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• W2044134902 abstract "Very long-chain (C24 to C34) polyunsaturated fatty acids (VLCPUFA) are important constituents of sphingomyelin (SM) and ceramide (Cer) in testicular germ cells. In the present paper we focused on the SM and Cer and their fatty acids in spermatozoa and their main regions, heads and tails. In bull and ram spermatozoa, SM was the third most abundant phospholipid and VLCPUFA were the major acyl groups (∼70%) of SM and Cer. In rat epididymal spermatozoa the SM/Cer ratio was low in the absence of and could be maintained high in the presence of the cation chelator EDTA, added to the medium used for sperm isolation. This fact points to the occurrence of an active divalent cation-dependent sphingomyelinase. Bull and rat sperm had an uneven head-tail distribution of phospholipid, with virtually all the VLCPUFA-rich SM located at the head, the lower SM content in the rat being determined by the lower sperm head/tail size ratio. Most of the SM from bull sperm heads was readily solubilized with 1% Triton X-100 at 4 °C. The detergent-soluble SM fraction was richer in VLCPUFA than the nonsoluble fraction and richer in saturated fatty acids. Cer was produced at the expense of SM, thus decreasing severalfold the SM/Cer ratio in rat spermatozoa incubated for 2 h in presence of the sperm-capacitating agents, calcium, bicarbonate, and albumin. The generation of Cer from SM in the sperm head surface may be an early step among the biochemical and biophysical changes known to take place in the spermatozoon in the physiological events preceding fertilization. Very long-chain (C24 to C34) polyunsaturated fatty acids (VLCPUFA) are important constituents of sphingomyelin (SM) and ceramide (Cer) in testicular germ cells. In the present paper we focused on the SM and Cer and their fatty acids in spermatozoa and their main regions, heads and tails. In bull and ram spermatozoa, SM was the third most abundant phospholipid and VLCPUFA were the major acyl groups (∼70%) of SM and Cer. In rat epididymal spermatozoa the SM/Cer ratio was low in the absence of and could be maintained high in the presence of the cation chelator EDTA, added to the medium used for sperm isolation. This fact points to the occurrence of an active divalent cation-dependent sphingomyelinase. Bull and rat sperm had an uneven head-tail distribution of phospholipid, with virtually all the VLCPUFA-rich SM located at the head, the lower SM content in the rat being determined by the lower sperm head/tail size ratio. Most of the SM from bull sperm heads was readily solubilized with 1% Triton X-100 at 4 °C. The detergent-soluble SM fraction was richer in VLCPUFA than the nonsoluble fraction and richer in saturated fatty acids. Cer was produced at the expense of SM, thus decreasing severalfold the SM/Cer ratio in rat spermatozoa incubated for 2 h in presence of the sperm-capacitating agents, calcium, bicarbonate, and albumin. The generation of Cer from SM in the sperm head surface may be an early step among the biochemical and biophysical changes known to take place in the spermatozoon in the physiological events preceding fertilization. In a number of mammals including humans a series of very long-chain polyunsaturated fatty acids (VLCPUFA), 2The abbreviations used are: VLCPUFA, very long-chain polyunsaturated fatty acids; Cer, ceramide; SM, sphingomyelin; SMase, sphingomyelinase; PL, phospholipid; PBS, phosphate-buffered saline; KR, Krebs-Ringer; GC-MS, gas chromatography-mass spectrometry; CGP, choline glycerophospholipid; EGP, ethanolamineglycerophospholipids; DPG, diphosphatidylglycerol; ros, rod outer segment(s); PC, phosphatidylcholine. 