Abstract
BackgroundDetergents are often used to isolate proteins, lipids as well as "detergent-resistant membrane domains" (DRMs) from cells. Different detergents affect different membrane structures according to their physico-chemical properties. However, the effects of different detergents on membrane lysis of boar spermatozoa and the lipid composition of DRMs prepared from the affected sperm membranes have not been investigated so far.ResultsSpermatozoa were treated with the selected detergents Pluronic F-127, sodium cholate, CHAPS, Tween 20, Triton X-100 and Brij 96V. Different patterns of membrane disintegration were observed by light and electron microscopy. In accordance with microscopic data, different amounts of lipids and proteins were released from the cells by the different detergents. The biochemical methods to assay the phosphorus and cholesterol contents as well as 31P NMR to determine the phospholipids were not influenced by the presence of detergents since comparable amounts of lipids were detected in the organic extracts from whole cell suspensions after exposure to each detergent. However, matrix-assisted laser desorption and ionization time-of-flight mass spectrometry applied to identify phospholipids was essentially disturbed by the presence of detergents which exerted particular suppression effects on signal intensities. After separation of the membrane fractions released by detergents on a sucrose gradient only Triton X-100 and sodium cholate produced sharp turbid DRM bands. Only membrane solubilisation by Triton X-100 leads to an enrichment of cholesterol, sphingomyelin, phosphatidylinositol and phosphatidylethanolamine in a visible DRM band accompanied by a selective accumulation of proteins.ConclusionThe boar sperm membranes are solubilised to a different extent by the used detergents. Particularly, the very unique DRMs isolated after Triton X-100 exposure are interesting candidates for further studies regarding the architecture of sperm.
Highlights
Detergents are often used to isolate proteins, lipids as well as "detergent-resistant membrane domains" (DRMs) from cells
This study demonstrates that residual amounts of detergents - which even remain in DRM lipids subsequent to extraction with organic solvents - essentially disturb the analysis of membrane lipids by MALDI-TOF mass spectrometry (MS)
As measures of the extraction efficiency, the cholesterol and phosphate contents in the organic sperm extracts were determined by biochemical assays
Summary
Detergents are often used to isolate proteins, lipids as well as "detergent-resistant membrane domains" (DRMs) from cells. The lipid composition, in particular the cholesterol moiety, the headgroup of phospholipids (PLs) and the number of double bonds in the acyl, alkyl or alkenyl residues are the main determinants of membrane flexibility, its fusion behaviour and the interaction with proteins This is important in the case of spermatozoa that possess a considerable content of highly unsaturated acyl (or alkyl and alkenyl) residues, in particular docosahexaenoyl (22:6) and docosapentaenoyl (22:5) residues [1]. The lower densities of these lipid-protein complexes with less incorporated detergent allow their isolation from other membrane parts by flotation on density gradients At present, it is still under discussion whether those DRMs reflect real organisational units of cellular membranes or represent only an artificial clustering upon their preparation with detergents
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