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

Abstract

IntroductionThe lateral distribution of lipids and proteins in cell membranes is known to be highly asymmetric (Edidin, 1993;Jacobson et al., 1995;Vereb et al., 2003). Elucidating how these heterogeneities are generated and maintained over relatively large distances against the randomising forces of diffusion is central to understanding many of the processes involved in cell differentiation and establishment of polarity.Uncontrolled mixing of membrane components is thought to be prevented by several complementary processes, the best known of which are interaction with the cytoskeleton, spontaneous self-association, selective internalisation and confinement behind diffusion barriers (Gumbiner and Louvard, 1985). Diffusion barriers have long been implicated in the asymmetric distribution of surface antigens, such as between the cell body and axon in neurons (Kobayashi et al., 1992), the inner and outer segments of retinal rod cells (Peters et al., 1983) and the apical and basolateral membranes in epithelial cells (Rodriguez-Boulan and Nelson, 1989), and are thought to be largely responsible for creating micrometer-sized macrodomains. Frequently, they have a physical basis in the form of electron-dense structures within the membrane, e.g. zona occludens between epithelial cells (Dragsten et al., 1981).However, confinement zones also exist at the nanometer scale. These were first suggested by low recovery rates during fluorescence photobleaching (FRAP) experiments and later demonstrated directly using laser tweezers to drag 40 nm gold particles across the cell surface (Edidin, 2003;Vereb et al., 2003). Recent work by Kusumi and co-workers (Fujiwara et al., 2002;Murase et al., 2004) using single particle tracking at very high video speeds have refined this idea into the 'picket and fence model', in which proteins and lipids are confined temporarily within 200-300 nm diameter compartments and effect long-range diffusion by 'jumping' between compartments. It is not altogether clear at present how the temporary confinement model accommodates lipid rafts, which are envisaged as dynamic structures varying in size from a few molecules to ~50 nm in diameter and are rich in cholesterol, glycosphingolipids and GPI-anchored proteins (Edidin, 2003). At all levels of scale, it seems that membrane compartments are influenced by the cytoskeleton, thereby affording a means of transducing signals from the cytoplasm to the cell surface and vice versa.The mammalian spermatozoon is an example of a polarised cell with a highly compartmentalised plasma membrane (Holt, 1984; Cardullo and Wolfe, 1990). During differentiation in the

FRAP analysis of molecular diffusion inside sea-urchin spermatozoa

“…By electron microscopy the 'neck' region between head and flagellum was shown to be densely packed in mammalian sperm (Pessh and Bergmann, 2006). In addition, the presence of diffusion barriers has been reported in the plasma membrane of mammalian sperm (James et al, 2004;Ladha et al, 1997;Mackie et al, 2001). Such evidence suggests that there could be an intracellular diffusion barrier around the neck region.…”

Protective effects of exogenous gangliosides on ROS-induced changes in human spermatozoa

“…13,77 The cryopreservation-induced damage of the sperm membrane manifests as changes in the organisation and lipid composition of the membrane, leading to changes in sperm permeability. 78,79 Several reports on mammalian sperm have demonstrated the protective capacity of phospholipid-based cryoprotectants. The most important role of phospholipids could be coating the cell membrane, resulting in reinforcement during cryostorage.…”

Characterization of the endocannabinoid system in boar spermatozoa and implications for sperm capacitation and acrosome reaction

“…With this knowledge we sought to investigate whether sperm cells of boar (Sus scropha) were able to bind and metabolize AEA, and whether this endocannabinoid might modulate their function. It should be recalled that boar physiology closely resembles that of humans (Logan and Sharma, 1999), and its spermatozoa are largely used as a model system for experiments on reproductive physiology of mammals (Gadella et al, 1995;James et al, 2004). However, we did not further extend this study to the metabolic enzymes that degrade (Dinh et al, 2002) and synthesize (Bisogno et al, 2003) 2-AG, because the properties of these recently discovered proteins are still under investigation.…”