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

Abstract

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.

Remodelling of Lipid Rafts during In vitro Capacitation and Acrosome Reaction of Ram Spermatozoa

“…One likely difference among these studies could be the fixation procedure [60], which might affect viability. In fact, it has been reported that GM1 moves rapidly from its position overlying the acrosome to the post-acrosomal area after cell death in unfixed or lightly fixed samples [59]. Our results show that GM1 is located at the postacrosomal region and tail of all ram spermatozoa.…”

“…Nevertheless, the reported results about the location of GM1 in sperm are contradictory. GM1 has been found at the postacrosome of epididymal mouse [57] and rat [58] spermatozoa, and at the acrosomal area of epididymal murine sperm [59]. Conversely, GM1 was reported as nonexistent or patchy and inconsistent throughout the whole sperm cell of human and boar .…”

The Multi‐PDZ domain protein MUPP1 as a lipid raft‐associated scaffolding protein controlling the acrosome reaction in mammalian spermatozoa

“…Albeit, it is currently not known this may be due to various and possibly complex tasks of MUPP1 in neurons and different subcellular localizations (Suzuki, 2002), whereas in mammalian spermatozoa, MUPP1 action may be limited to one of the few hallmarks of sperm function. DRM rafts, which have been found in mammalian sperm cells of several species (Travis et al, 2001;Trevino et al, 2001;Cross, 2004;Shadan et al, 2004;Sleight et al, 2005;van Gestel et al, 2005;Selvaraj et al, 2006;Thaler et al, 2006), are specifically enriched in cholesterol (Rajendran and Simons, 2005), and, thus, are particularly important during the posttesticular developmental process of capacitation, which is characterized by a net loss of cholesterol . The experimental approaches used in the present study to determine whether capacitation-dependent reorganization of plasma membrane microdomains (van Gestel et al, 2005) is accomplished by a shift of MUPP1 to other subcellular compartments, led to the key observation that, upon capacitation, MUPP1 was still associated with highly-ordered DRM subdomains (Fig.…”

Roles of gangliosides in mouse embryogenesis and embryonic stem cell differentiation

“…For example, it provides evidence for the existence of membrane sub-domains in living cells, which is still a matter of some controversy (Munro, 2003). Standing in contrast to reports on the segregation of GM1 in live sperm (Selvaraj et al, 2006) are studies suggesting that there is no barrier to the lateral diffusion of lipids in mature spermatozoa (Mackie et al, 2001).…”