Reorganization of lipid rafts during capacitation of human sperm

Ejaculated mammalian sperm must complete a final maturation, termed capacitation, before they can undergo acrosomal exocytosis and fertilize an egg. In human sperm, loss of sperm sterol is an obligatory, early event in capacitation. How sterol loss leads to acrosomal responsiveness is unknown. These experiments tested the hypothesis that loss of sperm sterol affects the organization of cold detergent-resistant membrane microdomains (lipid "rafts"). The GPI-linked protein CD59, the ganglioside GM1, and the protein flotillin-2 were used as markers for lipid rafts. In uncapacitated sperm, 51% of the CD59, 41% of the GM1, and 90% of the flotillin-2 were found in the raft fraction. During capacitation, sperm lost 67% of their 3beta-hydroxysterols, and the percentages of CD59 and GM1 in the raft fraction decreased to 34% and 31%, respectively. The distribution of flotillin-2 did not change. Preventing a net loss of sperm sterol prevented the loss of CD59 and GM1 from the raft fraction. Fluorescence microscopy showed CD59 and GM1 to be distributed over the entire sperm surface. Flotillin-2 was located mainly in the posterior head and midpiece. Patching using bivalent antibodies indicated that little of the GM1 and CD59 was stably associated in the same membrane rafts. Likewise, GM1 and flotillin-2 were not associated in the same membrane rafts. In summary, lipid rafts of heterogeneous composition were identified in human sperm and the two raft components, GM1 and CD59, showed a partial sterol loss-dependent shift to the nonraft domain during capacitation.     

Cholesterol content regulates acrosomal exocytosis by enhancing Rab3A plasma membrane association

The acrosome is an exocytic granule that overlies the spermatozoan nucleus. In response to different stimuli, it undergoes calcium-regulated exocytosis. Freshly ejaculated mammalian sperm are not immediately capable of undergoing acrosome reaction. The acquisition of this ability is called capacitation and involves a series of still not well-characterized changes in the sperm physiology. Plasma membrane cholesterol removal is one of the sperm modifications that are associated with capacitation. However, how sterols affect acrosomal exocytosis is unknown. Here, we show that short incubations with cyclodextrin, a cholesterol removal agent, just before stimulation promote acrosomal exocytosis. Moreover, the effect was also observed in permeabilized cells stimulated with calcium, indicating that cholesterol plays a direct role in the calcium-dependent exocytosis associated with acrosome reaction. Using a photo-inhibitable calcium chelator, we show that cholesterol affects an early event of the exocytic cascade rather than the lipid bilayers mixing. Functional data indicate that one target for the cholesterol effect is Rab3A. The sterol content does not affect the Rab3A activation-deactivation cycle but regulates its membrane anchoring. Western blot analysis and immunoelectron microscopy confirmed that cholesterol efflux facilitates Rab3A association to sperm plasma membrane. Our data indicate that the cholesterol efflux occurring during capacitation optimizes the conditions for the productive assembly of the fusion machinery required for acrosome reaction.     

Albumin-mediated changes in sperm sterol content during capacitation

The role of albumin in mouse sperm capacitation was studied in relation to its activities as a lipid-solubilizing protein and a sterol acceptor. Two bovine serum albumins (BSA) which supported capacitation, Fraction V and fatty acid-free, both contained cholesterol and phospholipid but were without detectable levels of serum high-density lipoprotein (HDL). The lipid content of BSA could be reduced by trichloroacetic acid (TCA) precipitation; however, removal of all detectable lipids required precipitation with ethanolic acetone and diethyl ether extraction. In medium supplemented with Fraction V, fatty acid-free, or TCA-precipitated BSA, mouse sperm were capacitated as evidenced by their ability to fertilize eggs, concomitant with decreases in total cellular sterol and increases in phospholipid content. Delipidated BSA, fractionated on Sephadex G-100 in guanidine HCl also supported capacitation and mediated a 20% decrease in sperm sterol content, while cellular phospholipid levels remained unchanged. When BSA was modified by cholesterol augmentation, fertilization was inhibited in a cholesterol dose-dependent manner. These findings suggest that modulation of sperm lipid levels comprises an event of capacitation and that albumin mediates this process through its activity as a sterol acceptor.     

