The lipid composition modulates the influence of the bovine seminal plasma protein PDC-109 on membrane stability

The bovine seminal plasma protein PDC-109 exerts an essential influence on the sperm cell plasma membrane during capacitation. However, by any mechanism, it has to be ensured that this function of the protein on sperm cells is not initiated too early, that is, upon ejaculation when PDC-109 and sperm cells come into first contact, but rather at later stages of sperm genesis in the female genital tract. To answer the question of whether changes of the bovine sperm lipid composition can modulate the effect of PDC-109 on sperm membranes, we have investigated the influence of PDC-109 on the integrity of (i) differently composed lipid vesicles and of (ii) membranes from human red blood cells and bovine spermatozoa. PDC-109 most effectively disturbed lipid membranes composed of choline-containing phospholipids and in the absence of cholesterol. The impact of the protein on lipid vesicles was attenuated in the presence of cholesterol or of noncholine-containing phospholipids, such as phosphatidylethanolamine or phosphatidylserine. An extraction of cholesterol from lipid or biological membranes using methyl-beta-cyclodextrin caused an increased membrane perturbation by PDC-109. Our results argue for a oppositional effect of PDC-109 during sperm cell genesis. We hypothesize that the lipid composition of ejaculated bull sperm cells allows a binding of PDC-109 without leading to an impairment of the plasma membrane. At later stages of sperm cell genesis upon release of cholesterol from sperm membranes, PDC-109 triggers a destabilization of the cells.     

Equine sperm membrane phase behavior: the effects of lipid-based cryoprotectants

The plasma membrane of sperm can undergo lipid phase separation during freezing, resulting in irreversible damage to the cell. The objective of our study was to examine the membrane phase behavior of equine spermatozoa in the absence and presence of lipid-based cryoprotectants. Biophysical properties of sperm membranes were investigated with Fourier-transform infrared spectroscopy. Compared to fresh untreated sperm, postthaw untreated sperm showed extensive lipid phase separation and rearrangement. In contrast, postthaw sperm that were cryopreserved in egg phosphatidylcholine (egg PC)- or soy phosphatidylcholine (soy PC)-based diluents showed similar lipid phase behavior to that of fresh, untreated sperm. Studies with a deuterium-labeled PC lipid (POPCd-31) suggest that exogenous lipid from the diluents are strongly associated with the sperm membrane, and scanning electron microscopy images of treated sperm show the presence of lipid aggregates on the membrane surface. Thus, the exogenous lipid does not appear to be integrated into the sperm membrane after cryopreservation. When compared to a standard egg-yolk-based diluent (INRA 82), the soy and egg PC media preserved viability and motility equally well in postthaw sperm. A preliminary fertility study determined that sperm cryopreserved in the soy PC-based medium were capable of fertilization at the same rate as sperm frozen in the conventional INRA 82 medium. Our results show that pure lipid-based diluents can prevent membrane damage during cryopreservation and perform as well as a standard egg-yolk-based diluent in preserving sperm viability, motility, and fertility.     

Évolution de la composante lipidique de la membrane plasmique des spermatozoïdes durant la maturation épididymaire

One aspect of mammalian post-testicular sperm maturation is the progressive change in their plasma membrane lipid composition. These modifications in lipids allow sperm cells to fuse with oocytes during fertilization. A significant share of these sperm lipid changes occurs during their descent through the epididymal tubule. It then continues within the female genital tract during the capacitation process, an essential prerequisite for acrosomic reaction and hence fertilization. This review presents what is known concerning the sperm plasma membrane lipid changes during epididymal maturation in various mammalian models. In the first section, after a brief presentation of the classic eukaryotic cell plasma membrane lipid organization, the emphasis is on the particularities of sperm plasma membrane lipids. The second section presents the different changes occurring in the three major classes of lipids (i.e. phospholipids, sterols and fatty acids) during the sperm’s epididymal descent. The final section briefly describes the mechanisms by which these lipid changes might happen in the epididymal lumen environment. The role played by lipid-rich vesicles secreted by the epididymal epithelium via apocrine secretory processes is highlighted.     

