Characterization of the proteomes associating with three distinct membrane raft sub-types in murine sperm

Abstract

Mammalian sperm are transcriptionally and translationally inactive. To meet changing needs in the epididymis and female tract, they rely heavily on post-translational modifications and protein acquisition/degradation. Membrane rafts are sterol and sphingolipid-enriched micro-domains that organize and regulate various pathways. Rafts have significance in sperm by transducing the stimulus of sterol efflux into changes in intracellular signaling that confer fertilization competence. We recently characterized three biochemically distinct sub-types of sperm rafts, and now present profiles for proteins targeting to and associating with these sub-types, along with a fraction largely comprised of "non-raft" domains. Proteomics analysis using a gel-based LC-MS/MS approach identified 190 strictly validated proteins in the raft sub-types. Interestingly, many of these are known to be expressed in the epididymis, where sperm membrane composition matures. To investigate potential roles for rafts in epididymal protein acquisition, we compared the expression and localization of two different sterol-interacting proteins, apolipoprotein-A1 (apoA1) and prominin-1 (prom1) in sperm from different zones. We found that apoA1 was gradually added to the plasma membrane overlying the acrosome, whereas prom1 was not, suggesting different mechanisms for raft protein acquisition. Our results define raft-associating proteins, demonstrate functional similarities and differences among raft sub-types, and provide insights into raft-mediated epididymal protein acquisition.

Fig. 1

Membrane organization and biochemical characteristics of raft sub-types in murine sperm. A schematic diagram of murine sperm shows the high degree of cell polarization and compartmentalization of the plasma membrane (A). The plasma membrane overlying the acrosome (APM) is highly enriched in G_M1, detected by FITC-CTB (B). Sterols and caveolin-1 are also segregated into the same area (C and D, [12, 14]). Biochemical analysis of membrane raft fractions demonstrated the presence of 3 different raft sub-types (fx 1-4, 5 and 6, and 7) characterized by reproducibly distinct lipid and protein compositions [summarized in panel E; “+” signs denote relative molar ratios (sterols or G_M1 versus phospholipid) or mass ratios (total protein:total lipid) in the sub-types [9]].

Fig. 2

Non-detergent based separation of membrane rafts and sample preparation for mass spectrometry. Sperm membranes were isolated by dounce homogenization and sonication and then spun down at 10,000 × g for 10 minutes. The supernatant was then centrifuged at 100,000 × g to yield a membrane pellet. This was mixed with 80% (w/v) sucrose to obtain a final sucrose concentration of 45%. The lysate was overlaid with a 10–30% linear sucrose density gradient and centrifuged at 100,000 × g for 28 hours. The supernatant was divided into 10 fractions from the top. Reproducibility of the raft separation was confirmed with refractometry (B) [9]. 1-D SDS-PAGE was utilized to separate proteins and the gel excised into 9 gel pieces (a–i) for tryptic digestion followed by mass spectrometry.

Fig. 3

Characterization of protein profiles among raft fractions. A Venn diagram showing overlap of identified proteins among three raft fractions (A–I), and raft versus non-raft fractions (A–II). Similarities of protein profiles were characterized manually among raft fractions (fx 1-4, 5 and 7) and between each raft sub-type (fx 1-4, 5 and 7) and non-raft fractions (fx 9). The distributions of isoelectric points (pI) and GRAVY (http://www.expasy.ch/tools/protparam.html) in raft sub-types and non-rafts are portrayed by use of a relative frequency histogram (B) and (C). The numbers of proteins categorized in the ranges are indicated above the bars. The proportions of negatively or positively charged proteins (pI < 7 or ≥ 7) at physiological pH were compared (B inset). Statistical analysis among groups was performed with chi-square test for independence or Fisher’s exact probability test when sample sizes smaller than 5 were analyzed. The asterisks denote significant difference for the raft sub-types when compared with fx 9 in the respective compartments (P < 0.05). Percentages of proteins with at least one transmembrane domain (TM) and protein expression in murine epididymides (D).

Fig. 4

Expression and localization of apoA1 and prom1 in murine sperm. Immunoblots of proteins from cauda epididymal sperm confirmed that apoA1 and prom1 were expressed at the appropriate molecular weights (A). ApoA1 was transferred to the sperm plasma membrane during epididymal transit (B–I), consistent with the results of immunoblots for this protein (C). However, localization of prom1 was conserved between different epididymal regions, showing the midpiece and the cytoplasmic droplet (arrows) (B–II).