Structural features of sterols required to inhibit human sperm capacitation

Ejaculated mammalian sperm must undergo a final maturation (capacitation) before they can acrosome-react and fertilize eggs. Loss of cholesterol is an essential step in the capacitation of human sperm. Experimentally maintaining a high level of cholesterol inhibits capacitation, but the mechanism is unknown. The present study investigated the structural features that are required for cholesterol's inhibitory activity. Human sperm also contain much desmosterol, which is lost from sperm during capacitation. Preventing the loss of desmosterol inhibited capacitation (as assessed by acrosomal responsiveness), with an effectiveness approximately equal to cholesterol's inhibitory activity. Other structural analogs were added to the incubation medium to replace sperm cholesterol and desmosterol. Most inhibited capacitation, including those that lacked cholesterol's 3beta-OH group (cholesteryl methyl ether and epicholesterol) and those with modified C17 groups (ergosterol and diosgenin). Two steroids did not inhibit capacitation well. Coprostanol, which has a nonplanar steroid nucleus, had low inhibitory activity that could be explained by an elevated endogenous cholesterol concentration. Epicoprostanol, which has a nonplanar ring structure and a 3alpha-OH group, promoted rather than inhibited capacitation. The inhibitory activity of the analogs was correlated with their ability to promote order of egg phosphatidylcholine as measured by fluorescence anisotropy. In summary, a planar ring structure is required for sterol inhibitory activity, but a 3beta-OH group and a saturated cholesterol-like aliphatic tail on C17 are not required. The present results support the hypothesis that sperm sterols block capacitation by increasing order of phospholipids.     

Isolation and proteomic analysis of mouse sperm detergent-resistant membrane fractions: evidence for dissociation of lipid rafts during capacitation

Mammalian sperm acquire fertilization capacity after residing in the female tract during a process known as capacitation. The present study examined whether cholesterol efflux during capacitation alters the biophysical properties of the sperm plasma membrane by potentially reducing the extent of lipid raft domains as analyzed by the isolation of detergent-resistant membrane fractions using sucrose gradients. In addition, this work investigated whether dissociation of the detergent-resistant membrane fraction during capacitation alters resident sperm raft proteins. Mouse sperm proteins associated with such fractions were studied by silver staining, tandem mass spectrometry, and Western blot analysis. Caveolin 1 was identified in sperm lipid rafts in multimeric states, including a high-molecular-weight oligomer that is sensitive to degradation under reducing conditions at high pH. Capacitation resulted in reduction of the light buoyant-density, detergent-resistant membrane fraction and decreased the array of proteins isolated within this fraction, including loss of the high-molecular-weight caveolin 1 oligomers. Proteomic analysis of sperm proteins isolated in the light buoyant-density fraction identified several proteins, including hexokinase 1, testis serine proteases 1 and 2, TEX101, hyaluronidase (PH20, SPAM1), facilitated glucose transporter 3, lactate dehydrogenase A, carbonic anhydrase IV, IZUMO, pantophysin, basigin, and cysteine-rich inhibitory secretory protein 1. Capacitation also resulted in a significant reduction of sperm labeling by the fluorescent lipid-analog DiIC16, indicating that capacitation alters the liquid-ordered domains in the sperm plasma membrane. The observations that capacitation alters the protein composition of the detergent-resistant membrane fractions is consistent with the hypothesis that cholesterol efflux during capacitation dissociates lipid raft constituents, initiating signaling events that lead to sperm capacitation.     

Identification of sterol acceptors that stimulate cholesterol efflux from human spermatozoa during in vitro capacitation

