Tris extender applied to ram sperm. The results showed that sperm motility and plasma membrane integrity were significantly improved (p<0.05) by supplementation with 10 μg/mL LTF compared to those in the control group. There was no significant difference in mitochondrial activity between the 0 μg/mL group and other groups (p>0.05). Supplementation of the cryoprotective extender with 10 μg/mL LTF led to decreased ROS levels compared with those in the control and other groups (p<0.05).ConclusionThe LTF is an important protein during cryopreservation, and the addition of 10 μg/mL LTF to a cryoprotective extender can significantly improve the function of frozen ram sperm.     

Seasonal variations in the composition of ram seminal plasma and its effect on frozen-thawed ram sperm

It has been proposed that seminal plasma (SP) in the extender or in post-thaw media can prevent and revert cold-shock damage in cryopreserved ram sperm; however, this was dependent on season. We evaluated sperm parameters from Frisian ram semen incubated for various intervals with SP from all seasons and stored at -18 or -196 degrees C. At both temperatures, SP from autumn or winter increased (P<0.05) sperm motility, whereas no SP, or SP from spring or summer, had no effect. However, neither viability nor membrane or acrosomal status were modified by SP. Thirteen SP proteins were bound to the sperm surface (16.1, 16.7, 17.4, 23.3, 25.2, 27.5, 35.0, 40.0, 49.0, 53.5, 55.5, 61.0, and 86.0kDa). The SP proteins that bound to sperm were affected by season, but not by conservation temperature. Sperm incubated with SP from autumn had increased concentrations of five proteins; two were identified (with specific antibodies) as RSVP14 and RSVP20. In conclusion, SP from autumn and winter improved sperm motility of frozen-thawed ram sperm, and storage of ram SP at -18 or -196 degrees C did not affect protein composition. The SP proteins that bound to the sperm surface may be responsible for sperm membrane stabilization and should be further investigated.     

Conserved ram seminal plasma proteins bind to the sperm membrane and repair cryopreservation damage

Whole seminal plasma (SP) enhances the function and fertility of frozen/thawed ram sperm. The objective of the current study was to investigate whether SP proteins capable of binding to molecules from the sperm plasma membrane were conserved among ram breeds, and whether these proteins were sufficient to overcome cryopreservation-induced reductions in sperm quality. Whole ram SP, obtained from rams of various breeds, improved progressive motility of frozen/thawed sperm at all times evaluated (P < 0.05); however, it did not improve total motility (15 min, P = 0.480; 30 min, P = 0.764; and 45 min, P = 0.795). To identify SP proteins responsible for this effect, a new method was developed to retain SP proteins that bound specifically to the sperm membrane by immobilization of sperm membrane proteins. These proteins specifically bound to the sperm surface, especially the acrosomal region. Lactotransferrin, epididymal secretory protein E1, Synaptosomal-associated protein 29, and RSVP-20 were identified (mass spectrometry) in this fraction. The retained SP proteins fraction repaired ultrastructural damage of frozen/thawed sperm and, with the addition of fructose, significantly improved motility of frozen/thawed sperm. We concluded that SP proteins that bound to the sperm membrane were conserved among ram breeds, and that when added to frozen/thawed semen (along with an energy source), they repaired ram sperm damage and enhanced sperm motility.     

Changes in sperm quality and lipid composition during cryopreservation of boar semen

Egg yolks are commonly used in diluents in order to improve the freezability of semen. Two aspects of the role of lipids in boar semen freezability are reported in this article. The first one concerns the eventual exchanges of lipid components between the spermatozoa and the yolk-based diluent during cryopreservation. Two types of yolk have been considered as ingredients in diluents for cryopreservation: yolks with a standard fatty acid composition and yolks enriched in docosahexaenoic acid (DHA). The relation between lipid exchanges and the quality of fresh semen is considered. The other aspect concerns the possibility to enhance the freezability of boar spermatozoa by altering the plasma membranes under the influence of dietary fatty acids. Spermatozoa were damaged significantly by the cryopreservation cycle in all experiments. Spermatozoa with the best fresh quality had accumulated the largest quantity of lipids upon thawing. A general decrease in the proportion of polyunsaturated fatty acids was observed after thawing. The yolks enriched in n-3 fatty acids failed to improve the quality of sperm following cryopreservation. The proportion of DHA was significantly higher in spermatozoan phospholipids from thawed cells that had been in contact with n-3 yolks. A significant reduction in cholesterol was observed in spermatozoa after the cryopreservation cycle, which correlated with an increased number of acrosome-reacted cells and changes in the parameters of motility. The addition of 3% fish oil to the daily boar ration significantly increased the content of DHA (from 33 to 45% of the total fatty acids) in the spermatozoa. Ejaculate concentrations were significantly increased in the experimental group. DHA-enriched semen did not show improved freezability, at least not as assessed by in vitro parameters.     