Composition lipidique membranaire durant la préparation de spermatozoïdes à la fécondation

The final modifications that the spermatozoa undergo correspond with the destabilization of their plasma membrane. This indispensable step facilitates the fusion of membranes and primes the signal transduction during fertilization. This destabilization is composed of a series of changes and modulation of the lipids in membranes such as cholestérol, phospholipids and glycolipids. Several differences exist in the lipid composition of the plasma, acrosome, nuclear and mitochondrial membranes of spermatozoa. The principal membrane phospholipids are phosphatidyl choline, phosphatidyl ethanolamine and sphingomyelin. Plasma membrane of sperm is also rich in polyunsaturated fatty acids (PUFA) linked to phospholipids. Such as C₁₈∶2n?6, C₂₀∶4n?6 and large amounts of docosahexaenoic acid (C₂₂∶6n?6). The amount of membrane lipids in human sperm varies considerably between patients. This variation, could influence certain functional properties of the sperm cells such as their ability to undergo capacitation, the acrosome reaction and the fusion between sperm and oocyte membranes. The lipid composition of the human sperm cell can be altered during the process of freezing-thawing. A significant decrease in phospholipids (phosphatidyl choline, phosphatidyl ethanolamine), and PUFA in particular docosahexaenoic acid and arachidonic acid was observed. Human spermatozoa have a molar cholestérol/phopholipid ratio ≤1.0, and reduces during capacitation due to loss of cholestérol. In addition, the decrease in the levels of cholestérol and the methylation of phospholipids is involved in the modification of membrane fluidity and in the maturation of the sperm plasma membrane receptors. Therefore it seems that the methylation is important for the fusion between sperm and oocyte membranes. Intrinsic sperm phospholipase A2 also plays a role in the destabilization of the plasma membrane by producing of lysophospholipid. Therefore this enzyme and free fatty acids are believed to play a role in the acrosome reaction, an indispensable event facilitating the fusion between sperm and oocyte membranes.     

Role of seminal plasma in damage to turkey spermatozoa during in vitro storage

In vitro storage of turkey spermatozoa is performed without consideration of the potential role of seminal plasma on sperm functions. We report the effects of seminal plasma on membrane permeability, lipid metabolism, energy status, motility and fertility of turkey spermatozoa stored at 4 or 20 degrees C. Phospholipid content (1077 nmol/10(9) spz versus 1219 nmol/10(9) spz at 48 h) and membrane permeability of spermatozoa were significantly damaged by the presence of seminal plasma after 48 h of storage at 4 degrees C, whereas damage to ATP content and fertility occurred earlier damaged by this presence (fertility after 24h storage 51% with seminal plasma versus 71% without). At 20 degrees C, seminal plasma decreased the phospholipid content of spermatozoa in the first hour of storage (1326 nmol/10(9) spz versus 1636 nmol/10(9) spz). Twenty-four hours later, this effect was masked by intense lipid peroxidation. These results show that seminal plasma is deleterious to storage of turkey spermatozoa at 4 degrees C and is involved in phospholipid metabolism of spermatozoa. Lipid peroxidation could be responsible for the acceleration of the degradation of sperm phospholipids during storage at 20 degrees C. However, lipid peroxidation seems not to be active at 4 degrees C. In this case, we suggest that phospholipase activation may contribute to sperm degradation, especially in the presence of seminal plasma.     

Capacitation-dependent reorganization of microdomains in the apical sperm head plasma membrane: functional relationship with zona binding and the zona-induced acrosome reaction

For sperm to successfully fertilize an oocyte, it needs to pass through certain steps prior to, during and after initial recognition of the zona pellucida (ZP). During capacitation, the surface of the sperm head becomes remodelled, priming it to bind to the ZP and subsequently to undergo the ZP-induced acrosome reaction. During capacitation, sperm ZP-binding proteins are ordered in functional protein complexes that only emerge at the apical tip of the sperm head plasma membrane; this is also functionally the exclusive sperm surface area involved in primary ZP binding. After primary ZP binding, the same area is probably involved in the induction of the acrosome reaction. A combination of biochemical and proteomic membrane protein techniques have enabled us to dissect and highly purify the apical sperm plasma membrane area from control and capacitated sperm cells. The actual ZP-binding proteins identified predominantly belonged to the sperm membrane-associated family members of spermadhesins (AQN-3) and were present in the aggregating lipid ordered membrane microdomains (lipid rafts) that emerged during in vitro capacitation in the apical ridge area of the sperm head plasma membrane. This clustering of these rafts was dependent on the presence of bicarbonate (involved in protein kinase A activation) and on the presence of albumin (involved in cholesterol removal). Remarkably, cholesterol removal was restricted to the non-raft membrane fraction of the sperm plasma membrane, but did not cause any depletion of cholesterol in the raft membrane fraction. Interestingly, sperm SNARE proteins (both VAMP from the outer acrosomal membrane, as well syntaxin from the apical sperm head plasma membrane) shared lateral redistribution properties, along with the ZP-binding protein complex and raft marker proteins. All of these were recovered after capacitation in detergent-resistant membrane preparations from sperm thought to represent membrane lipid rafts. We inferred that the capacitation-dependent formation of an aggregated lipid ordered apical ridge surface area in the sperm head plasma membrane was not only relevant for ZP-binding, but also for the ZP-induced acrosome reaction.     

