Mechanisms underlying the micron‐scale segregation of sterols and GM1 in live mammalian sperm

We demonstrate for the first time that a stable, micron‐scale segregation of focal enrichments of sterols exists at physiological temperature in the plasma membrane of live murine and human sperm. These enrichments of sterols represent microheterogeneities within this membrane domain overlying the acrosome. Previously, we showed that cholera toxin subunit B (CTB), which binds the glycosphingolipid, G_M1, localizes to this same domain in live sperm. Interestingly, the G_M1 undergoes an unexplained redistribution upon cell death. We now demonstrate that G_M1 is also enriched in the acrosome, an exocytotic vesicle. Transfer of lipids between this and the plasma membrane occurs at cell death, increasing G_M1 in the plasma membrane without apparent release of acrosomal contents. This finding provides corroborative support for an emerging model of regulated exocytosis in which membrane communications might occur without triggering the “acrosome reaction.” Comparison of the dynamics of CTB‐bound endogenous G_M1 and exogenous BODIPY–G_M1 in live murine sperm demonstrate that the sub‐acrosomal ring (SAR) functions as a specialized diffusion barrier segregating specific lipids within the sperm head plasma membrane. Our data show significant differences between endogenous lipids and exogenous lipid probes in terms of lateral diffusion. Based on these studies, we propose a hierarchical model to explain the segregation of this sterol‐ and G_M1‐enriched domain in live sperm, which is positioned to regulate sperm fertilization competence and mediate interactions with the oocyte. Moreover, our data suggest potential origins of subtypes of membrane raft microdomains enriched in sterols and/or G_M1 that can be separated biochemically. J. Cell. Physiol. 218: 522–536, 2009. © 2008 Wiley‐Liss, Inc.