Ulrike Jakop

Changes of murine sperm phospholipid composition during epididymal maturation determined by MALDI-TOF mass spectrometry

After leaving the testis, spermatozoa undergo several important steps of biochemical maturation during the passage through the epididymis, increasing their motility and fertilizing ability. These changes comprise (among others) the modification of the phospholipid composition of the sperm membrane. This process is thought to be important for the achievement of motility and fertilizing capacity. The lipids of the sperm membrane are characterized by a significant content of unsaturated fatty acyl residues, resulting in a high sensitivity against oxidative stress. This is evidenced by the appearance of lysolipids, for example, lysophosphatidylcholine, which acts like a detergent and is normally present in only very small amounts in biological membranes. The epididymis represents a tubular system comprising three main parts (caput, corpus, and cauda), through which the spermatozoa are consecutively transported undergoing distinct maturation stages. Using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, we established three striking differences in the lipid composition of murine spermatozoa from the different epididymal regions: in comparison to the caput sperm, sperm from the cauda are characterized by (1) a higher degree of unsaturation (PC 18:0/22:5 and 18:0/22:6 vs. 18:0/20:4 and 18:0/18:1), (2) an enhanced plasmalogen content, and (3) an enhanced content of lysolipids. These changes are likely to be of physiological relevance and potentially useful as diagnostic markers of sperm maturation and acquisition of motility.

A simple method to identify ether lipids in spermatozoa samples by MALDI-TOF mass spectrometry

AbstractPlasmalogens (alkenylacyl glycerophospholipids) are important lipid constituents of many tissues and cells (e.g., selected spermatozoa). Since the molecular weights of plasmalogens overlap with that of diacyl- or alkyl acyl lipids, sophisticated mass spectrometry (MS; including MS/MS) analysis is normally used for the unequivocal identification of plasmalogens. We will show here that a simple matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (without MS/MS capability) in combination with acidic hydrolysis and subsequent derivatization with 2,4-dinitrophenylhydrazine (DNPH) and/or digestion with phospholipase A2 (PLA2) is sufficient to determine the contributions of ether lipids in spermatozoa extracts. As neither diacyl nor alkylacyl lipids are sensitive to acids and do not react with DNPH, the comparison of the mass spectra before and after treatment with acids and/or DNPH addition readily provides unequivocal information about the plasmalogen content. Additionally, the released aldehydes are readily converted into the 2,4-dinitrophenylhydrazones and can be easily identified in the corresponding negative ion mass spectra. Finally, PLA2 digestion is very useful in confirming the presence of plasmalogens. The suggested method was validated by analyzing roe deer, bovine, boar, and domestic cat spermatozoa extracts and comparing the results with isolated phospholipids. Figureᅟ

MALDI-TOF mass spectrometry as a simple tool to determine the phospholipid/glycolipid composition of sperm: Pheasant spermatozoa as one selected example

Cellular membranes are composed of highly variable lipid molecules, mainly cholesterol and phospholipids (PLs). The cholesterol moiety and the saturation degree of the fatty acyl residues of PL determine the fluidity of the membrane, which is particularly important for sperm because they have to undergo characteristic membrane-dependent processes (acrosomal exocytosis and fusion with the oocyte). Glycolipids are an essential part of the membrane surface acting as key mediators in the interactions of sperm with components of the female genital tract. Although the lipid composition of many mammalian spermatozoa has already been determined, the lipid composition of avian spermatozoa has scarcely been investigated. Using spermatozoa extracts of the ring-necked pheasant (Phasianus colchicus) as a selected example, this work demonstrates that matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a simple and fast method to determine spermatozoal lipid compositions. The lipid compositions of pheasant spermatozoa have not yet been investigated. In addition to common membrane (primarily diacyl) PL (sphingomyelin, phosphatidylcholine, phosphatidylinositol and phosphatidylethanolamine), remarkable variation of different sulfoglycolipids (sulfogalactocerebrosides) was identified. This is in strong contrast to all other animal species investigated so far which nearly exclusively contain the sulfoglycolipid seminolipid (sulfogalactoalkylacylglycerol). We emphasize that the MALDI MS approach allows the characterization of sulfoglycolipids of sperm within a few minutes without the necessity for previous chromatographic separation.