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 Tm's 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.     

Prostasomes, dérivés actifs de l’oxygène et sperme humain

Prostasomes are particular lipid vesicles secreted by the human prostate and found in the semen. No specific action has yet been attributed to prostasomes, but they appear to act at various levels. For example, prostasomes enhance sperm motility in vitro and participate in the immunomodulation properties of seminal plasma. Excessive production of reactive oxygen species (ROS) in human semen has a negative influence on the functional capacities of spermatozoa. The presence of leukocytes in semen is associated with increased production of ROS that can be harmful to sperm cells, under certain conditions. Previous results tend to suggest a possible role of prostasomes on ROS production in human semen. After reviewing the literature concerning the structural and functional characteristics of prostasomes and the role of ROS in human semen, we report our results concerning the influence of prostasomes on ROS production and the consequences on semen. We have demonstrated that prostasomes exert an antioxidant function in human semen. This function is effective both on polymorphonuclear neutrophils and on sperm cells. The mechanism of action of prostasomes is unusual, as they act on ROS production mainly on the plasma membranes of neutrophils. They induce a decrease of NADPH-oxidase activity associated with rigidification of the plasma membrane. Prostasomes protect the functional capacities of spermatozoa during an oxidative stress created by the presence of NADPH in the incubation medium.     

Effects of metabolic rate and sperm competition on the fatty‐acid composition of mammalian sperm

The sperm membrane is a key structure affecting sperm function and thus reproductive success. Spermatozoa are highly specialized and differentiated cells that undergo a long series of processes in the male and female reproductive tracts until they reach the site of fertilization. During this transit, the sperm membrane is prone to damage such as lipid peroxidation. The characteristics and performance of the sperm membrane are strongly determined by the fatty‐acid composition of membrane phospholipids. Polyunsaturated fatty‐acids (PUFAs) are the most prone to lipid peroxidation. Lipid peroxidation and other types of oxidative damage increase with higher metabolism and with higher levels of sperm competition due to the increased ATP production to fuel higher sperm velocities. Consequently, we hypothesized that, in order to avoid oxidative damage, and the ensuing impairment of sperm function, sperm cells exhibit a negative relationship between PUFA content and mass‐specific metabolic rate (MSMR). We also hypothesized that higher sperm competition leads to a reduction in the proportion of sperm PUFAs. We performed a comparative study in mammals and found that high MSMR and high levels of sperm competition both promote a decrease in the proportion of PUFAs that are more prone to lipid peroxidation. The negative relationship between MSMR and these PUFAs in sperm cells is surprising, because a positive relationship is found in all other cell types so far investigated. Our results support the idea that the effects of MSMR and sperm competition on sperm function can operate at very different levels.     

Diffusion and regionalization in membranes of maturing ram spermatozoa

An essential feature of the "fluid mosaic model" (Singer, S. J., and G. L. Nicolson , 1972, Science (Wash. DC)., 175:720-731) of the cell plasma membrane is the ability of membrane lipids and proteins to diffuse laterally in the plane of the membrane. Mammalian sperm are capable of overcoming free random diffusion and restricting specific membrane components, both lipid and protein, to defined regions of the sperm's surface. The patterns of these regionalizations evolve with the processes of sperm differentiation: spermatogenesis, epididymal maturation, and capacitation. We have used the technique of fluorescence recovery after photobleaching to measure the diffusion of the lipid analogue 1,1'- dihexadecyl 3,3,3',3'- tetramethylindocarbocyanine perchlorate ( C16dil ) on the different morphological regions of testicular and ejaculated ram spermatozoa. We have found: (a) that the major morphologically distinct regions (head, midpiece, and tail) of the plasma membrane of both testicular and ejaculated spermatozoa are also physically distinct as measured by C16dil diffusibility; (b) that despite regional differences in diffusibility there is exchange of this lipid analogue by lateral diffusion between the major morphological regions of the plasma membrane; and (c) that epididymal maturation results in changes in C16dil diffusibility in the different regions of the sperm plasma membrane. In particular, the plasma membranes of the anterior and posterior heads become physically distinct.     