The nature of cholesterol-binding proteins acting upon human spermatozoa during in vitro capacitation was determined by measuring the efflux of [³H]cholesterol and of [³H]cholesteryl sulfate from labeled spermatozoa. Efflux of [³H]sterols was stimulated when the labeled gametes were incubated in Ham's F-10 medium supplemented with female serum or follicular fluid. Upon centrifugation of capacitated spermatozoa and application of the supernatant to density-gradient ultracentrifugation for lipoprotein analysis, both [³H]cholesterol and [³H]cholesteryl sulfate were found to be carried by very-low-density lipoproteins (VLDL), low-density lipoproteins (VLDL), high-density lipoproteins (HDL), as well as the albumin fraction (d > 1.21) in serum. When the capacitation medium was supplemented with follicular fluid, the [³H]sterols were bound to HDL's and to the albumin fraction; when the latter fraction was analysed by molecular sieve chromatography, 60–70% of the radioactivity eluted in fractions with a mean molecular weight corresponding to that of human serum albumin. Sperm cholesterol efflux was also stimulated when serum or follicular fluid was added to a simplified medium (50 mM Tris-HCl, 0.56% NaCl, pH 7.8); efflux of [³H]cholesterol from labeled gametes progressed in a time-dependent manner, but was low in the absence of serum components. The [³H]cholesterol/cholesterol ratios were higher in the albumin and HDL fractions, indicating some degree of specificity of these sterol acceptors. It was observed that follicular fluid albumin has a [³H]sterol binding capacity that is 2—3-fold higher than that of serum albumin. Commercial human serum albumin also promoted sperm cholesterol efflux. These results provide new information concerning those components of follicular fluid which may play a role in human sperm capacitation and provide further support for the concept that loss of cholesterol from the sperm plasma membrane is an important component of the capacitation process.     

Lipid changes of goat sperm plasma membrane during epididymal maturation

Highly purified plasma membranes of maturing goat caput-, corpus- and cauda-epididymal spermatozoa were isolated by aqueous two-phase polymer methods and their lipid constituents were analysed. Phospholipid (approx. 75% w/w), neutral lipid (approx. 15% w/w) and glycolipid (approx. 10% w/w) were the major sperm membrane lipids. There was a significant decrease in the total lipids (approx. 25% w/w), phospholipid (approx. 30% w/w) and glycolipid (approx. 80% w/w) contents of sperm membrane during epididymal maturation. On the contrary, the mature cauda-sperm membrane showed greater (approx. 50% w/w) neutral lipid content than that of the immature caput sperm. Phosphatidylcholine (PC), phosphatidylethanolamine (PE) and sphingomyelin were the phospholipids of the sperm membrane, the former two being the major lipids. Both PC and PE fractions consisted of three species--diacyl, alkylacyl and alkenylacyl forms, the last one being the dominant species in both PC and PE. Of all the phospholipids, diacyl PE decreased most strikingly (approx. 65% w/w) during sperm maturation. The neutral lipid fraction contained sterols, wax esters, 1-O-alkyl-2,3-diacylglycerol, triacylglycerol and fatty acids. Sterols represented nearly 75% w/w of the neutral lipids and cholesterol was the major component (approx. 95% w/w) of the sterol fraction. The sperm maturity was associated with marked increase of sterol (approx. 60% w/w) and steryl ester (approx. 200% w/w) and decrease (approx. 50-65% w/w) of the other membrane-bound neutral lipids. The glycolipid was identified as monogalactosyldiacylglycerol. The fatty acid profile of the various membrane lipids underwent marked alteration during the epididymal transit of the male gametes. Cholesterol/phospholipid and saturated/unsaturated fatty acid ratios increased greatly in the maturing sperm membrane. The altered lipid profile of the mature sperm membrane leads to changes in its fluidity that play an important role in determining the structure and functions of the biomembrane.     

Sphingomyelin modulates capacitation of human sperm in vitro

Ejaculated mammalian sperm must mature (capacitate) before they can undergo acrosomal exocytosis and fertilize an egg. Loss of sperm sterols is an early step in capacitation. Because sphingomyelin slows cholesterol efflux from other cells, the role of sphingomyelin in capacitation was tested. Human sperm were exposed to sphingomyelinase and then incubated for as long as 24 h. The ability of sperm to acrosome-react in response to progesterone was tested to measure capacitation. Sphingomyelinase-treated sperm became responsive to progesterone approximately 10 h earlier than control sperm. Sphingomyelinase also increased spontaneous acrosomal exocytosis. The effects of sphingomyelinase were accompanied by accelerated losses of the inhibitory sterols, cholesterol and desmosterol. To test whether sphingomyelinase-generated ceramide might promote capacitation, sperm were incubated for 8 h with the cell-permeable ceramide N:-hexanoylsphingosine (25 microM) or with solvent. Ceramide increased the incidence of progesterone-responsive sperm and, at later times, spontaneously reacted sperm. N:-Hexanoylsphinganine, an inactive control ceramide, had no effect. These results suggest that sphingomyelin in the sperm influences the rate of capacitation by slowing the loss of sterols, and that exogenous sphingomyelinase accelerates capacitation by speeding the loss of sterols and by generating ceramide.     

