Do not touch! The surface of a living cell is soft and responsive and therefore high‐resolution imaging of the cell membrane has not been possible to date. Now noncontact imaging of protein complexes in the plasma membrane of living cells has been demonstrated (see picture) and has been used to follow the cells' structural reorganization. This breakthrough opens up a wealth of new experiments in membrane and cell biology.

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.

A long-standing problem in epididymal physiology is the fate of unejaculated spermatozoa in the cauda epididymidis under conditions such as congenital absence of the vas deferens, long-term vasectomy, or castration. There is no convincing evidence for significant absorption of spermatozoa, defective or otherwise, by spermiophagy or dissolution in the epididymis of normal animals. Spermiophagy by epithelial cells or intraluminal macrophages may take place if the duct ruptures and granulomas form (e.g., after experimental ligation), although there is no quantitative information on the rate of sperm removal by this means. In one animal model (the rabbit), the epididymis is unusually resistant to granuloma formation and has provided unique insights into a phenomenon that is suggested to be present in all species. Spermatozoa retained in the rabbit cauda epididymidis by placing ligatures on the vas deferens and corpus epididymidis degenerate after several weeks but do not decrease significantly in numbers. After castration, however, they die very rapidly and >90% disappear. It is hypothesized that, in the normal androgen-maintained epididymis, degradative pathways are present in the luminal fluid that are constitutively inhibited by survival signals emanating from the epithelium. In the absence of androgen, the intraluminal mileau changes and death signals predominate that activate degradative pathways via the ubiquitin-proteasome system, DNAses, etc., to mediate dissolution of sperm organelles and nucleoprotein. It is suggested that the latter condition is the default situation and is only prevented by the stimulatory action of androgens on the epididymal epithelium.

We have developed a new method, using a nanopipette, for controlled voltage-driven delivery of individual fluorescently labeled probe molecules to the plasma membrane which we used for single-molecule fluorescence tracking (SMT). The advantages of the method are 1), application of the probe to predefined regions on the membrane; 2), release of only one or a few molecules onto the cell surface; 3), when combined with total internal reflection fluorescence microscopy, very low background due to unbound molecules; and 4), the ability to first optimize the experiment and then repeat it on the same cell. We validated the method by performing an SMT study of the diffusion of individual membrane glycoproteins labeled with Atto 647-wheat germ agglutin in different surface domains of boar spermatozoa. We found little deviation from Brownian diffusion with a mean diffusion coefficient of 0.79 +/- 0.04 microm(2)/s in the acrosomal region and 0.10 +/- 0.02 microm(2)/s in the postacrosomal region; this difference probably reflects different membrane structures. We also showed that we can analyze diffusional properties of different subregions of the cell membrane and probe for the presence of diffusion barriers. It should be straightforward to extend this new method to other probes and cells, and it can be used as a new tool to investigate the cell membrane.

1. Protein synthesis has been investigated in different regions of the rat epididymis by measuring incorporation of [35S]methionine in tissue minces incubated in vitro followed by analysis of labelled proteins on polyacrylamide gels containing sodium dodecyl sulphate. Rates of synthesis were highest in the proximal cauda > distal cauda > initial segment > ductuli efferentes > corpus > distal caput > proximal caput. One protein (mol.wt. 23 000) characterized the initial segment, three proteins (mol.wts. 18 500, 19 000 and 32 000) the caput and one protein (mol.wt. 47 000) the cauda. 2. After castration, [35S]methionine incorporation in all regions of the epididymis was reduced to < 10% of that in normal animals but could be restored to control levels within 5 days by testosterone treatment. Other steroids (corticosterone, oestrogen or progesterone) were ineffective. 3. The synthesis of the 18 500, 19 000, and 32 000 mol.wt. proteins in the caput and the 47 000 mol.wt. protein in the cauda were preferentially regulated by androgens, whilst the synthesis of 23 000 and approx. 80 000 mol.wt. proteins in the initial segment was dependent upon factors present in testicular fluid. 4. The androgen-dependent and testicular fluid-dependent proteins were major components of epididymal secretion. Purification and characterization of the 18 500, 19 000, 23 000 and 32 000 mol.wt. proteins showed them to be acidic glycoproteins with a carbohydrate content of 7.6-13.2%. The 47 000 mol.wt. protein, on the other hand, is highly basic. 5. A possible role for these proteins in the acquisition of motility, fertilizing capacity and storage of spermatozoa in the epididymis is discussed.

1. The distribution and properties of superoxide dismutase were examined in mammalian semen, and the enzyme was used to investigate the role of superoxides in metal-ion-catalysed lipid-peroxidation reactions in spermatozoa. 2. Superoxide dismutase activity was detected in seminal plasma and spermatozoa from all species studied, exceptionally high activity being found in donkey semen. The enzyme is easily solubilized from spermatozoa, as 85-90% of the total activity is released by cold shock, a relatively mild form of cellular damage. 3. Purification and characterization of the enzyme from supernatant fractions prepared from cold-shocked boar spermatozoa showed it to be cyanide-sensitive, to have a mol.wt. of 31 000, a pI of 5.9 and to contain 1.85 g-atoms of copper and 1.91 g-atoms of zinc per mol of protein. However, extensive sonication of spermatozoa released a small amount of a cyanide-insensitive enzyme, presumably a mangano superoxide dismutase, from the mitochondrial matrix. 4. The presence of superoxide dismutase in spermatozoa, either intracellularly or extracellularly, did not inhibit ascorbate/Fe2+-catalysed lipid-peroxidation reactions, suggesting that superoxides are not essential intermediates in this system.

The accurate discrimination of the aerodynamic parameters affecting the flight of sports balls is essential in the product development process. Aerodynamic studies reported to date have been limited, primarily because of the inherent difficulty of making accurate measurements on a moving or spinning ball. Manufacturers therefore generally rely on field trials to determine ball performance, but the approach is time-consuming and subject to considerable variability. The current paper describes the development of a method for mounting stationary and spinning footballs in a wind tunnel to enable accurate force data to be obtained. The technique is applied to a number of footballs with differing constructions and the results reported. Significant differences in performance are noted for both stationary and spinning balls and the importance of the ball orientation to the flow is highlighted. To put the aerodynamic data into context the results are applied in a flight model to predict the potential differences in the behaviour of each ball in the air. The aerodynamic differences are shown to have a considerable effect on the flight path and the effect of orientation is shown to be particularly significant when a ball is rotating slowly. Though the techniques reported here are applied to a football they are equally applicable to other ball types.

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