Interleukin (IL)-1, IL-6, and IL-8 predict mucosal toxicity of vaginal microbicidal contraceptives

Inflammation of the female reproductive tract increases susceptibility to HIV-1 and other viral infections and, thus, it becomes a serious liability for vaginal products. Excessive release of proinflammatory cytokines may alter the mucosal balance between tissue destruction and repair and be linked to enhanced penetration and replication of viral pathogens upon chemical insult. The present study evaluates four surface-active microbicide candidates, nonoxynol-9 (N-9), benzalkonium chloride (BZK), sodium dodecyl sulfate, and sodium monolaurate for their activity against human sperm and HIV, and their capacity to induce an inflammatory response on human vaginal epithelial cells and by the rabbit vaginal mucosa. Spermicidal and virucidal evaluations ranked N-9 as the most potent compound but were unable to predict the impact of the compounds on vaginal cell viability. Interleukin (IL)-1 release in vitro reflected their cytotoxicity profiles more accurately. Furthermore, IL-1 concentrations in vaginal washings correlated with cumulative mucosal irritation scores after single and multiple applications (P < 0.01), showing BZK as the most damaging agent for the vaginal mucosa. BZK induced rapid cell death, IL-1 release, and IL-6 secretion. The other compounds required either more prolonged or repeated contact with the vaginal epithelium to induce a significant inflammatory reaction. Increased IL-8 levels after multiple applications in vivo identified compounds with the highest cumulative mucosal toxicity (P < 0.01). In conclusion, IL-1, IL-6, and IL-8 in the vaginal secretions are sensitive indicators of compound-induced mucosal toxicity. The described evaluation system is a valuable tool in identifying novel vaginal contraceptive microbicides, selecting out candidates that may enhance, rather than decrease, HIV transmission.     

Membrane processes and biophysical characterization of living cells decorated with chromatic polydiacetylene vesicles

The structural complexity of the cell membrane makes analysis of membrane processes in living cells, as compared to model membrane systems, highly challenging. Living cells decorated with surface-attached colorimetric/fluorescent polydiacetylene patches might constitute an effective platform for analysis and visualization of membrane processes in situ. This work examines the biological and chemical consequences of plasma membrane labeling of promyelocytic leukemia cells with polydiacetylene. We show that the extent of fusion between incubated lipid/diacetylene vesicles and the plasma membrane is closely dependent upon the lipid composition of both vesicles and cell membrane. In particular, we find that cholesterol presence increased bilayer fusion between the chromatic vesicles and the plasma membrane, suggesting that membrane organization plays a significant role in the fusion process. Spectroscopic data and physiological assays show that decorating the cell membrane with the lipid/diacetylene patches reduces the overall lateral diffusion within the membrane bilayer, however polydiacetylene labeling does not adversely affect important cellular metabolic pathways. Overall, the experimental data indicate that the viability and physiological integrity of the surface-engineered cells are retained, making possible utilization of the platform for studying membrane processes in living cells. We demonstrate the use of the polydiacetylene-labeled cells for visualizing and discriminating among different membrane interaction mechanisms of pharmaceutical compounds.     

Biophysical Investigation of the Membrane-Disrupting Mechanism of the Antimicrobial and Amyloid-Like Peptide Dermaseptin S9

Dermaseptin S9 (Drs S9) is an atypical cationic antimicrobial peptide with a long hydrophobic core and with a propensity to form amyloid-like fibrils. Here we investigated its membrane interaction using a variety of biophysical techniques. Rather surprisingly, we found that Drs S9 induces efficient permeabilisation in zwitterionic phosphatidylcholine (PC) vesicles, but not in anionic phosphatidylglycerol (PG) vesicles. We also found that the peptide inserts more efficiently in PC than in PG monolayers. Therefore, electrostatic interactions between the cationic Drs S9 and anionic membranes cannot explain the selectivity of the peptide towards bacterial membranes. CD spectroscopy, electron microscopy and ThT fluorescence experiments showed that the peptide adopts slightly more β-sheet and has a higher tendency to form amyloid-like fibrils in the presence of PC membranes as compared to PG membranes. Thus, induction of leakage may be related to peptide aggregation. The use of a pre-incorporation protocol to reduce peptide/peptide interactions characteristic of aggregates in solution resulted in more α-helix formation and a more pronounced effect on the cooperativity of the gel-fluid lipid phase transition in all lipid systems tested. Calorimetric data together with ²H- and ³¹P-NMR experiments indicated that the peptide has a significant impact on the dynamic organization of lipid bilayers, albeit slightly less for zwitterionic than for anionic membranes. Taken together, our data suggest that in particular in membranes of zwitterionic lipids the peptide binds in an aggregated state resulting in membrane leakage. We propose that also the antimicrobial activity of Drs S9 may be a result of binding of the peptide in an aggregated state, but that specific binding and aggregation to bacterial membranes is regulated not by anionic lipids but by as yet unknown factors.     