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.     

A molecular membrane model of sperm capacitation and the acrosome reaction of mammalian spermatozoa

The abundance of data pertaining to the metabolism of lipids in relation to mammalian fertilization has warranted an effort to assemble a molecular membrane model for the comprehensive visualization of the biochemical events involved in sperm capacitation and the acrosome reaction. Derived both from earlier models as well as from current concepts, our membrane model depicts a lipid bilayer assembly of space-filling molecular models of sterols and phospholipids in dynamic equilibrium with peripheral and integral membrane proteins. A novel feature is the possibility of visualizing individual lipid molecules such as phosphatidylcholine, phosphatidylethanolamine, lysophospholipids, fatty acids, and free or esterified cholesterol. The model illustrates enzymatic reactions which are believed to regulate the permeability and integrity of the plasma membrane overlying the acrosome during interactions between the male gamete and capacitation factors present in fluids of the female genital tract. The use of radioactive lipids as molecular probes for monitoring the metabolism of cholesterol and phosphatidylcholine revealed the presence of (1) steroid sulfatase in hamster cumulus cells, (2) lecithin: cholesterol acyltransferase in human follicular fluid, (3) phospholipase A₂, and (4) lysophospholipase in human spermatozoa. These enzymatic reactions can be integrated into a pathway that provides a link between the concepts of lysophospholipid accumulation in the sperm membranes and alteration of the cholesterol/phospholipid ratio as factors involved in the preparation of the membranes for the acrosome reaction. Capacitation is viewed as a reversible phenomenon which, upon completion, results in a decrease in negative surface charge, an efflux of membrane cholesterol, and an influx of calcium between the plasma and outer acrosomal membranes. Triggered by the entry of calcium, the acrosome reaction involves phospholipase A₂ activation followed by a transient accumulation of unsaturated fatty acids and lysophospholipids implicated in membrane fusion which occurs during the formation of membrane vesicles in spermatozoa undergoing the acrosome reaction.     

Plant sterol or stanol esters retard lesion formation in LDL receptor-deficient mice independent of changes in serum plant sterols

Statins do not always decrease coronary heart disease mortality, which was speculated based on increased serum plant sterols observed during statin treatment. To evaluate plant sterol atherogenicity, we fed low density lipoprotein-receptor deficient (LDLr(+/-)) mice for 35 weeks with Western diets (control) alone or enriched with atorvastatin or atorvastatin plus plant sterols or stanols. Atorvastatin decreased serum cholesterol by 22% and lesion area by 57%. Adding plant sterols or stanols to atorvastatin decreased serum cholesterol by 39% and 41%. Cholesterol-standardized serum plant sterol concentrations increased by 4- to 11-fold during sterol plus atorvastatin treatment versus stanol plus atorvastatin treatment. However, lesion size decreased similarly in the sterol plus atorvastatin (-99% vs. control) and the stanol plus atorvastatin (-98%) groups, with comparable serum cholesterol levels, suggesting that increased plant sterol concentrations are not atherogenic. Our second study confirms this conclusion. Compared with lesions after a 33 week atherogenic period, lesion size further increased in controls (+97%) during 12 more weeks on the diet, whereas 12 weeks with the addition of plant sterols or stanols decreased lesion size (66% and 64%). These findings indicate that in LDLr(+/-) mice 1) increased cholesterol-standardized serum plant sterol concentrations are not atherogenic, 2) adding plant sterols/stanols to atorvastatin further inhibits lesion formation, and 3) plant sterols/stanols inhibit the progression or even induce the regression of existing lesions.     

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.     