2The abbreviations used are: VLCPUFA, very long-chain polyunsaturated fatty acids; Cer, ceramide; SM, sphingomyelin; SMase, sphingomyelinase; PL, phospholipid; PBS, phosphate-buffered saline; KR, Krebs-Ringer; GC-MS, gas chromatography-mass spectrometry; CGP, choline glycerophospholipid; EGP, ethanolamineglycerophospholipids; DPG, diphosphatidylglycerol; ros, rod outer segment(s); PC, phosphatidylcholine. i.e. n-6 and n-3 tetraenoic, pentaenoic and hexaenoic fatty acids with up to 32 or 34 carbon atoms, depending on the species, was characterized in the sphingomyelin (SM) from testis and spermatozoa (1Poulos A. Johnson D.E. Beckman K. White I.G. Easton C. Biochem. J. 1987; 248: 961-964Crossref PubMed Scopus (57) Google Scholar, 2Robinson B.S. Johnson D.W. Poulos A. J. Biol. Chem. 1992; 268: 1746-1751Abstract Full Text PDF Google Scholar). In the testis of various mammals, we focused on the fatty acids of the ceramide (Cer), a lipid molecule with which SM bears a close precursor-product relationship, showing that SM and Cer species containing VLCPUFA are a specific feature of cells of the spermatogenic lineage (3Furland N.E. Zanetti S.R. Oresti M.G. Maldonado E.N. J. Biol. Chem. 2007; 282: 18141-18150Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Because these testicular cells are predecessors of spermatozoa, the question arose as to the quantitative importance of these molecules in spermatozoa, where they could play a role in sperm functions related to fertilization.Transit through the epididymis is a crucial phase in sperm maturation. Spermatozoa exiting the testis are immotile, unable to bind to eggs and to undergo the acrosomal reaction in vitro in response to commonly used stimuli. By the time they reach the region of cauda epididymis, sperm cells have acquired their progressive motility and their ability to bind, penetrate, and fertilize eggs (4Williams R.M. Graham J.K. Hammerstedt R.H. Biol. Reprod. 1991; 44: 1080-1091Crossref PubMed Scopus (40) Google Scholar). One of the questions we addressed was whether epididymal maturation gives rise to spermatozoa with a larger or a smaller proportion of SM and Cer containing these VLCPUFA as opposed to other fatty acids.Spermatozoa are functionally regionalized cells. Sperm-oocyte interactions are head-related functions, energy production for motion is a main role of mitochondria located in the midpiece, and motility is a function associated with the contractile proteins located to the tail. We next addressed the distribution of VLCPUFA-containing species of SM and Cer in sperm tails and heads and employed a nonionic detergent, Triton X-100, to gain information on the behavior of the lipids present in the plasma membrane of sperm heads, focusing on SM and its fatty acids.As a prerequisite to fertilization, mammalian spermatozoa must undergo the time-dependent priming process known as capacitation, as a result of which sperm change their motility patterns and migrate to the site of fertilization, in the process becoming competent to undergo the acrosomal reaction. This reaction entails a specific fusion event in the spermatozoal head whereby the plasma membrane of the apical region and the outer membrane of the underlying acrosome fuse at many points, creating vesiculations and fenestrations that enable exocytosis of the acrosomal constituents. The release of hydrolytic enzymes is one of the factors facilitating penetration of the egg coat, thus allowing the post-acrosomal membrane of a spermatozoon to merge with the oolemma. The molecular mechanisms underlying these processes have been studied in great molecular detail over the years (see Refs. 5Yaganimachi R. Knobil E. Neill J.D. The Physiology of Reproduction. 2nd Ed. Raven Press, New York1994: 189-317Google Scholar, 6Baldi E. Luconi M. Bonaccorsi M. Forti G. Front. Biosci. 2001; 5: 110-123Crossref Google Scholar, 7Flesh F.M. Gadella B.M. Biochim. Biophys. Acta. 2000; 1469: 197-235Crossref PubMed Scopus (430) Google Scholar, 8Martinez P. Morros A. Front. Biosci. 