Endosomes differ from plasma membranes in the phospholipid molecular species composition

125I-Labeled epidermal growth factor was incorporated into and highly concentrated in endosomes of Chinese hamster V79-UF cells during incubation at 37 degrees C for 8 min after binding to its receptors on the cell surface at 4 degrees C. From the labeled cells, endosomes were isolated by isopycnic centrifugation on a Percoll density gradient and then sucrose density gradients. The isolated endosomes were mostly free from contamination by Golgi, endoplasmic reticulum, lysosome, plasma membrane and mitochondria. Endosome membranes were found to differ from plasma membranes in the phospholipid composition. Sphingomyelin and phosphatidylserine were enriched in endosomes, compared with plasma membranes. Diacylphosphatidylcholine and diacylphosphatidylethanolamine were major phospholipids of the membranes in both organelles. The contents of molecular species of diacylphosphatidylcholine and diacylphosphatidylethanolamine with two monoenoic fatty acids were lower in endosomes than in plasma membranes. The differences in the polar head group and molecular species compositions of phospholipids between endosomes and plasma membranes did not change, regardless of whether or not the proportions of phospholipid molecular species in plasma membranes changed. The significance of the lipids in endosomes is discussed.     

Enzyme and phospholipid asymmetry in liver microsomal membranes

The transverse distribution of enzyme proteins and phospholipids within microsomal membranes was studied by analyzing membrane composition after treatment with proteases and phospholipases. Upon trypsin treatment of closed microsomal vesicles, NADH- and NADPH-cytochrome c reductases as well as cytochrome b5 were solubilized or inactivated, while cytochrome P-450 was partially inactivated. When microsomes were exposed to a concentration of deoxycholate which makes them permeable to macromolecules but does not disrupt the membrane, the detergent alone was sufficient to release four enzymes: nucleoside diphosphatase, esterase, beta-glucuronidase, and a portion of the DT-diaphorase. Introduction of trypsin into the vesicle lumen inactivated glucose-6-phosphatase completely and cytochrome P-450 partially. The rest of this cytochrome, ATPase, AMPase, UDP-glucuronyltransferase, and the remaining 50% of DT-diaphorase activity were not affected by proteolysis from either side of the membrane. Phospholipase A treatment of intact microsomes in the presence of albumin hydrolyzed all of the phosphatidylethanolamine, phosphatidylserine, and 55% of the phosphatidylcholine. From this observation, it was concluded that these lipids are localized in the outer half of the bilayer of the microsomal membrane; Phosphatidylinositol, 45% of the phosphatidylcholine, and sphingomyelin are tentatively assigned to the inner half of this bilayer. It appears that the various enzyme proteins and phospholipids of the microsomal membrane display an asymmetric distribution in the transverse plane.     

Altered phospholipid composition of plasma membranes from thrombin-stimulated human platelets

The individual phospholipid concentrations, and their respective fatty acid distributions, in whole platelet lysates and plasma membranes derived from unstimulated and thrombin-stimulated intact human platelets were studied. This was of interest, since previous work had led to the suggestion that altered phospholipid concentrations in plasma membranes of intact stimulated cells may be of importance in mediating cellular responses. The concentrations (nmol/mg protein) of phosphatidylinositol in whole platelet lysates and plasma membranes derived from thrombin-activated platelets decreased by 37 and 45%, respectively, a compared to their corresponding controls. As well, concentrations of plasma membrane phosphatidylcholine and phosphatidylethanolamine in thrombin-stimulated platelets decreased by 20 and 9%, respectively, when compared with their control values. The amounts of phosphatidylserine and sphingomyelin in whole platelet lysates and plasma membranes were unchanged by exposure to thrombin. Fatty acid analyses revealed that thrombin stimulation of intact human platelets induced a decrease in the arachidonate content (from 37.7 to 33.1 wt.% of total fatty acid) of plasma membrane phosphatidylinositol. Similar shifts in the wt% of arachidonic acid in plasma membrane phosphatidylcholine were found. These results indicate that thrombin stimulation of intact human platelets produces a significant decrease in the mass of phosphatidylinositol in plasma membranes and raises the suggestion that the preferential depletion of the plasma membrane in arachidonoyl-containing phosphatidylinositol may be of importance in mediating cellular responses to external stimuli.     

