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Bicarbonate stimulated phospholipid scrambling induces cholesterol redistribution and enables cholesterol depletion in the sperm plasma membrane

¹ Department of Biochemistry and Cell Biology, Utrecht University, Utrecht, 3584 CM, The Netherlands
² Department of Farm Animal Health, Graduate School of Animal Health, Utrecht University, Utrecht, 3584 CL, The Netherlands
³ Department of Molecular Cell Biology, Institute of Biomembranes, Utrecht University, Utrecht, 3584 CH, The Netherlands

View larger version (15K): [in a new window]   Fig. 1. Effects of bicarbonate, albumin, and extracellular calcium on relative cholesterol levels in boar sperm cells incubated for 2 hours in HBT. (a) The relative amount of cholesterol (mol%) is expressed in sperm samples that have been subjected to incubation in HBT (depleted of CaCl2) containing the indicated additions (i.e. combinations of 1 mM EGTA, 2 mM CaCl2, 15 mM NaHCO3, 0.3% (w/v) BSA; sperm samples were washed through Percoll prior to lipid extraction). Mean values of three independent experiments (measured in triplicate) are indicated (error bars represent s.d. values; , P<0.001). (b) Absolute amounts (µg/109 cells) of the major lipid classes in the corresponding samples that were incubated in HBT (open bars) and HBT-Bic supplemented with 0.3% (w/v) albumin (filled bars). Major lipid classes: cholesterol (chol), phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylserine (PS), sulfogalactosyl glycerol (SGG), sphingomyelin (SM). Mean values of three independent experiments (measured in triplicate) are indicated (error bars represent s.d. values, , P<0.001). (c) Relative composition (mol%) of the most abundant lipid classes that were extracted from plasma membrane fractions derived from sperm suspensions incubated in HBT (open bars), HBT-Bic (hatched bars) and HBT-Bic supplemented with 0.3% (w/v) albumin (filled bars). Mean values of one experiment measured in triplicate.

View larger version (56K): [in a new window]   Fig. 2. Flow cytometric analysis and sorting of low and high M540 fluorescent viable sperm cell subpopulations. Sperm cells were incubated, analyzed and sorted using a FACS Vantage SE as described in Materials and Methods. Sperm-specific events with fluorescent properties in M540 fluorescence (membrane fluidity) and Yo-Pro 1 fluorescence (viability) were continuously recorded and sperm cells in specified regions (gray circles) were sorted and collected at room temperature (a). Within 10 minutes, the sorted and collected sperm cells were re-analyzed for low M540 fluorescence (b) as well as for high M540 fluorescence (c) demonstrating that cells remained viable and did not alter M540 fluorescent properties. The sorting efficiency was >99% for the low M540 and >95% for the high M540 fluorescent sperm subpopulations, respectively. Under a confocal microscope the unsorted sperm cells contained low and high M540 fluorescent cells (d), whereas the subpopulation sorted for low M540 fluorescence showed very dim fluorescence (e), and the subpopulation sorted for high M540 fluorescence showed bright fluorescence (f).

View larger version (15K): [in a new window]   Fig. 3. Composition of lipid classes in viable sperm cell subpopulations with low or high M540 fluorescence. (a) Separation of lipid classes on HPLC as detected by evaporative light scattering detection. Lipid classes were identified by comparison with lipid standards and online electrospray ionization mass spectrometry as triacylglycerols and cholesterol esters (1); cholesterol (2); diacylglycerol (3); ceramides (4); phosphoglycerolipids (5-7) with head-group ethanolamine (PE) (5), choline (PC) (6), serine (PS) (7); sphingomyelin (SM) (8); seminolipid (SGG) and phosphatidylinositol (PI) (9); and lysoPC (10). (b) Online identification of individual molecular species during the elution of PC. The distribution across the mass spectrum results from the variety in fatty radyl chain length, the degree of unsaturation and the type of linkage at the sn-1 position (ester versus ether) of the glycerol backbone. Peaks are labelled with their nominal masses (bottom) or the total number of carbon atoms in the radyl groups and the type of sn-1 linkage (top). PtdCho, 1,2 diacyl phosphatidylcholine; AlkCho, 1-alkyl 2-acyl phosphatidylcholine; PlasCho, 1-alk-1'-enyl 2-acyl phosphatidylcholine (Plasmalogen PC). The italic numbers indicate the total number of unsaturations (double bonds) in the fatty radyl chains. The top line was recorded during elution of lipids from low M540 fluorescent cells, the bottom line during elution of lipids from high M540 fluorescent cells. Note the occurrence of isotope peaks at odd m/z ratios, due to the natural occurrence of approximately 1% [13C]. Experiments were performed three times with similar results.

View larger version (126K): [in a new window]   Fig. 4. Ultrastructural localization of filipin sterol complexes on freeze fracture replicas of the head plasma membrane of fixed boar sperm cells. (a) Freeze fracture replica of a large proportion of the plasma membrane of a boar sperm head with filipin labeling type A (Fig. 7A), with a high density of cholesterol-filipin complexes in the apical and equatorial subdomain and lower density in the post-acrosomal subdomain. (b) As in (a), but showing filipin labeling type B (Fig. 7B), with high density of cholesterol-filipin complexes in the pre-equatorial subdomain but no labeling in the equatorial and post-equatorial subdomain. The particles on this replica represent filipin cholesterol complexes. (c) Freeze-fracture replica of a non-labeled sperm cell from a DMSO control experiment showing the absence of the complexes, the small particles represent membrane-associated proteins. Note the pronounced neck region labeling in both labeled replica.