1996; 1: 103-117Crossref PubMed Google Scholar for reviews).Capacitation increases sperm plasma membrane lipid disorder, reducing the stability of such membrane in preparation for the hyperactivated motility and membrane fusion events that follow (8Martinez P. Morros A. Front. Biosci. 1996; 1: 103-117Crossref PubMed Google Scholar). One of the first lipid changes shown to occur in the spermatozoal membrane in this context is a significant loss of cholesterol (9Davis B.K. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 7560-7564Crossref PubMed Scopus (246) Google Scholar). More recently, the normal transbilayer asymmetry of four phospholipids (PL) including SM in the sperm plasma membrane was shown to change as a result of capacitation (10Gadella B.M. Harrison R.A.P. Development. 2000; 127: 2407-2420Crossref PubMed Google Scholar). The basic facts that SM has a high affinity for cholesterol, that both lipids are abundant in the sperm plasma membrane, and that this membrane has an active neutral sphingomyelinase (SMase) activity (11Hinkovska V.T. Petkova D.H. Koumanov K.S. Biochem. Cell Biol. 1987; 65: 525-528Crossref PubMed Scopus (21) Google Scholar) capable of producing Cer in situ suggest that the SM-Cer pair may well be involved in sperm membrane functions. Given the increasing importance being accorded to SM and its hydrolysis in the generation of biologically active, and membrane disturbing, membrane-bound metabolites, especially Cer, investigation of SM in regard to sperm functions is of obvious interest.Exposure of human spermatozoa to an exogenous SMase abbreviates by several hours the loss of sterols and sperm capacitation, measured as the ability of sperm to acrosome (react in response to progesterone), incubation of sperm with C6-Cer mimicking in part the SMase effects (12Cross N.L. Biol. Reprod. 2000; 63: 1129-1134Crossref PubMed Scopus (41) Google Scholar). The acrosomal exocytosis triggered by Ca2+ in the presence of a calcium ionophore is enhanced after incubation of boar spermatozoa with the analogue C2-Cer and with a ceramidase inhibitor, the results suggesting that Cer may be involved in the mechanisms underlying the acrosomal reaction (13Murase T. Imaeda N. Kondoh N. Tsubota T. J. Reprod. Dev. 2004; 50: 667-674Crossref PubMed Scopus (14) Google Scholar). The nature of the endogenous SM and Cer of spermatozoa and how the SM/Cer ratio is affected during normal sperm functioning remain to be investigated.In this work we studied SM and Cer, focusing on their fatty acids, in bull and ram spermatozoa isolated from fresh semen and in rat spermatozoa isolated from cauda epididymis. Beyond the exceptionality of their fatty acids, study of these lipids showed that conspicuous modifications in the SM/Cer ratio occur not only when spermatozoa are incubated in capacitating conditions, but even spontaneously when spermatozoa are isolated after a few simple steps. This biochemical information is of interest to both biophysicists and gamete physiologists, because the in situ conversion SM → Cer is likely to result in consequential changes in sperm membrane physicochemical properties and functions.EXPERIMENTAL PROCEDURESSpermatozoa—Freshly ejaculated semen was collected from fertile Shorthorn bulls in a local breeding station, transported to the laboratory in a Dewar glass at 25 °C, and rapidly centrifuged at room temperature at 600 × g to separate the seminal plasma. The cells were then gently suspended in phosphate-buffered saline (PBS) (95 mm NaCl, 2.7 mm KCl, 1.2 mm KH2PO4, 8.1 mm Na2HPO4, pH 7.4) and centrifuged; this procedure was repeated twice to remove traces of seminal plasma (and its lipids) from sperm samples. Semen from Australian Merino rams was collected during the reproductive season and treated similarly. Rat spermatozoa were obtained from 4–5-month-old Wistar rats.For rat spermatozoa isolation, caudal epididymi were excised, freed of their fat pad and blood vessels, and transferred to small dishes containing PBS. A few incisions were made with a scalpel blade in the sperm-rich area of