Unique species of ceramide (Cer) with very-long-chain polyunsaturated fatty acid (VLCPUFA), mainly 28-32 carbon atoms, 4-5 double bonds, in nonhydroxy and 2-hydroxy forms (n-V Cer and h-V Cer, respectively), are generated in rat spermatozoa from the corresponding sphingomyelins during the acrosomal reaction. The aim of this study was to determine the properties of these sperm-distinctive ceramides in Langmuir monolayers. Individual Cer species were isolated by HPLC and subjected to analysis of surface pressure, surface potential, and Brewster angle microscopy (BAM) as a function of molecular packing. In comparison with known species of Cer, n-V Cer and h-V Cer species showed much larger mean molecular areas and increased molecular dipole moments in liquid expanded phases, which suggest bending and partial hydration of the double bonded portion of the VLCPUFA. The presence of the 2-hydoxyl group induced a closer molecular packing in h-V Cer than in their chain-matched n-V Cer. In addition, all these Cer species showed liquid-expanded to liquid-condensed transitions at room temperature. Existence of domain segregation was confirmed by BAM. Additionally, thermodynamic analysis suggests a phase transition close to the physiological temperature for VLCPUFA-Cers if organized as bulk dispersions.

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Sphingomyelin (SM) is a ubiquitous sphingolipid component of cell membranes in vertebrate tissues. Like phosphatidylcholine (PC), it has phosphorylcholine as the head group, its hydrophobic moieties being a long-chain or a very-long-chain fatty acid amide-bound to the long-chain base sphingosine. Molecular species naturally occurring in mammalian SM often contain saturated (16:0, 18:0, 24:0) or monounsaturated (18:1, 22:1, 24:1) fatty acids ( 1 ).The testis and spermatozoa of various mammals including man contain SM species with an infrequent series of very-long-chain polyunsaturated fatty acids (VLCPUFAs) ( 2 ). These are elongated versions of ordinary 20-or 22-carbon atom tetraenoic, pentaenoic, and hexaenoic fatty acids of the n-3 or the n-6 series, according to the mammalian species. Whereas SMs from bovine and ovine spermatozoa are exceedingly rich in n-3 VLCPUFA with up to 34 carbon atoms, those from rodent germ and sperm cells mostly contain tetraenoic and pentaenoic fatty acids of the n-6 series with 28 to 32 carbon atoms, representative components being 28:4n-6, 30:5n-6, and 32:5n-6 ( 2, 3 ).In some mammals including the rat, a unique series of molecular species of SM containing 2-hydroxylated derivatives of the mentioned VLCPUFA also occurs in testis and spermatozoa ( 4 ). In adult rat testis, the SM species having nonhydroxy VLCPUFA and 2-hydroxy VLCPUFA (n-V SM and h-V SM, respectively) are exclusive components of spermatogenic cells, as are the corresponding ceramides containing Abstract In rat germ cells and spermatozoa, sphingomyelin (SM) contains molecular species with nonhydroxy (n) and 2-hydroxy (h) very-long-chain polyunsaturated fatty acids (V), the most abundant being SMs with (n-and h-) 28:4n-6, 30:5n-6, and 32:5n-6 as acyl chains. The aim of this study was to gain information about their thermotropic behavior and interactions with other lipids. After isolation from rat testis, multilamellar and giant unilamellar vesicles from these SMs were examined using fl uorescent probes.

Molecular species of sphingomyelin (SM) with nonhydroxy (n) and 2-hydroxy (h) very long chain polyunsaturated fatty acids (n- and h-28:4, 30:5, and 32:5) abound in rat spermatogenic cells and spermatozoa. These SMs are located on the sperm head, where they are converted to the corresponding ceramides (Cer) after the completion of the acrosomal reaction, as induced in vitro. The aim of this study was to look into the surface properties of these unique SM species and how these properties change by the SM → Cer conversion. After isolation by HPLC, these SMs were organized in Langmuir films and studied alone, in combination with different proportions of Cer, and during their conversion to Cer by sphingomyelinase. Compression isotherms for all six SMs under study were compatible with a liquid-expanded (LE) state and showed large molecular areas. Only the longest SMs (n-32:5 and h-32:5 SM) underwent a phase transition upon cooling. Interestingly, the abundant h-28:4 Cer exhibited a highly compressible liquid-condensed (LC) phase compatible with a high conformational freedom of Cer molecules but with the characteristic low diffusional properties of the LC phase. In mixed films of h-28:4 SM/h-28:4 Cer, the components showed favorable mixing in the LE phase. The monolayer exhibited h-28:4 Cer-rich domains both in premixed films and when formed by the action of sphingomyelinase on pure h-28:4 SM films. Whereas the SMs from sperm behaved in a way similar to that of shorter acylated SMs, the corresponding Cers showed atypical rheological properties that may be relevant to the membrane structural rearrangements that take place on the sperm head after the completion of the acrosomal reaction.

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The sphingolipids (SLs) of rodent spermatogenic cells (spermatocytes, spermatids) and spermatozoa contain nonhydroxylated and 2-hydroxylated versions of very-long-chain (C26-C32) PUFAs (n-V and h-V, respectively) not present in Sertoli cells (SCs). Here, we investigated the expression of selected fatty acid elongases [elongation of very-long-chain fatty acid protein ()], with a focus on , and a fatty acid 2-hydroxylase () in rat testes with postnatal development and germ cell differentiation. Along with and, was actively transcribed in the adult testis. mRNA levels were high in immature testes and SCs, though the protein was absent. The Elovl4 protein was a germ cell product. All cells under study elongated [H]arachidonate to tetraenoic and pentaenoic C24 PUFA, but only germ cells produced C26-C32 PUFAs. Spermatocytes displayed the highest Elovl4 protein levels and enzymatic activity. mRNA was produced exclusively in germ cells, mostly round spermatids. As a protein, Fa2h was mainly concentrated in late spermatids, in the step of spermiogenesis in which they elongate and their heads change shape. The expression of Elovl4 and Fa2h thus correlate with the abundance of n-Vs and h-Vs in the SLs of rat spermatocytes and spermatids, respectively.

The mitochondrion is an essential organelle enclosed by two membranes whose functionalities depend on their very specific protein and lipid compositions. Proteins from the outer mitochondrial membrane (OMM) are specialized in mitochondrial dynamics and mitophagy, whereas proteins of the inner mitochondrial membrane (IMM) have dedicated functions in cellular respiration and apoptosis. As for lipids, the OMM is enriched in glycerophosphatidyl choline but cardiolipin is exclusively found within the IMM. Though the lipid topology and distribution of the OMM and IMM are known since more than four decades, little is known about the interfacial and dynamic properties of the IMM and OMM lipid extracts. Here we build monolayers, supported bilayers and giant unilamellar vesicles (GUVs) of native OMM and IMM lipids extracts from porcine heart. Additionally, we perform a comparative analysis on the interfacial, phase immiscibility and mechanical properties of both types of extract. Our results show that IMM lipids form more expanded and softer membranes than OMM lipids, allowing a better understanding of the physicochemical and biophysical properties of mitochondrial membranes.