Assessment of post-thawed ram sperm viability after incubation with seminal plasma

A suggested alternative to improve post-thawed ram semen quality is the addition of seminal plasma (SP). This is thought to be capable of improving sperm resistance to thermal shock, reverting cryocapacitation and helping sperm survival. The aim of this study was to evaluate the effect of frozen-thawed ram semen incubation with SP on mitochondrial activity, acrosomal membrane integrity, necrosis and apoptosis. Frozen/thawed semen was divided into two groups: the SP Group and the control group. After 0, 30 and 60 min, fluorescent probes were added to aliquots from each treatment group and evaluated using flow cytometry. There was no difference between treatment groups in almost all viability parameters evaluated, with exception of the apoptosis, which was found increased in SP group. The increase in incubation period resulted in a decreased percentage of sperm with high mitochondrial membrane potential and acrosomal membrane integrity and an increased percentage of necrotic and apoptotic sperm cells. In conclusion, the present study showed that addition of seminal plasma after thawing cryopreserved ram sperm had no identifiable beneficial effect on sperm quality.     

Effects of cryopreservation procedures on sperm membranes

Empirical approaches to semen cryopreservation have resulted in the production of young in a broad range of species. However, acceptable levels of fertility in most domestic animal species has not been achieved. In this review, an attempt has been made to describe the complexity of the sperm plasma membrane and the many steps in a cryopreservation procedure where membrane perturbations can occur. Improvement in sperm cryopreservation procedures will require a careful consideration of the complexity of the sperm plasma membrane, the interaction of its components and the influence of cooling, freezing and thawing on these interactions.     

Diffusion barriers in ram and boar sperm plasma membranes: directionality of lipid diffusion across the posterior ring

The plasma membrane of mammalian spermatozoa, like that of other differentiated cells, is compartmentalized into discrete regions or domains that are biochemically and functionally distinct from one another. Physical structures within the membrane, such as the posterior ring at the juncture of the sperm head and tail, have long been thought to act as diffusion barriers to help segregate important molecules required for fertilization within specific domains and to regulate migration of molecules between domains. In this investigation, we used a quantitative photobleaching technique (video-FRAP) to assess the efficacy of the posterior ring as a barrier to exchange of lipids between the postacrosomal and midpiece plasma membranes. A lipid reporter probe (1,1'-diduodecyl-3,3,3', 3'-tetramethylindocarbocyanine; DiIC(12)) was incorporated into the plasma membrane of live ram and boar spermatozoa, and the directionality of its diffusion across the posterior ring was measured by line-profile analysis. Results showed that DiIC(12) was able to traverse the posterior ring from the direction of the postacrosomal plasma membrane and to diffuse onto the midpiece plasma membrane. These results suggest that the posterior ring is not an immutable barrier to lipid exchange in mature spermatozoa and that there are other mechanisms for maintaining in-plane lipid asymmetry, such as differential phase behavior and interaction with the submembranous cytoskeleton.     

Functional and pathological significance of phospholipid asymmetry in erythrocyte membranes

The normal asymmetric distribution of phospholipids in the plasma membrane is perturbed in erythrocytes from patients with chronic myelogenous leukemia. Since experimentally-produced lipid-symmetric erythrocytes are more interactive with cells of the reticuloendothelial system than are their lipid-asymmetric counterparts, the biological recognition of chronic myelogenous leukemia erythrocytes by the reticuloendothelial system was examined. With one exception, all erythrocyte samples from patients with chronic/benign chronic myelogenous leukemia were more adherent to endothelial cells and more readily phagocytosed by macrophagesin vitro than were normal erythrocytes. Thus, these naturally occurring pathological erythrocytes display the same dysfunctional intercellular interactions as the laboratory models.     

Application of seminal plasma in sex-sorting and sperm cryopreservation

Substantial dilution of boar semen during processing decreased the concentration of seminal plasma, perhaps contributing to the decline in sperm quality after cryopreservation and sex-sorting. Results of replacing seminal plasma in investigations from many laboratories have been contradictory. Results and discussion here suggest that whereas membrane status can be influenced by seminal plasma, the action of its various components, both positive and negative, is determined in part by the membrane status of the spermatozoa to which it is being exposed. Although progress has been made in identifying components of seminal plasma responsible for its protective effect (notably PSP-I/II spermadhesin for sex-sorted boar spermatozoa), little is known (in any species) regarding how external factors may influence their levels, and their functionality, in seminal plasma. It is noteworthy that seminal plasma is beneficial to post-thaw quality of sex-sorted ram spermatozoa only when added before freezing, not after thawing. Therefore, the action of seminal plasma and its components is dependent on sperm-related factors, in particular the type of processing to which they have been previously exposed. Further research is needed to unravel these biological complexities, and then characterise and synthesise useful proteins within seminal plasma.     