each epididymis. The organs thus treated were gently incubated for 15 min at 34 °C to allow release and diffusion of spermatozoa and then removed. The spermatozoa were obtained from the supernatants by centrifugation at 600 × g. They were then resuspended in 65% (w/v) sucrose at 4 °C and purified in a discontinuous density gradient, made up of 70 and 75% (w/v) sucrose in PBS. These steps decreased the yield but increased the purity of the epididymal spermatozoa samples, in comparison with the procedures used in an earlier report (14Aveldaño M.I. Rotstein N.P. Vermouth N.T. Biochem. J. 1992; 283: 235-241Crossref PubMed Scopus (67) Google Scholar). When present, the metal-ion chelator EDTA was incorporated at a concentration of 2.5 mm in the medium used for sperm isolation, including the initial PBS and the sucrose solutions. It was necessary to pool spermatozoa isolated from the epididymi of several rats to obtain sufficient material for analysis of SM, Cer, and their fatty acids.Sperm Heads and Tails—To separate sperm heads from tails, spermatozoa from freshly ejaculated bull semen or from the epididymi of several rats were gently suspended in PBS containing 1 mm phenylmethylsulfonyl fluoride, and the samples were sonicated at 30-s intervals using a model 250 Branson sonifier (15Gitlits V.M. Toh B.H. Loveland K.L. Sentry J.W. Eur. J. Cell Biol. 2000; 79: 104-111Crossref PubMed Scopus (54) Google Scholar). The resulting mixture of heads and tails was pelleted at 600 × g at 4 °C, resuspended in 65% sucrose, and loaded onto a step gradient made up of 70 and 75% (w/v) sucrose in PBS-phenylmethylsulfonyl fluoride. The tubes were centrifuged at 104,000 × g at 4 °C for 60 min. The head fraction was collected as a pellet. The fraction at the 65–70% sucrose interface containing the tail-rich fraction was collected, resuspended, and purified by a further centrifugation (104,000 × g, 4 °C, 60 min) on a 65–75% sucrose gradient. The purity of the fractions was verified by light microscopy.Detergent Solubilization of Head Phospholipids—A bull sperm head fraction prepared as just described was solubilized with a nonionic detergent to study the fatty acids of the SM present in the detergent-soluble and nonsoluble fractions. The following procedure was chosen in view of its successful application to the study of the localization of enzymes in human spermatozoal parts (16Horowitz J.A. Toeg H. Orr G.A. J. Biol. Chem. 1984; 259: 832-838Abstract Full Text PDF PubMed Google Scholar) and because it has been reported to completely remove the plasma membrane from whole, intact spermatozoa (17Fisch J.D. Behr B. Conti M. Human Reprod. 1998; 13: 1248-1254Crossref PubMed Scopus (90) Google Scholar). Briefly, the pellet of sperm heads was suspended and homogenized in a hypotonic solution (58.5 mm NaCl, 4.8 mm KCl, 1 mm MgCl2, 58.5 mm KH2PO4, and 50 mm Tris-HCl, pH 7.4), containing 2 mm phenylmethylsulfonyl fluoride. The resulting homogenate was centrifuged at 40,000 × g for 15 min to separate the sperm cytosolic fraction. The pellet was resuspended in 1% Triton X-100 (Sigma) prepared in the same saline buffer. The suspension was agitated gently for 10 min and then centrifuged at 40,000 × g for 15 min. This produced a detergent-soluble supernatant and a detergent-insoluble pellet. A temperature of 4 °C was maintained throughout.Incubations of Rat Spermatozoa—Spermatozoa were isolated from the epididymi of a dozen rats in PBS containing or lacking EDTA, pelleted at 600 × g, and washed once in the same medium to reduce in the latter group possible divalent cations from the epididymal tissue. After discarding the supernatants, the cells were gently resuspended in a Krebs-Ringer (KR)-based medium (95 mm NaCl, 2.7 mm KCl, 1.2 mm KH2PO4, 8.1 mm Na2HPO4, and 5.5 mm glucose, pH 7.4) in three different conditions. The cells that had been isolated in media containing EDTA were resuspended in KR containing 2.5 mm EDTA. The cells that had not been in contact with this chelator were divided into equal groups, pelleted, and resuspended in KR containing 2 mm CaCl2, 25 mm NaHCO3, and 0.4% fatty acid-free bovine serum albumin (Sigma), a medium classically used to stimulate sperm capacitation (18Toyoda Y. Chang M.C. J. Reprod. Fertil. 