Spermatozoal response to osmotic stress

The process of sperm cryopreservation imparts on sperm cells the stress of low-temperature and drastic osmotic change. Damage to the cell plasma membrane results in cell injury in a number of cellular structures and associated functions. Studies in the author's laboratory have focused upon the various mechanisms of osmotic and thermal injury including plasma membrane lipid structure, mitochondrial membrane potential, and intracellular signaling. We have determined that cryoinjury to sperm, as for somatic cells, is a multi-factorial event and some of these events are reversible while some are not.     

Membrane processes and biophysical characterization of living cells decorated with chromatic polydiacetylene vesicles

The structural complexity of the cell membrane makes analysis of membrane processes in living cells, as compared to model membrane systems, highly challenging. Living cells decorated with surface-attached colorimetric/fluorescent polydiacetylene patches might constitute an effective platform for analysis and visualization of membrane processes in situ. This work examines the biological and chemical consequences of plasma membrane labeling of promyelocytic leukemia cells with polydiacetylene. We show that the extent of fusion between incubated lipid/diacetylene vesicles and the plasma membrane is closely dependent upon the lipid composition of both vesicles and cell membrane. In particular, we find that cholesterol presence increased bilayer fusion between the chromatic vesicles and the plasma membrane, suggesting that membrane organization plays a significant role in the fusion process. Spectroscopic data and physiological assays show that decorating the cell membrane with the lipid/diacetylene patches reduces the overall lateral diffusion within the membrane bilayer, however polydiacetylene labeling does not adversely affect important cellular metabolic pathways. Overall, the experimental data indicate that the viability and physiological integrity of the surface-engineered cells are retained, making possible utilization of the platform for studying membrane processes in living cells. We demonstrate the use of the polydiacetylene-labeled cells for visualizing and discriminating among different membrane interaction mechanisms of pharmaceutical compounds.     

Characterization of sperm plasma membrane properties after cholesterol modification: consequences for cryopreservation of rainbow trout spermatozoa

During cryopreservation, the cell plasma membrane faces severe perils, including lipid phase separation, solute effects, and osmotic stresses associated with ice crystallization. How the initial biophysical properties of the plasma membrane can be modulated before cryopreservation in order to influence cellular resistance to the freeze-thaw stress is addressed in this study. Rainbow trout (Oncorhynchus mykiss) spermatozoa were chosen because the lack of an acrosome in this species suppresses potential interactions of cryopreservation with capacitation. Methyl-beta cyclodextrin-induced modulation of membrane cholesterol revealed the presence of a significant cholesterol exchangeable pool in the trout sperm plasma membrane, as membrane cholesterol content could be halved or doubled with respect to the basic composition of the cell without impairing fresh sperm motility and fertilizing ability. Biophysical properties of the sperm plasma membrane were affected by cholesterol changes: membrane resistance to a hypo-osmotic stress increased linearly with membrane cholesterol whereas membrane fluidity, assessed with DPH (1,6-diphenyl-1,3,5-hexatriene) and with several spin-labeled analogues of membrane lipids, decreased. Phosphatidyl serine translocation between the bilayers was slowed at high cholesterol content. The increased cohesion of fresh trout sperm plasma membrane as cholesterol increased did not improve the fertilizing ability of frozen-thawed sperm whereas the lowest cholesterol contents impaired this parameter of sperm quality. Our study demonstrated that cholesterol induced a stabilization of the plasma membrane in rainbow trout spermatozoa, but this stabilization before cryopreservation brought no improvement to the poor freezability of this cell.     