View larger version (151K): [in a new window]   Fig. 5. Ultrastructural localization of filipin sterol complexes on freeze-fracture replicas of the head plasma membrane of fixed boar sperm cells. Freeze-fracture replica of a sperm cell in which the fracture plane is running across through the sperm head. Filipin/sterol complexes are only visible at sites where the plasma membrane is in the fracture plane, whereas acrosomal and nuclear membranes as well as the protamine condensed DNA (organized into multilammelar sheets) (Balhorn et al., 1999) are devoid of these particles.

View larger version (20K): [in a new window]   Fig. 6. Excitation and emission scans of filipin complexed to boar sperm cells. Sperm cells were labeled with filipin (see Materials and Methods) and suspensions of 106 labeled cells/ml were pipetted in quarts cuvettes and placed in a fluorimeter. The excitation of complexed filipin was detected at an emission wavelength of 480 nm over the range of 275-400 nm (solid line). The emission of complexed filipin was detected at 357 nm excitation (one of the excitation peaks) at a range of 400-600 nm (broken line).

View larger version (106K): [in a new window]   Fig. 7. Fluorescent labeling of filipin sterol complexes on fixed boar sperm cells. Filipin-labeled cells were mounted in coverslips and sealed with nail-polish. The filipin fluorescence was observed under 340-380 nm excitation and fluorescence signals >425 nm were selected by the emission filter. (A) Filipin labeling of a sperm cell from the low M540 fluorescent sperm subpopulation (identical to pattern A; Fig. 4a). (B) As in A but from the high M540 fluorescent subpopulation (identical to pattern B; Fig. 4b). (C) Filipin labeling was absent in the sperm head of cells that were capacitated in HBT-Bic supplemented 0.3% (w/v) albumin (pattern C). Note the labeling of the neck region of the sperm cell (out of focus in B), all cells depicted did not contain a cytoplasmic droplet as checked by phase-contrast microscopy.

View larger version (24K): [in a new window]   Fig. 8. The effect of bicarbonate and albumin on cholesterol and phospholipid scrambling in incubated boar sperm samples. Ejaculates from six different boars were incubated in HBT, HBT supplemented with 0.3% (w/v) albumin (HBT-BSA), for 2 hours. Cholesterol was fluorescently visualized by filipin and phospholipid scrambling by M540 (see Materials and Methods). Sperm cells showing the filipin pattern depicted in Fig. 7C (pattern C) were scored as cells featuring cholesterol efflux (filled bar), whereas filipin pattern B (Fig. 7B) were scored as cells featuring cholesterol redistribution (hatched bar). Sperm cells showing high M540 fluorescence (Fig. 2f) were scored as cells that underwent phospholipid scrambling (open bar). After each incubation condition, 200 cells were counted in triplicate, the resulting mean values were calculated from all six boars. From these data mean values with s.d. are expressed (n=6).

View larger version (15K): [in a new window]   Fig. 9. Morphology scores of the boar samples after a two hour incubation in HBT-Bic supplemented with 0.3% (w/v) BSA. The filipin and M540 response was determined as described in Fig. 8. The sperm samples were also subjected to morphological examination: Sperm cells were diluted, fixed and examined under a phase-contrast microscope. Ejaculates from ten boars were collected (three ejaculates from each boar) and counted in triplicate. The percentage of sperm cells with a normal mature morphology are expressed on the x-axis whereas the percentage of sperm cells with the bicarbonate response is indicated on the y-axis: , cells that stained positively with M540; , cells with filipin response (either labeling patterns B or C; Fig. 7). 200 cells were counted in triplicate and mean values with s.d values (n=10) are expressed for morphology (horizontal error bar) and for M540 or filipin response (vertical error bar).

View larger version (63K): [in a new window]   Fig. 10. Model for biphasic modulation of cholesterol in bicarbonate-stimulated sperm cells. (A) In the absence of high levels of bicarbonate (i.e. in epididymal or seminal fluids) phospholipid scrambling is blocked. Cholesterol has a wide-spread lateral localization in the sperm head plasma membrane; caveolin-1-mediated raft formation does not take place. (B) In the presence of high levels of bicarbonate a soluble adenylate cyclase is activated by bicarbonate (entry by the bicarbonate chloride exchanger), which triggers production of cAMP and activates protein kinase A (Cheng et al., 2000). This pathway leads to the activation of phospholipid scrambling in the apical plasma membrane of the sperm head (Gadella and Harrison 2000; Gadella et al., 1999b) via a yet unclear pathway. This apical scrambling coincides with the concentration of cholesterol in this area (M540 correlated with both responses). The preliminary finding that caveolin-1 is present in sperm cells makes it possible that the scrambling surface area (concentration of cholesterol) is forming a membrane raft. Note that phospholipid scrambling-induced membrane blebbing may be compensated with the caveolin-1-induced membrane invagination. If a membrane raft is formed, a scavenger receptor (SRB-1) may be able to transport cholesterol out of the apical plasma membrane of the sperm head (Fielding and Fielding, 2000). (C) This will only lead to a decrease of cellular cholesterol if an acceptor of cholesterol (e.g. BSA) is present.