1974; 36: 9-22Crossref PubMed Scopus (319) Google Scholar) or KR not containing these three factors. The three groups of cells were incubated for 2 h at 37 °C under a 5% CO2, 95% air atmosphere. After incubations, aliquots were fixed and stained with Coomassie Blue for assessment of acrosomal status as described by Larson and Miller (19Larson J.L. Miller D.J. Mol. Reprod. Dev. 1999; 52: 445-449Crossref PubMed Scopus (169) Google Scholar). Sperm counting was carried out using a Neubauer hemocytometric chamber.Lipid Separation—Most of the solvents used in this work were high pressure liquid chromatography grade (JT Baker; UVE, Dorwill, Argentina). After preparation of lipid extracts, centrifugation, and partition (20Bligh E.G. Dyer W.J. Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (42174) Google Scholar), the organic phases containing the lipids were recovered, and the solvents were evaporated under N2. The aliquots were taken for measuring total lipid phosphorus content in samples (21Rouser G. Fleischer S. Yamamoto A. Lipids. 1970; 5: 494-496Crossref PubMed Scopus (2859) Google Scholar). For analysis of phospholipid class composition, aliquots of the extracts were spotted on high performance TLC plates (Merck) and resolved using chloroform/methanol/acetic acid/water (50:37.5:3.5:2, by volume), after presaturating the plates with the solvent vapors for 15 min (22Holub B.J. Skeaff C.M. Methods Enzymol. 1987; 141: 234-422Crossref PubMed Scopus (173) Google Scholar). After separation, the phospholipid spots were located with the aid of iodine vapors, scraped from the plates, and quantified by phosphorus analysis (21Rouser G. Fleischer S. Yamamoto A. Lipids. 1970; 5: 494-496Crossref PubMed Scopus (2859) Google Scholar).To isolate Cer for fatty acid analysis, lipid extracts from spermatozoa or their parts were spotted (as bands) on TLC plates (500 μm, silica gel G) under N2, along with commercial standards (Sigma). Ceramides were resolved using chloroform/methanol/ammonia/water (90:10:05:0.5, by volume) (3Furland N.E. Zanetti S.R. Oresti M.G. Maldonado E.N. J. Biol. Chem. 2007; 282: 18141-18150Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar) as solvent. This solvent was run up to the middle of the plates, dried under N2, and followed by a run up to the top of the plates with hexane/ether (80:20, by volume), to separate other neutral lipids (not described here). The silica support containing the total polar lipids remaining at the origin of these plates was collected for preparative isolation of SM. The PL were eluted by three successive washings of this lipid-laden silica with chloroform/methanol/water (1:1:0.2, by volume). The eluates were collected, and the solvents then were partitioned by the addition of 0.8 volumes of water. The organic phases were dried under N2 and subjected to TLC to isolate SM, using chloroform: methanol:acetic acid:0.15 m NaCl (50:25:8:2.5, by volume) (23Brown E. Subbaiah P. Lipids. 1994; 29: 825-829Crossref PubMed Scopus (70) Google Scholar) as solvent, in this case without presaturating the tank atmosphere with the solvent vapors.Lipid bands were located under ultraviolet light after spraying the TLC plates with 2′,7′-dichlorofluorescein in methanol, exposing them to NH3 vapors in a closed tank, and then spraying with water. The zones containing SM and Cer were scraped into tubes for further elution, performed as detailed above. The eluted SM and Cer were taken to dryness and treated (under N2) with 0.5 n NaOH in anhydrous methanol at 50 °C for 10 min to remove any potential lipid contaminant with ester-bound fatty acids. After