Studies on the mechanism of capacitation. II. Evidence for lipid transfer between plasma membrane of rat sperm and serum albumin during capacitation in vitro

1. Evidence has been provided for the transfer of phosphatidyl[14C]choline and [3H]cholesterol between bovine serum albumin and cauda epididymal rat spermatozoa in Krebs-Ringer bicarbonate medium, which can promote sperm capacitation. 2. An analysis of the lipid composition in both albumin and spermatozoa revealed that phospholipid levels decreased in the protein and increased by roughly comparable amounts in sperm cells during incubation in vitro. 3. Cholesterol (free + ester) increased in albumin and decreased in spermatozoa. Changes in the amount of esterified cholesterol were solely responsible for the increase associated with albumin, whereas whole sperm cell extracts showed a significant decline in free cholesterol. 4. The composition of albumin-bound fatty acids did not alter appreciably as a result of incubation with spermatozoa. 5. Rates of [14C]palmitic acid utilization by spermatozoa suggest that lipid synthesis accounted for less than 5% of the changes observed under the conditions of this study. 6. These results are interpreted as broadly supporting our previous proposal that lipid exchange between albumin and sperm cells is implicated in sperm capacitation in vitro. Specifically, the results are compatible with the idea that a decreased cholesterol/phospholipid ratio in the sperm plasma membrane facilitates this transformation.     

Cholesterol efflux alters lipid raft stability and distribution during capacitation of boar spermatozoa

A reduction in plasma membrane cholesterol is one of the early events that either triggers or is closely associated with capacitation of mammalian spermatozoa. In this investigation, we have examined the effects of cholesterol efflux on tyrosine phosphorylation, lipid diffusion, and raft organization in boar spermatozoa. Results show that a low level of cholesterol efflux, mediated by 5 mM methyl-beta-cyclodextrin (MBCD), enhances capacitation and induces phosphorylation of two proteins at 26 and 15 kDa without affecting sperm viability. Lipid diffusion rates under these conditions are largely unaffected except when cholesterol efflux is excessive. Low-density Triton X100-insoluble complexes (lipid rafts) were isolated from spermatozoa and found to have a restricted profile of proteins. Capacitation-associated cholesterol efflux has no effect on raft composition, but cholesterol depletion destabilizes them completely and phosphorylation is suppressed. During MBCD-mediated capacitation, the distribution of GM1 gangliosides on spermatozoa changes in a sequential manner from overlying the sperm tail to clustering on the sperm head. It is concluded that there is a safe window for removal of plasma membrane cholesterol from spermatozoa within which protein phosphorylation and polarized migration of lipid rafts take place. A preferential loss of cholesterol from the nonraft pool may be the stimulus that promotes raft clustering over the anterior sperm head.     

Segregation of micron-scale membrane sub-domains in live murine sperm

Lipid rafts, membrane sub-domains enriched in sterols and sphingolipids, are controversial because demonstrations of rafts have often utilized fixed cells. We showed in living sperm that the ganglioside G(M1) localized to a micron-scale membrane sub-domain in the plasma membrane overlying the acrosome. We investigated four models proposed for membrane sub-domain maintenance. G(M1) segregation was maintained in live sperm incubated under non-capacitating conditions, and after sterol efflux, a membrane alteration necessary for capacitation. The complete lack of G(M1) diffusion to the post-acrosomal plasma membrane (PAPM) in live cells argued against the transient confinement zone model. However, within seconds after cessation of sperm motility, G(M1) dramatically redistributed several microns from the acrosomal sub-domain to the post-acrosomal, non-raft sub-domain. This redistribution was not accompanied by movement of sterols, and was induced by the pentameric cholera toxin subunit B (CTB). These data argued against a lipid-lipid interaction model for sub-domain maintenance. Although impossible to rule out a lipid shell model definitively, mice lacking caveolin-1 maintained segregation of both sterols and G(M1), arguing against a role for lipid shells surrounding caveolin-1 in sub-domain maintenance. Scanning electron microscopy of sperm freeze-dried without fixation identified cytoskeletal structures at the sub-domain boundary. Although drugs used to disrupt actin and intermediate filaments had no effect on the segregation of G(M1), we found that disulfide-bonded proteins played a significant role in sub-domain segregation. Together, these data provide an example of membrane sub-domains extreme in terms of size and stability of lipid segregation, and implicate a protein-based membrane compartmentation mechanism.     

