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Lipids are critical regulators of mammalian sperm function, first helping prevent premature acrosome exocytosis, then enabling sperm to become competent to fertilize at the right place/time through the process of capacitation, and ultimately triggering acrosome exocytosis. Yet because they do not fit neatly into the "DNA--RNA-protein" synthetic pathway, they are understudied and poorly understood. Here, we focus on three lipids or lipid classes-cholesterol, phospholipids, and the ganglioside G(M1)--in context of the modern paradigm of acrosome exocytosis. We describe how these various- species are precisely segregated into membrane macrodomains and microdomains, simultaneously preventing premature exocytosis while acting as foci for organizing regulatory and effector molecules that will enable exocytosis. Although the mechanisms responsible for these domains are poorly defined, there is substantial evidence for their composition and functions. We present diverse ways that lipids and lipid modifications regulate capacitation and acrosome exocytosis, describing in more detail how removal of cholesterol plays a master regulatory role in enabling exocytosis through at least two complementary pathways. First, cholesterol efflux leads to proteolytic activation of phospholipase B, which cleaves both phospholipid tails. The resultant changes in membrane curvature provide a mechanism for the point fusions now known to occur far before a sperm physically interacts with the zona pellucida. Cholesterol efflux also enables G(M1) to regulate the voltage-dependent cation channel, Ca(V)2.3, triggering focal calcium transients required for acrosome exocytosis in response to subsequent whole-cell calcium rises. We close with a model integrating functions for lipids in regulating acrosome exocytosis.

Asian elephant spermatozoa are sensitive to chilling and do not respond well to cryopreservation. The objectives of the present study were to: (1) determine whether cholesterol content can be modified by preincubation of Asian elephant spermatozoa with cholesterol-loaded cyclodextrin (CLC); and (2) assess the effects of CLC concentration(s), temperature at time of glycerol addition (22°C vs 4°C) and dilution medium on post-thaw sperm survival. Spermatozoa incubated with ≥1.5 mg CLC exhibited increased (P < 0.05) cholesterol concentrations. Pretreatment of spermatozoa with 1.5 mg CLC resulted in improvements (P < 0.05) in all post-thaw parameters. Glycerol addition at 4°C also improved all post-thaw parameters compared with 22°C. Dilution of thawed spermatozoa in an egg yolk-based medium improved (P < 0.05) motility compared with Ham's F-10 culture medium. In summary, our findings indicate that modifying cholesterol content within the plasma membrane improves the cryosurvival of Asian elephant spermatozoa. The development of an improved cryopreservation method that includes modification of membrane cholesterol and the addition of glycerol at 4°C, as reported in the present study, is an important step towards utilisation of cryopreserved spermatozoa in captive management of this species.

Our earlier studies have shown that Kir2.x channels are suppressed by an increase in the level of cellular cholesterol, whereas cholesterol depletion enhances the activity of the channels. In this study, we show that Kir2.1 and Kir2.3 channels have double-peak distributions between cholesterol-rich (raft) and cholesterol-poor (non-raft) membrane fractions, indicating that the channels exist in two different types of lipid environment. We also show that whereas methyl-beta-cyclodextrin-induced cholesterol depletion removes cholesterol from both raft and non-raft membrane fractions, cholesterol enrichment results in cholesterol increase exclusively in the raft fractions. Kinetics of both depletion-induced Kir2.1 enhancement and enrichment-induced Kir2.1 suppression correlate with the changes in the level of raft cholesterol. Furthermore, we show not only that cholesterol depletion shifts the distribution of the channels from cholesterol-rich to cholesterol-poor membrane fractions but also that cholesterol enrichment has the opposite effect. These observations suggest that change in the level of raft cholesterol alone is sufficient to suppress Kir2 activity and to facilitate partitioning of the channels to cholesterol-rich domains. Therefore, we suggest that partitioning to membrane rafts plays an important role in the sensitivity of Kir2 channels to cholesterol.

This study investigates how changes in the level of cellular cholesterol affect inwardly rectifying K+ channels belonging to a family of strong rectifiers (Kir2). In an earlier study we showed that an increase in cellular cholesterol suppresses endogenous K+ current in vascular endothelial cells, presumably due to effects on underlying Kir2.1 channels. Here we show that, indeed, cholesterol increase strongly suppressed whole-cell Kir2.1 current when the channels were expressed in a null cell line. However, cholesterol level had no effect on the unitary conductance and only little effect on the open probability of the channels. Moreover, no cholesterol effect was observed either on the total level of Kir2.1 protein or on its surface expression. We suggest, therefore, that cholesterol modulates not the total number of Kir2.1 channels in the plasma membrane but rather the transition of the channels between active and silent states. Comparing the effects of cholesterol on members of the Kir2.x family shows that Kir2.1 and Kir2.2 have similar high sensitivity to cholesterol, Kir2.3 is much less sensitive, and Kir2.4 has an intermediate sensitivity. Finally, we show that Kir2.x channels partition virtually exclusively into Triton-insoluble membrane fractions indicating that the channels are targeted into cholesterol-rich lipid rafts.

Protein tyrosine phosphorylation is an important intracellular event accompanying the in-vitro capacitation of mouse, bovine and human spermatozoa. Here, we demonstrate that bovine serum albumin (BSA) and NaHCO(3) are required for protein tyrosine phosphorylation in ejaculated human spermatozoa. The absence of protein tyrosine phosphorylation in media minus these two constituents could be recovered by addition to the media of cAMP analogues and/or phosphodiesterase inhibitors. Since BSA is postulated to modulate capacitation by removal of cholesterol from the sperm plasma membrane, we determined whether cholesterol release leads to changes in protein tyrosine phosphorylation. Incubation of spermatozoa in media containing BSA resulted in the release of significant amounts of cholesterol when compared with media devoid of BSA. Preloading BSA with cholesterol-SO(4) inhibited protein tyrosine phosphorylation, as well as capacitation, and this inhibitory effect was overcome by the addition of dibutyryl cAMP plus isobutylmethylxanthine (IBMX). The functional significance of BSA-mediated cholesterol release, protein tyrosine phosphorylation and capacitation was confirmed by examining the effects of the cholesterol-binding heptasaccharides, methyl-beta-cyclodextrin or OH-propyl-beta-cyclodextrin. Both cyclodextrins caused cholesterol efflux from the spermatozoa, increased protein tyrosine phosphorylation, and stimulated capacitation. Therefore, cholesterol release is associated with the activation of a signal transduction pathway involving protein kinase A and tyrosine kinase second messenger systems, and resulting in protein tyrosine phosphorylation and capacitation.