alkaline treatment, chloroform and 0.5 n HCl were added to the methanol, the organic phase was rapidly recovered and dried, and the lipids were separated again by TLC.Fatty Acid Analysis—The fatty acid composition of SM and Cer was determined by gas-liquid chromatography of their fatty acid methyl ester derivatives. These were prepared by warming the dry lipid samples and placing them overnight at 45 °C in Teflon®-lined screw capped tubes in the presence of 0.5 n H2SO4 in anhydrous methanol (24Christie W.W. Lipid Analysis.2nd Ed. Pergamon Press, Oxford, UK1982: 51-61Google Scholar) (under N2). Methyl heneicosanoate was added as an internal standard for quantitative analysis (2 μg/sample to measure Cer fatty acids). Before GC, the fatty acid methyl ester samples with the internal standard were routinely purified by TLC (using hexane/ether, 95:5, by volume) on silica gel G plates that had been previously washed (with methanol/ethyl ether, 75:25, by volume). The methyl esters were located after spraying the plates with 2′,7′-dichlorofluorescein, scraped into tubes, and recovered after thoroughly mixing the silica support with methanol: water:hexane (1:1:1, by volume), carrying out three successive hexane extractions.The VLCPUFA of SM and Cer were identified by procedures and criteria detailed in previous work, including GC-MS. The VLCPUFA of ram spermatozoa have been characterized in detail by GC-MS by Poulos et al. (1Poulos A. Johnson D.E. Beckman K. White I.G. Easton C. Biochem. J. 1987; 248: 961-964Crossref PubMed Scopus (57) Google Scholar). The major VLCPUFA of bull SM and Cer were the same as those characterized in ram and were also identical to the VLCPUFA previously characterized by GC-MS in bovine retina rod outer segment phosphatidylcholine (25Aveldaño M.I. Sprecher H. J. Biol. Chem. 1987; 262: 1180-1186Abstract Full Text PDF PubMed Google Scholar). Those of rat spermatozoa were the same as those previously identified by GC-MS in the SM of rat testis (2Robinson B.S. Johnson D.W. Poulos A. J. Biol. Chem. 1992; 268: 1746-1751Abstract Full Text PDF Google Scholar) and in SM and Cer of rat seminiferous tubules (3Furland N.E. Zanetti S.R. Oresti M.G. Maldonado E.N. J. Biol. Chem. 2007; 282: 18141-18150Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar).For fatty acid composition analysis, a Varian 3700 gas chromatograph and a Varian Star Chromatography Work station (version 4.51) were used. The instrument was equipped with two (2 mm × 2 m) glass columns packed with 15% SP 2330 on Chromosorb WAW 100/120 (Supelco, Inc. CA) and two flame ionization detectors, operated in the dual-differential mode. The column oven temperature was programmed from 150 to 230 °C at a rate of 5 °C/min. This final temperature was then held constant for 30 min (rat) to 50 min (bull and ram) per run to allow VLCPUFA enough time to emerge from the column. Injector and detector temperatures were 220 and 230 °C, respectively, and N2 (30 ml/min) was the carrier gas.The fatty acid compositions of SM and Cer are expressed on a weight percentage basis in accordance with the described procedures. SM and Cer amounts are expressed as the sum of their respective fatty acid amounts, with no corrections for the presence of the sphingoid base in the two lipids or of phosphorylcholine in SM. SM/Cer ratios were calculated by dividing the sums of their respective total fatty acids (proportional to mole ratios). To simplify the presentation of the fatty acid data in the figures, the acyl groups of SM and Cer are grouped into saturated, monoenoic, dienoic, and very long-chain polyenoic fatty acids and appear as their sums.RESULTSSM and Cer of Whole Spermatozoa—After inspection of all major polar and neutral lipid classes separated by TLC, SM, and Cer were found to be the only lipids to contain VLCPUFA in spermatozoa. We unexpectedly found that VLCPUFA were major fatty acid components of both sphingolipids in fresh