Changes in the lipid content of boar sperm plasma membranes during epididymal maturation

Plasma membranes of boar sperm from caput, corpus and cauda of the epididymis were purified by differential- and sucrose-density equilibrium centrifugation and were found to yield a single band at a density of 1.13 g/cm3. This fraction was enriched in acid and alkaline phosphatase, 5'-nucleotidase and (Na+ + K+)-ATPase activities, whereas it contained minimal amounts of hyaluronidase and N-acetylglucosaminidase and no succinic acid dehydrogenase activities. The plasma membrane of caput, corpus and cauda sperm had the same phospholipid/protein and cholesterol/phospholipid ratios but yielded different amounts of protein and individual lipid classes. Several changes in the plasma membrane were observed during transit of sperm through the epididymis. Within the phospholipid class a decrease in the percentage of phosphatidylethanolamine, phosphatidylserine and phosphatidylinositol was detected accompanied by an increase in amount of phosphatidylcholine, sphingomyelin and polyphosphoinositides. In the other lipid classes there was a decrease in the amount of free fatty acid and the major glycolipid. The amount of cholesterol decreased, while the amount of desmosterol and cholesterol sulfate increased. There was an increase in the amount of diacylglycerol. In addition, the changes in the fatty acid composition of the total membrane lipid and each phospholipid were determined. The above changes in the lipid composition of the plasma membrane during epididymal maturation may help to explain the decreased resistance to cold shock and changes in membrane fluidity of sperm during transit in the epididymis.     

Idebenone improves quality of ram sperm by mitigating oxidative stress during cryopreservation

The present study was designed to test the effect of different levels of idebenone, a potent antioxidant on the quality of ram semen at post thaw. Eighteen (18) ejaculates were collected and extended with tris extender supplemented with no antioxidant (CON), with 2 μM idebenone (Id2), 5 μM idebenone (Id5), 7.5 μM idebenone (Id7.5) and 10 μM idebenone (Id10). The sperm quality was determined in terms of percent sperm motility, live sperm percentage, percent hypoosmotic swelling test (HOST) positive spermatozoa and percent intact acrosome (PIA). Moreover, malondialdehyde (MDA) level, an end product of lipid peroxidation (LPO) was also measured at post thaw both in seminal plasma and sperm cell. At post thaw, the percent sperm motility was significantly higher (p < 0.05) for Id10 as compared to Id2, Id5, Id7.5 and control. The live sperm percentage was non-significantly (p > 0.05) higher for Id10 as compared to control, Id5 and Id7.5 but significantly higher than Id2. The percent HOST positive spermatozoa was significantly higher (p < 0.05) for Id10 than control, Id2 and Id5. The MDA level in seminal plasma was significantly lower (p < 0.05) for Id10 than control and Id2. The MDA level in spermatozoa did show similar trend as in seminal plasma. Further, all the sperm parameters at all idebenone levels declined significantly from pre freeze to post thaw. In conclusion, idebenone at 10 μM level improved post thaw sperm quality by mitigating peroxidative stress, hence could be considered as a promising antioxidant additive for cryopreservation of ram semen.     

Cholesterol asymmetry in synaptic plasma membranes

Lipids are essential for the structural and functional integrity of membranes. Membrane lipids are not randomly distributed but are localized in different domains. A common characteristic of these membrane domains is their association with cholesterol. Lipid rafts and caveolae are examples of cholesterol enriched domains, which have attracted keen interest. However, two other important cholesterol domains are the exofacial and cytofacial leaflets of the plasma membrane. The two leaflets that make up the bilayer differ in their fluidity, electrical charge, lipid distribution, and active sites of certain proteins. The synaptic plasma membrane (SPM) cytofacial leaflet contains over 85% of the total SPM cholesterol as compared with the exofacial leaflet. This asymmetric distribution of cholesterol is not fixed or immobile but can be modified by different conditions in vivo: (i) chronic ethanol consumption; (ii) statins; (iii) aging; and (iv) apoE isoform. Several potential candidates have been proposed as mechanisms involved in regulation of SPM cholesterol asymmetry: apoE, low-density lipoprotein receptor, sterol carrier protein-2, fatty acid binding proteins, polyunsaturated fatty acids, P-glycoprotein and caveolin-1. This review examines cholesterol asymmetry in SPM, potential mechanisms of regulation and impact on membrane structure and function.