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.

Background: The observed age of menarche has fallen, which may have important adverse social and health consequences. Increased exposure to endocrine-disrupting compounds (EDCs) has been associated with adverse reproductive outcomes.Objective: Our objective was to assess the relationship between EDC exposure and the age of menarche in adolescent girls.Methods: We used data from female participants 12–16 years of age who had completed the reproductive health questionnaire and laboratory examination for the Centers for Disease Control and Prevention’s National Health and Nutrition Examination Survey (NHANES) for years 2003–2008 (2005–2008 for analyses of phthalates and parabens). Exposures were assessed based on creatinine-corrected natural log urine concentrations of selected environmental chemicals and metabolites found in at least 75% of samples in our study sample. We used Cox proportional hazards analysis in SAS 9.2 survey procedures to estimate associations after accounting for censored data among participants who had not reached menarche. We evaluated body mass index (BMI; kilograms per meter squared), family income-to-poverty ratio, race/ethnicity, mother’s smoking status during pregnancy, and birth weight as potential confounders.Results: The weighted mean age of menarche was 12.0 years of age. Among 440 girls with both reproductive health and laboratory data, after accounting for BMI and race/ethnicity, we found that 2,5-dichlorophenol (2,5-DCP) and summed environmental phenols (2,5-DCP and 2,4-DCP) were inversely associated with age of menarche [hazard ratios of 1.10; 95% confidence interval (CI): 1.01, 1.19 and 1.09; 95% CI: 1.01, 1.19, respectively]. Other exposures (total parabens, bisphenol A, triclosan, benzophenone-3, total phthalates, and 2,4-DCP) were not significantly associated with age of menarche.Conclusions: Our findings suggest an association between 2,5-DCP, a potential EDC, and earlier age of menarche in the general U.S. population.

Despite enormous interest in membrane raft microdomains, no studies in any cell type have defined the relative compositions of the raft fractions on the basis of their major components-sterols, phospholipids, and proteins-or additional raft-associating lipids such as the ganglioside, G M1 . Our previous localization data in live sperm showed that the plasma membrane overlying the acrosome represents a stabilized platform enriched in G M1 and sterols. These findings, along with the physiological requirement for sterol efflux for sperm to function, prompted us to characterize sperm membrane fractions biochemically. After confirming limitations of commonly-used detergent-based approaches, we utilized a non-detergent-based method, separating membrane fractions that were reproducibly distinct based on sterol, G M1 , phospholipid and protein compositions (both mass amounts and molar ratios). Based on fraction buoyancy and biochemical composition, we identified at least three highly reproducible subtypes of membrane raft. Electron microscopy revealed that raft fractions were free of visible contaminants and were separated by buoyancy rather than morphology. Quantitative proteomic comparisons and fluorescence localization of lipids suggested that different organelles contributed differentially to individual raft sub-types, but that multiple membrane microdomain sub-types could exist within individual domains. This has important implications for scaffolding functions broadly associated with rafts. Most importantly, we show that the common practice of characterizing membrane domains as either "raft" or "non-raft" oversimplifies the actual biochemical complexity of cellular membranes.

ABSTRACT:We previously showed that in live murine and bovine sperm heads, the ganglioside G M1 localizes to the sterol-rich plasma membrane overlying the acrosome (APM). Labeling G M1 using the pentameric cholera toxin subunit B (CTB) induced a dramatic redistribution of signal from the APM to the sterol-poor postacrosomal plasma membrane (PAPM) upon sperm death. We now show a similar phenomenon in the flagellum where CTB induces G M1 redistribution to sterol-poor membrane subdomains of the annulus and flagellar zipper. Because sterol efflux from the plasma membrane is required for capacitation, we examined whether G M1 localization might be useful to detect membrane changes associated with capacitation and/or acrosomal exocytosis. First, incubation of murine and bovine sperm with their respective stimuli for capacitation did not change G M1 distribution in live cells. However, incubation of sperm of both species with specific stimuli for capacitation, followed by the use of specific fixation conditions, induced reproducible, stimulus-specific patterns of G M1 distribution. By assessing changes in G M1 distribution in response to progesterone-induced AE, we show that these patterns reflect the response of murine sperm populations to capacitating stimuli. These data suggest that G M1 localization can be used as a diagnostic tool for evaluating sperm response to stimuli for capacitation and/or AE. Such information could be useful when deciding between technologies of assisted reproduction or when screening for male fertility. Furthermore, stimulus-specific changes in G M1 distribution showed that sperm could respond to NaHCO 3 or mediators of sterol efflux independently, thereby refining existing models of capacitation.