Biochemical characterization of membrane fractions in murine sperm: identification of three distinct sub-types of membrane rafts

Abstract

Despite enormous interest in membrane raft micro-domains, 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 sub-types 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 micro-domain 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.

Fig. 1

Comparison of membrane protein solubility when extracted with TX-100 versus CHAPS. Total sperm membranes (lanes A) were extracted using 0.5% TX-100 or 20 mM CHAPS for 15 min at 4°C and then separated into detergent-soluble and -insoluble fractions by centrifugation (lanes B and lanes C, respectively). Equal amounts of protein were separated by SDS-PAGE and analyzed by immunoblotting with the indicated antisera. Note the difference in partitioning of GLUT 3 between the two detergents.

Fig. 2

Quantitative biochemical characterization of membrane fractions isolated from murine sperm without detergent. Fractions of sperm membranes were separated based on their relative buoyancies. The fractions were collected from the sucrose density gradient and numbered 1–10, with fraction 10 representing the highest density, bottom fraction. Measurement of refractive indices of the fractions showed a highly reproducible and highly linear separation (A; mean values: fx1--11.2%, fx2--12.5%, fx3--14.6%, fx4--17.3%, fx5--20.2%, fx6--23.2%, fx7--26.1%, fx8--28.5%, fx9--30.9%). Quantification of mass amounts of sterol (B), G_M1 (C), phospholipid (D) and protein (E) content were performed on 7 membrane fractions as described, and the results were normalized in each trial to 1 × 10⁹ sperm. All data were expressed as mean ± SEM (n=9–10). The different letters denote significant differences between the fractions where found (P < 0.05). Immunoblotting showed a representative distribution of caveolin-1 and α-tubulin through the gradient. Caveolin-1 was present in high abundance in fraction 10, but is not shown in this panel to get a clear image of the neighboring lane.

Fig. 3

Ratiometric comparisons of the components of the membrane fractions isolated from murine sperm without detergent. After quantitative analyses, mass amounts were converted into mole units using the average molecular masses. The ratios of sterol/phospholipid (A), G_M1/phospholipid (B) and G_M1/sterol (C) were estimated. Panel A contains the ratios for sterol/phospholipid for the various fractions. Note that the ratio of G_M1/phospholipid in fx1-4 (0.44) and fx 7 (0.25) were higher than in the other fxs (which ranged from 0.03–0.07). A ratio of total protein/total lipid was estimated using the sum of phospholipid plus sterol as an estimation of total lipid in each fraction (D). Because of variation in molecular masses between different proteins and species of lipids, this final ratiometric comparison was made using mass amounts. Note that the total protein/total lipid ratio was higher in fx -4 (10.4) than in other fxs (which ranged from 2.2–4.2). The broken line in each panel demarcates the boundary between raft and non-raft fractions typically used when separating membrane fractions by buoyancy on a step gradient and also reflects the point of a 1:1 molar ratio of sterol:PL. This boundary was consistent with the data shown here, in that fractions that were more buoyant (fractions 1–7) had enrichments of sterols and/or G_M1 relative to phospholipid. All results were expressed as mean ± SEM. The different letters denote significant differences between the fractions where found (P < 0.05).

Fig. 4

Morphological examination of membrane vesicles found in the various fractions obtained without detergent. Membrane vesicles from the 4 fractions analyzed were negative stained and subjected to electron microscopy as described. Images of vesicles from 4 fractions are depicted: pooled fractions 1–4 (A); fraction 5 (B); fraction 7 (C); and fraction 9 (D). Morphologically, the vesicles were similar in size in all fractions, with fraction 9 having increased abundance of smaller vesicles. Contamination of vesicles with other discernible sub-cellular structures was not visible in fractions 1–7; rare linear structures consistent with fragments of cytoskeletal elements of the sperm flagellum were seen in fraction 8, and these increased slightly in frequency in fraction 9.

Fig. 5

Localization of G_M1- and sterol-enriched membranes in murine germ cells. Testis paraffin sections were subjected to antigen retrieval, and then labeled with CTB-647. SYTOX Green Dye was used for nuclear staining (A, a, d, and g). G_M1 localized to the acrosomal membranes in round spermatids of golgi phase (A, b–c), cap phase (A, e–f), and acrosome phase (A, h–i), but was not observed in pachytene spermatocytes (A, f and i). G_M1 enriched membranes were also observed in condensed spermatids (arrowheads, A, a–c). Focal enrichments of sterols (B) were localized using PFO-D4 to a structure at the opposite pole from the acrosomal membranes, consistent with the golgi in location and appearance (compare B, a–b). Hoechst 33342 was used for nuclear staining (B, c). The relative lack of co-localization of G_M1 and sterols in the membranes of the developing acrosome were corroborated using CTB-488 and filipin (compare C, a–b). Merged images of CTB and sterol probes are shown in (B, d and C, c). DIC images of these cap phase spermatids are shown in (B, e and C, d). The bar is 10 μm in all panels.