The effect of sterol structure on membrane lipid domains reveals how cholesterol can induce lipid domain formation

Detergent-insoluble membrane domains, enriched in saturated lipids and cholesterol, have been implicated in numerous biological functions. To understand how cholesterol promotes domain formation, the effect of various sterols and sterol derivatives on domain formation in mixtures of the saturated lipid dipalmitoylphosphatidylcholine (DPPC) and a fluorescence quenching analogue of an unsaturated lipid was compared. Quenching measurements demonstrated that several sterols (cholesterol, dihydrocholesterol, epicholesterol, and 25-hydroxycholesterol) promote formation of DPPC-enriched domains. Other sterols and sterol derivatives had little effect on domain formation (cholestane and lanosterol) or, surprisingly, strongly inhibit it (coprostanol, androstenol, cholesterol sulfate, and 4-cholestenone). The effect of sterols on domain formation was closely correlated with their effects on DPPC insolubility. Those sterols that promoted domain formation increased DPPC insolubility, whereas those sterols that inhibit domain formation decreased DPPC insolubility. The effects of sterols on the fluorescence polarization of diphenylhexatriene incorporated into DPPC-containing vesicles were also correlated with sterol structure. These experiments indicate that the effect of sterol on the ability of saturated lipids to form a tightly packed (i.e., tight in the sense that the lipids are closely packed with one another) and ordered state is the key to their effect on domain formation. Those sterols that promote tight packing of saturated lipids promote domain formation, while those sterols that inhibited tight packing of saturated lipids inhibited domain formation. The ability of some sterols to inhibit domain formation (i.e., act as "anti-cholesterols") should be a valuable tool for examining domain formation and properties in cells.     

Bovine seminal plasma phospholipid-binding proteins stimulate phospholipid efflux from epididymal sperm.

Several studies have shown that sperm capacitation was accompanied by a change in the lipid composition of the sperm membrane. In cattle, the major proteins of (bovine)seminal plasma (BSP proteins: BSP-A1/A2, BSP-A3, and BSP-30-kDa) potentiate sperm capacitation induced by high-density lipoprotein (HDL). Our recent studies indicate that these proteins and HDL stimulate sperm cholesterol efflux during capacitation. In order to gain more insight into the mechanisms of BSP-mediated sperm capacitation, we studied whether or not BSP proteins induce phospholipid efflux from epididymal sperm membrane. By direct determination of choline phospholipids on unlabeled epididymal sperm, the results show that sperm incubated in the presence of BSP-A1/A2 protein lost 34.4% of their choline phospholipids compared with the control (11.5%). Similar results were obtained using labeled epididymal sperm. Labeling was carried out by incubating washed epididymal sperm for 1 h with medium containing [(3)H]palmitic acid. The majority of the label was incorporated into sperm phosphatidylcholine. Studies of sperm phospholipid efflux were done by incubating the labeled sperm with purified BSP proteins, delipidated BSA, or bovine seminal ribonuclease (RNase, control protein). When labeled ([(3)H]phospholipid) epididymal sperm were incubated with BSP proteins (20-120 microg/ml) for 8 h, the sperm lost [(3)H]phospholipid in a dose-dependent manner (maximum efflux of approximately 30%). After the incubation with BSP proteins, the efflux particles were fractionated by size-exclusion chromatography. Analysis of the fractions obtained showed that the [(3)H]phospholipid was associated with BSP proteins. BSA (6 mg/ml) stimulated a specific phospholipid efflux of approximately 22%. In contrast, bovine RNase (120 microg/ml) did not stimulate phospholipid efflux. These results indicate that BSP proteins participate in the sperm cholesterol and phospholipid efflux that occurs during capacitation.     

