The equatorial subsegment in mammalian spermatozoa is enriched in tyrosine phosphorylated proteins.

The equatorial subsegment (EqSS) was originally identified by atomic force microscopy as a discrete region within the equatorial segment of Artiodactyl spermatozoa. In this investigation, we show that the EqSS is enriched in tyrosine phosphorylated proteins and present preliminary evidence for its presence in mouse and rat spermatozoa. The anti-phosphotyrosine monoclonal antibody (McAb) 4G10 bound strongly and discretely to the EqSS of permeabilized boar, ram, and bull spermatozoa. It also bound to a small patch on the posterior acrosomal region of permeabilized mouse and rat spermatozoa, suggesting that the EqSS is not restricted to the order Artiodactyla. An anti-HSPA1A (formerly Hsp70) antibody recognized the EqSS in boar spermatozoa. Immunogold labeling with McAb 4G10 localized the tyrosine phosphorylated proteins to the outer acrosomal membrane. This was verified by freeze-fracture electron microscopy, which identified the EqSS in three overlying membranes, the plasma membrane, outer acrosomal membrane, and inner acrosomal membrane. In all five species, tyrosine phosphorylated proteins became restricted to the EqSS during sperm maturation in the epididymis. The major tyrosine phosphorylated proteins in the EqSS of boar and ram spermatozoa were identified by mass spectrometry as orthologs of human SPACA1 (formerly SAMP32). Immunofluorescence with a specific polyclonal antibody localized SPACA1 to the equatorial segment in boar spermatozoa. We speculate that the EqSS is an organizing center for assembly of multimolecular complexes that initiate fusion competence in this area of the plasma membrane following the acrosome reaction.

Supramolecular organization of the sperm plasma membrane during maturation and capacitation.

AIM: In the present study, a variety of high resolution microscopy techniques were used to visualize the organization and motion of lipids and proteins in the sperm's plasma membrane. We have addressed questions such as the presence of diffusion barriers, confinement of molecules to specific surface domains, polarized diffusion and the role of cholesterol in regulating lipid rafts and signal transduction during capacitation. METHODS: Atomic force microscopy identified a novel region (EqSS) within the equatorial segment of bovine, porcine and ovine spermatozoa that was enriched in constitutively phosphorylated proteins. The EqSS was assembled during epididymal maturation. Fluorescence imaging techniques were then used to follow molecular diffusion on the sperm head. RESULTS: Single lipid molecules were freely exchangeable throughout the plasma membrane and showed no evidence for confinement within domains. Large lipid aggregates, however, did not cross over the boundary between the post-acrosome and equatorial segment suggesting the presence of a molecular filter between these two domains. CONCLUSION: A small reduction in membrane cholesterol enlarges or increases lipid rafts concomitant with phosphorylation of intracellular proteins. Excessive removal of cholesterol, however, disorganizes rafts with a cessation of phosphorylation. These techniques are forcing a revision of long-held views on how lipids and proteins in sperm membranes are assembled into larger complexes that mediate recognition and fusion with the egg.

Compartmentalisation of the sperm plasma membrane: a FRAP, FLIP and SPFI analysis of putative diffusion barriers on the sperm head.

Spermatozoa are highly polarised cells with a compartmentalised distribution of lipids and proteins in their plasma membrane. It is not known how these compartments are stably maintained in what is essentially a fluid environment. In this investigation we have examined the hypothesis that intramembranous diffusion barriers selectively retain some components within compartments, while allowing free passage of others. A fluorescence loss in photobleaching analysis of the behaviour of the lipid reporter probe 1,1'-dihexadecyl-3,3,3'3'-tetramethyindocarbocyanine (DiIC16) on the head of boar spermatozoa revealed that it was freely diffusing between all three compartments (anterior acrosome, equatorial segment and postacrosome). Spermatozoa also contained rapidly diffusing particles of DiIC16 over the anterior acrosome and equatorial segment. These particles, approximately 200 nm in diameter, were tracked in real time and their trajectories analysed by mean square displacement. Particle diffusion was essentially random over the anterior acrosome and equatorial segment but showed a periodicity in jump sizes and diffusion coefficients suggestive of microheterogeneities. Particles did not exchange between the equatorial segment and postacrosome, indicating a barrier at the junction between these two compartments. No barrier was detected between the equatorial segment and anterior acrosome. A model is proposed in which a molecular 'filter' is present at the equatorial segment-postacrosomal boundary that allows free passage of single molecules but not molecular complexes. Passage of heterogeneous complexes, such as lipid rafts, requires disassembly and reassembly on either side of the filter.

Lipid diffusion in sperm plasma membranes exposed to peroxidative injury from oxygen free radicals.