Fatty acid and peptide profiles in plasma membrane and membrane rafts of PUFA supplemented RAW264.7 macrophages

The eukaryotic cell membrane possesses numerous complex functions, which are essential for life. At this, the composition and the structure of the lipid bilayer are of particular importance. Polyunsaturated fatty acids may modulate the physical properties of biological membranes via alteration of membrane lipid composition affecting numerous physiological processes, e.g. in the immune system. In this systematic study we present fatty acid and peptide profiles of cell membrane and membrane rafts of murine macrophages that have been supplemented with saturated fatty acids as well as PUFAs from the n-3, the n-6 and the n-9 family. Using fatty acid composition analysis and mass spectrometry-based peptidome profiling we found that PUFAs from both the n-3 and the n-6 family have an impact on lipid and protein composition of plasma membrane and membrane rafts in a similar manner. In addition, we found a relation between the number of bis-allyl-methylene positions of the PUFA added and the unsaturation index of plasma membrane as well as membrane rafts of supplemented cells. With regard to the proposed significance of lipid microdomains for disease development and treatment our study will help to achieve a targeted dietary modulation of immune cell lipid bilayers.     

Improving sperm cryopreservation with antifreeze proteins: effect on gilthead seabream (Sparus aurata) plasma membrane lipids

Changes in the plasma membrane lipid composition have been related to a decrease in sperm quality during cryopreservation. Antifreeze proteins (AFPs) have been tested in different species because of their ability to depress the freezing point and their potential interaction with membranes, but controversial effects were reported. In the present study we analyzed separately the lipid composition of two sperm membrane domains, head plasma membrane (HM) and flagellar membrane (FM), after cryopreservation with an extender containing 5% dimethyl sulfoxide (DMSO) either alone or with AFPI or AFPIII (1 μg/ml). We used sperm from a teleost, Sparus aurata, because the lack of acrosome avoids changes of lipid profiles due to capacitation process or acrosomal losses during freezing/thawing. Comparing with the control (cryopreservation with 5% DMSO alone), the addition of AFPIII increased the velocity, linearity of movement, and percentage of viable cells. In addition, freezing with DMSO alone increased the phosphatidyl-serine content as well as the saturated fatty acids and decreased the unsaturated ones (mainly polyunsaturated) both in HM and FM. These changes in the lipid components were highly avoided with the addition of AFPIII. HM had a higher amount of saturated fatty acids than FM and was more affected by cryopreservation without AFPs. The percentage of viable cells was positively correlated with the amount of unsaturated fatty acids in the HM, whereas the motility parameters were positively correlated with both FM and HM amount of unsaturated fatty acids. AFPs, especially AFPIII, seem to have interacted with unsaturated fatty acids, stabilizing the plasma membrane organization during cryopreservation and contributing to improve sperm quality after thawing.     

Bicarbonate Induces Membrane Reorganization and CBR1 and TRPV1 Endocannabinoid Receptor Migration in Lipid Microdomains in Capacitating Boar Spermatozoa

Mammalian spermatozoa acquire full fertilizing ability only after a morphofunctional maturation called "capacitation." During this process the high level of bicarbonate present within the upper female genital tract or in culture medium induces a marked reorganization of sperm membranes characterized by a biphasic behavior: In a few minutes, it promotes membrane phospholipid scrambling preliminary to the apical translocation of sterol that, 2-4?h later, enables spermatozoa to recognize zona pellucida after albumin-mediated cholesterol extraction. In the present research it was demonstrated that spermatozoa incubated with bicarbonate in protein-free media underwent a marked reorganization of lipid microdomains present in a detergent-resistant membrane fraction (DRM) isolated by ultracentrifugation on sucrose density gradient. In fact, bicarbonate exposed sperm (ES) cells, compared with ejaculated spermatozoa (nonexposed sperm [nES] cells), displayed an increase in protein DRM content and, in particular, in Cav-1 and CD55, markers of caveolae and lipid rafts, as well in acrosin-2, a marker of the outer acrosomal membrane (OAM). Moreover, the amount of certain proteins involved in capacitation, such as the endocannabinoid system receptors cannabinoid receptor type 1 (CBR1) and transient receptor potential cation channel 1 (TRPV1), increased in DRM obtained from ES. These data allow us to hypothesize that sperm membrane reorganization takes place even in the absence of extracellular proteins; that not only the plasma membrane but also the OAM participate in this process; and that important molecules playing a key role in inside-out signaling, such as the endocannbinoid receptors TRPV1 and CBR1, are involved in this event, with potentially important consequences on sperm function.     

脂筏的结构与功能

脂筏是膜脂双层内含有特殊脂质及蛋白质的微区.小窝是脂筏的一种类型,由胆固醇、鞘脂及蛋白质组成,以小窝蛋白为标记蛋白.脂筏的组分和结构特点有利于蛋白质之间相互作用和构象转化,可以参与信号转导和细胞蛋白质运转.一些感染性疾病、心血管疾病、肿瘤、肌营养不良症及朊病毒病等可能与脂筏功能紊乱有着密切的关系.