Phospholipase B Is Activated in Response to Sterol Removal and Stimulates Acrosome Exocytosis in Murine Sperm

Despite a strict requirement for sterol removal for sperm to undergo acrosome exocytosis (AE), the mechanisms by which changes in membrane sterols are transduced into changes in sperm fertilization competence are poorly understood. We have previously shown in live murine sperm that the plasma membrane overlying the acrosome (APM) contains several types of microdomains known as membrane rafts. When characterizing the membrane raft-associated proteomes, we identified phospholipase B (PLB), a calcium-independent enzyme exhibiting multiple activities. Here, we show that sperm surface PLB is activated in response to sterol removal. Both biochemical activity assays and immunoblots of subcellular fractions of sperm incubated with the sterol acceptor 2-hydroxypropyl-β-cyclodextrin (2-OHCD) confirmed the release of an active PLB fragment. Specific protease inhibitors prevented PLB activation, revealing a mechanistic requirement for proteolytic cleavage. Competitive inhibitors of PLB reduced the ability of sperm both to undergo AE and to fertilize oocytes in vitro, suggesting an important role in fertilization. This was reinforced by our finding that incubation either with protein concentrate released from 2-OHCD-treated sperm or with recombinant PLB peptide corresponding to the catalytic domain was able to induce AE in the absence of other stimuli. Together, these results lead us to propose a novel mechanism by which sterol removal promotes membrane fusogenicity and AE, helping confer fertilization competence. Importantly, this mechanism provides a basis for the newly emerging model of AE in which membrane fusions occur during capacitation/transit through the cumulus, prior to any physical contact between the sperm and the oocyte''s zona pellucida.     

Serum sterols during stanol ester feeding in a mildly hypercholesterolemic population

We investigated the changes of cholesterol and non-cholesterol sterol metabolism during plant stanol ester margarine feeding in 153 hypercholesterolemic subjects. Rapeseed oil (canola oil) margarine without (n = 51) and with (n = 102) stanol (2 or 3 g/day) ester was used for 1 year. Serum sterols were analyzed with gas-liquid chromatography. The latter showed a small increase in sitostanol peak during stanol ester margarine eating. Cholestanol, campesterol, and sitosterol proportions to cholesterol were significantly reduced by 5-39% (P < 0.05 or less for all) by stanol esters; the higher their baseline proportions the higher were their reductions. The precursor sterol proportions were significantly increased by 10- 46%, and their high baseline levels predicted low reduction of serum cholesterol. The decrease of the scheduled stanol dose from 3 to 2 g/day after 6-month feeding increased serum cholesterol by 5% (P < 0. 001) and serum plant sterol proportions by 8-13% (P < 0.001), but had no consistent effect on precursor sterols. In twelve subjects, the 12-month level of LDL cholesterol exceeded that of baseline; the non-cholesterol sterol proportions suggested that stimulated synthesis with relatively weak absorption inhibition contributed to the non-responsiveness of these subjects. In conclusion, plant stanol ester feeding lowers serum cholesterol in about 88% of subjects, decreases the non-cholesterol sterols that reflect cholesterol absorption, increases the sterols that reflect cholesterol synthesis, but also slightly increases serum plant stanols. Low synthesis and high absorption efficiency of cholesterol results in the greatest benefit from stanol ester consumption.     

Optimisation of plant sterols incorporation in human keratinocyte plasma membrane and modulation of membrane fluidity.

The in vitro effects of plant sterols were investigated with regard to their uptake and membrane lipid fluidity in human keratinocytes. Among the different media tested to transport sterols (liposomes, micelles and organic solvents), the best results in terms of incorporation and viability were obtained by the use of the organic solvents dimethylsulfoxide and ethanol. After 48 h incubation exogenous sterol can account for about 30% of the total cell sterol content. The total sterol amount in plasma membranes increased 2-fold after incubation with cholesterol, whereas it was not altered when phytosterols were incorporated. The incorporation of cholesterol, sitosterol and stigmasterol led to an increase in the percent of unsaturated fatty acid C18:1 in the plasma membrane. The effect of this uptake on membrane fluidity was studied by means of fluorescence polarisation using DPH and TMA-DPH as fluorescent probes. Whereas cholesterol and sitosterol had no significant effect on the DPH fluorescence anisotropy (rs), the presence of stigmasterol induced a 12% decrease of rs reflecting an increase in membrane fluidity. We can conclude from this study that in the presence of sitosterol, the mean fluidity of the membrane is regulated whereas stigmasterol triggers a looseness of molecular packing of phospholipids acyl chains, in accordance with previous results obtained on purely lipid model membranes.