Unsaturated lipids in sperm plasma membranes are very susceptible to peroxidation when exposed to reactive oxygen species (ROS). In this investigation we have incubated ram spermatozoa in the presence of two ROS generating systems, ascorbate/FeSO4 and potassium peroxychromate (K3CrO8), and examined their effects on membrane fluidity by measuring fluorescence recovery after photobleaching (FRAP) of a lipid reporter probe 5-(N-octadecanoyl)-aminofluorescein (ODAF). Peroxidation was monitored by malonaldehyde formation and changes in fluorescence emission of 4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-undecanoic acid (C11-BODIPY(581/591)). Ascorbate/FeSO4-induced peroxidation was inhibited by Vitamin E, butylated hydroxyanisole (BHA), 1,4-diazobicyclo(2,2,2)octane (DABCO), and to a lesser extent by ethanol. Added superoxide dismutase (SOD), gluthathione peroxidase (GPX), and catalase were ineffective scavengers. K3CrO8 induced very rapid peroxidation that could be delayed, but not prevented, by Vitamin E, BHT, DABCO, ethanol, and mannitol; once again SOD, GPX, and catalase were ineffective scavengers. Neither peroxidation with ascorbate/FeSO4 nor K3CrO8, or added H2O2 or malonaldehyde perturbed ODAF diffusion in any region of the sperm plasma membrane. Vitamin E tended to enhance diffusion rates. Exogenous cumene hydroperoxide, however, reduced ODAF diffusion to low levels on the sperm head. These results suggest that the adverse effects of ROS on spermatozoa are more likely to be caused by direct oxidation of proteins and membrane permeabilisation than disturbance of lipid fluidity.

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.

Kinetics of inhibition of sperm beta-acrosin activity by suramin.

Sperm beta-acrosin activity is inhibited by suramin, a polysulfonated naphthylurea compound with therapeutic potential as a combined antifertility agent and microbicide. A kinetic analysis of enzyme inhibition suggests that three and four molecules of suramin bind to one molecule of ram and boar beta-acrosins respectively. Surface charge distribution models of boar beta-acrosin based on its crystal structure indicate several positively charged exosites that represent potential 'docking' regions for suramin. It is hypothesised that the spatial arrangement and distance between these exosites determines the capacity of beta-acrosin to bind suramin.

Post-testicular development of a novel membrane substructure within the equatorial segment of ram, bull, boar, and goat spermatozoa as viewed by atomic force microscopy.

Atomic force microscopy has been used to investigate changes in the plasma membrane overlying the head region of mammalian spermatozoa (bull, boar, ram, goat, stallion, mouse, and monkey) during post-testicular development, after ejaculation, and after exocytosis of the acrosomal vesicle. On ejaculated ram, bull, boar, and goat spermatozoa the postacrosomal plasma membrane has a more irregular surface than that covering the acrosome. The equatorial segment, by contrast, is relatively smooth except for an unusual semicircular substructure within it that has a coarse uneven appearance. This substructure (referred to as the equatorial subsegment) is situated adjacent to the boundary between the postacrosomal region and the equatorial segment itself and seems to be confined to the order Artiodactyla as it has not been observed on stallion, mouse, or monkey spermatozoa. The equatorial subsegment develops during epididymal maturation, and following induction of the acrosome reaction with Ca(2+) ionophore A23187, its topography changes from a finely ridged appearance to that resembling truncated papillae. A monoclonal antibody to the equatorial subsegment binds only to permeabilized spermatozoa, suggesting that the subsegment is related to the underlying perinuclear theca that surrounds the sperm nucleus. A role for the equatorial subsegment in mediating fusion with the oolemma at fertilization is discussed.

Lipid diffusion in the plasma membrane of mouse spermatozoa: changes during epididymal maturation, effects of pH, osmotic pressure, and knockout of the c-ros gene.

It is well known that the plasma membranes of mammalian spermatozoa undergo extensive remodeling during maturation in the epididymal duct. In this investigation, we have used fluorescence recovery after photobleaching (FRAP) techniques to: 1) measure rates of lipid diffusion in the plasma membrane of mouse spermatozoa at different stages of maturation; 2) examine the effects of varying external conditions found in the epididymal duct (pH, temperature, and osmotic pressure) on lipid diffusion in mature sperm; and 3) investigate the effects of the c-ros null mutation that causes tail angulation in cauda spermatozoa after ejaculation as a result of cell swelling due to altered membrane function. Our results show that lipid diffusion (as measured using reporter probes 5-(N-octadecanoyl)aminofluorescein [ODAF] and 2-(6-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoyl-1-hexadecanoyl-sn-glycero-3-phosphocholine [NBD-C6-PC]) is several times faster across the membrane on the sperm head than on the tail and that it increases significantly during passage from caput to cauda. Temperature variations between 20 degrees C and 37 degrees C have a substantial effect on diffusion coefficients, with the sperm head being more responsive than the tail. Changes in external pH (6.5-8.5) or osmotic pressure (202-389 mOsm/kg), however, have little relative effect on lipid diffusion on any region of the sperm. The rate of diffusion of 22-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3beta-ol (NBD-cholesterol) is 10-fold higher across the head plasma membrane than across the tail and does not change significantly during epididymal maturation. Similarly, lipid diffusion in hairpin-shaped cauda sperm from c-ros (-/-) males is not significantly different from (+/+) controls. These results suggest that temperature and compositional changes are 2 of the important factors that regulate the dynamics of lipid molecules in the mouse sperm plasma membrane.