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 3 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 2 different sterol-interacting proteins, apolipoprotein-A1 and prominin-1 in sperm from different zones. We found that apolipoprotein-A1 was gradually added to the plasma membrane overlying the acrosome, whereas prominin-1 was not, suggesting different mechanisms for raft protein acquisition. Our results define raft-associating proteins, demonstrate functional similarities and differences among raft subtypes, and provide insights into raft-mediated epididymal protein acquisition.

Phospholipase B Is Activated in Response to Sterol Removal and Stimulates Acrosome Exocytosis in Murine Sperm

“…Our previous finding that PLB is present in sperm membrane rafts (18) suggested the possible involvement of this enzyme. Although functional roles for PLB remain unclear in any mammalian cell, PLB plays a central role in membrane fusion for several pathogenic bacteria and fungi (19,47,48).…”

“…In our proteomic characterization of these raft subtypes, we identified PLB on the basis of multiple specific peptide sequences (18). PLB is an integral membrane enzyme that is able to hydrolyze both the sn-1 and sn-2 acyl ester bonds of glycerophospholipids, displaying calcium-independent PLA 1 , PLA 2 , and lysophospholipase activities (19).…”

Comparison of Membrane Characteristics between Freshly Ejaculated and Cryopreserved Sperm in the Chicken

“…Isolation of sperm membranes was performed as described previously (Asano et al, 2010). In brief, fresh and frozenthawed sperm (1×10 8 cells) were subjected to dounce homogenization and sonication using a BH-200P (TOMY SEIKO, Tokyo, Japan).…”

Isolation and Proteomic Characterization of the Mouse Sperm Acrosomal Matrix

“…The data set comprises 11 previously published data sets. After conversion to Mouse Genome Informatics (MGI) ID's, 820 unique genes from (26) were merged with 324 from (27) (36). The combined unique genes from all 11 data sets resulted in a total sperm proteome database consisting of 1162 proteins.…”