Lipid dynamics in boar sperm studied by advanced fluorescence imaging techniques

The (re)organization of membrane components is of special importance to prepare mammalian sperm to fertilization. Establishing suitable methods to examine physico-chemical membrane parameters is of high interest. We characterized the behavior of fluorescent (NBD) analogs of sphingomyelin (SM), phosphatidylserine (PS), and cholesterol (Ch) in the acrosomal and postacrosomal macrodomain of boar sperm. Due to their specific transverse membrane distribution, a leaflet-specific investigation of membrane properties is possible. The behavior of lipid analogs in boar sperm was investigated by fluorescence lifetime imaging microscopy (FLIM), fluorescence recovery after photobleaching (FRAP), and fluorescence correlation spectroscopy (FCS). The results were compared with regard to the different temporal and spatial resolution of the methods. For the first time, fluorescence lifetimes of lipid analogs were determined in sperm cell membrane and found to be in a range characteristic for the liquid-disordered phase in artificial lipid membranes. FLIM analyses further indicate a more fluid microenvironment of NBD-Ch and NBD-PS in the postacrosomal compared to the acrosomal region. The concept of a more fluid cytoplasmic leaflet is supported by lower fluorescence lifetime and higher average D values (FCS) for NBD-PS in both head compartments. Whereas FLIM analyses did not indicate coexisting distinct liquid-ordered and -disordered domains in any of the head regions, comparisons between FRAP and FCS measurements suggest the incorporation of NBD-SM as well as NBD-PS in postacrosomal subpopulations with different diffusion velocity. The analog-specific results indicate that the lipid analogs used are suitable to report on the various physicochemical properties of different microenvironments.

The membrane phospholipid composition of honeybee (Apis mellifera) workers reflects their nutrition, fertility, and vitellogenin stores

Pollen feeding in the honeybee (Apis mellifera) is dependent on the caste, ethotype, and dominance status of individuals. Pollen is the near-exclusive external source of lipids for the colony. Lipids from pollen are rich in polyunsaturated fatty acyl residues, which, because of their sensitivity to lipid peroxidation, have been suggested to limit the lifespan of individual bees. We here investigated whether the spectrum of phosphatidylcholine (PC), the main class of membrane-constituting lipids in insects, is indeed, connected to pollen feeding. We further studied the relationship between pollen feeding, PC spectra, and fat-body stores of vitellogenin, an indicator of potential longevity in bees. For this, we determined the pollen consumption, PC spectra, abdominal vitellogenin stores, and behavioral as well as reproductive status of individuals in queenless groups of workers. In contrast to earlier studies, we found that reproduction in workers is not universally linked to trophallactic dominance alone, but can be accompanied by strong pollen consumption. Pollen consumption seemed connected to a strong remodeling of tissue PC spectra. There was no systematic link between these spectra and trophallactic activity, but individuals with strongly deviating spectra also showed extreme behavioral profiles. Abdominal vitellogenin was strongly and positively linked to pollen-influenced PC spectra and the prevalence of polyunsaturated fatty acyl residues, contradicting the hypothesis that pollen consumption is systematically leading to a shorter lifespan in workers. Our results suggest that the association between pollen consumption, short lifespan, and functional sterility that differentiates workers from queens cannot in all cases be extended to the situation within the worker caste.

The Phospholipid Composition of Kangaroo Spermatozoa Verified by Mass Spectrometric Lipid Analysis

Cryopreservation of kangaroo sperm has not been successful so far, and yet there is no promising cryopreservation protocol for these cells available. However, conservation of gametes is extremely important, particularly in the context of preserving endangered species. As spermatozoa are comprised of different membrane systems, the composition of these membranes might account for difficulties in cryopreservation. Lipids, as the main components, affect the physical properties of biological membranes and play a major role in sperm maturation. Therefore, knowledge of the lipid composition is crucial for any further step toward the preservation of the species. We used MALDI-TOF, ESI-IT, tandem mass spectrometry, and thin layer chromatography to investigate the lipid composition of epididymal spermatozoa of four different kangaroo species. Spectra of these species were very similar with respect to the identified lipid species. Tremendous changes in the lipid composition during the transit of sperm from caput to cauda epididymis could be seen, specifically an increase in poly-unsaturated fatty acids, ether lipids, and plasmalogens, as well as a reduction in mono- and di-unsaturated fatty acids. Additionally, phosphatidylcholines containing docosatrienoic acid (22:3), a heretofore unknown fatty acid for sperm membranes, showed the highest abundance in kangaroo sperm.