spermatozoa from adult, fertile bulls (≥70%) (Table 1). Ram spermatozoa, analyzed for comparison with bulls, had even higher percentages of VLCPUFA in the two lipids. In rams, there was a net predominance of n-3 hexaenoic VLCPUFA, the three major fatty acids of SM and Cer being 32:6n-3, 30:6n-3, and 34:6n-3 in agreement with the GC-MS tracings shown in Ref. 1Poulos A. Johnson D.E. Beckman K. White I.G. Easton C. Biochem. J. 1987; 248: 961-964Crossref PubMed Scopus (57) Google Scholar. In bulls, the same VLCPUFA occurred, although a higher percentage of VLCPUFA of the n-6 series than in rams were observed.TABLE 1Fatty acid composition of sphingomyelin and ceramide from testis and spermatozoaFatty acidBull testisBull spermatozoaRam spermatozoaSMCerSMCerSMCer14:00.65 ± 0.090.37 ± 0.090.21 ± 0.050.27 ± 0.240.40 ± 0.010.44 ± 0.0514:10.12 ± 0.030.07 ± 0.070.06 ± 0.010.08 ± 0.030.150.08 ± 0.0415:00.35 ± 0.070.52 ± 0.200.09 ± 0.030.07 ± 0.020.20 ± 0.010.29 ± 0.1515:10.10 ± 0.030.08 ± 0.110.03 ± 0.020.01 ± 0.010.09 ± 0.010.16 ± 0.0616:040.55 ± 2.3222.42 ± 2.8610.23 ± 1.615.55 ± 0.8014.36 ± 0.284.28 ± 1.0316:10.55 ± 0.391.45 ± 0.180.26 ± 0.130.34 ± 0.080.76 ± 0.061.35 ± 0.3017:01.56 ± 0.160.97 ± 0.080.18 ± 0.030.14 ± 0.070.31 ± 0.040.26 ± 0.0617:10.10 ± 0.060.55 ± 0.370.04 ± 0.020.11 ± 0.090.15 ± 0.030.17 ± 0.0618:07.96 ± 0.639.45 ± 1.321.51 ± 0.316.28 ± 1.985.11 ± 0.033.76 ± 1.6118:10.71 ± 0.414.67 ± 0.920.21 ± 0.100.49 ± 0.081.15 ± 0.031.64 ± 0.1419:00.34 ± 0.080.55 ± 0.700.05 ± 0.030.09 ± 0.040.16 ± 0.010.16 ± 0.0118:20.17 ± 0.131.78 ± 2.360.03 ± 0.020.08 ± 0.060.44 ± 0.020.40 ± 0.1220:01.02 ± 0.130.98 ± 0.960.61 ± 0.201.80 ± 0.113.39 ± 0.042.57 ± 1.0720:10.04 ± 0.030.34 ± 0.150.02 ± 0.010.04 ± 0.030.040.05 ± 0.0221:00.26 ± 0.030.090.05 ± 0.020.05 ± 0.010.08 ± 0.010.0922:06.06 ± 0.434.71 ± 0.322.63 ± 0.764.18 ± 0.422.02 ± 0.011.19 ± 0.2522:10.44 ± 0.040.46 ± 0.270.16 ± 0.040.40 ± 0.150.18 ± 0.0323:02.91 ± 0.232.46 ± 0.280.45 ± 0.100.46 ± 0.140.090.1123:10.94 ± 0.131.57 ± 0.500.24 ± 0.050.070.0624:09.20 ± 0.4911.63 ± 1.732.02 ± 0.691.76 ± 0.450.48 ± 0.020.62 ± 0.0124:116.11 ± 2.0816.62 ± 3.141.88 ± 0.392.71 ± 0.751.08 ± 0.050.64 ± 0.0224:20.30 ± 0.040.34 ± 0.341.10 ± 0.172.03 ± 0.480.10 ± 0.030.06 ± 0.0126:01.31 ± 0.311.04 ± 0.430.88 ± 0.060.57 ± 0.070.14 ± 0.050.05 ± 0.0424:4n-60.35 ± 0.060.41 ± 0.360.20 ± 0.050.0124:5n-60.69 ± 0.120.81 ± 0.390.22 ± 0.060.22 ± 0.080.020.04 ± 0.0224:5n-30.15 ± 0.050.20 ± 0.270.24 ± 0.080.11 ± 0.110.0126:3 + 24:60.25 ± 0.130.52 ± 0.443.59 ± 0.360.09 ± 0.040.200.2426:4n-60.14 ± 0.140.28 ± 0.055.16 ± 0.220.07 ± 0.0126:5n-60.24 ± 0.090.140.36 ± 0.080.33 ± 0.090.27 ± 0.090.20 ± 0.1026:5n-30.00 ± 0.000.48 ± 0.030.03 ± 0.0328:3 + 26:60.03 ± 0.010.01 ± 0.040.91 ± 0.050.51 ± 0.070.020.0128:4n-60.87 ± 0.211.95 ± 0.2718.79 ± 0.3724.67 ± 0.602.05 ± 0.042.91 ± 0.2328:5n-60.08 ± 0.050.12 ± 0.130.10 ± 0.010.15 ± 0.0828:5n-30.30 ± 0.140.47 ± 0.131.17 ± 0.181.71 ± 0.231.71 ± 0.061.92 ± 0.5028:6n-30.01 ± 0.000.04 ± 0.060.09 ± 0.0530:4n-60.24 ± 0.090.44 ± 0.2313.84 ± 1.928.86 ± 0.720.78 ± 0.010.83 ± 0.0530:5n-60.02 ± 0.010.19 ± 0.093.42 ± 0.743.53 ± 0.810.07 ± 0.010.14 ± 0.1630:5n-30.09 ± 0.050.19 ± 0.100.95 ± 0.180.60 ± 0.220.72 ± 0.010.71 ± 0.0830:6n-31.30 ± 0.142.90 ± 0.524.28 ± 0.994.73 ± 0.9913.56 ± 0.0615.40 ± 1.5232:4n-60.70 ± 0.180.17 ± 0.0632:5n-60.02 ± 0.010.03 ± 0.026.64 ± 2.034.54 ± 1.680.08 ± 0.020.08 ± 0.0432:6n-32.75 ± 0.436.92 ± 0.6116.09 ± 2.6513.72 ± 2.8440.62 ± 0.1051.11 ± 2.2634:5n-60.23 ± 0.321.88 ± 0.820.89 ± 0.1534:6n-30.46 ± 0.161.05 ± 0.143.59 ± 0.482.44 ± 0.299.03 ± 0.077.56 ± 0.12VLCPUFA8.23 ± 1.3416.49 ± 2.0077.29 ± 2.8572.27 ± 2.9069.23 ± 0.1081.39 ± 4.90 Open table in a new tab SM amounted to 13–15% of the total PL present in bull (Fig. 1) and ram spermatozoa, thus the 70–80% VLCPUFA in this lipid class represents values ranging from 9 to 12% of the total fatty acids of bull sperm cells. Because Cer had a similarly high percentage of VLCPUFA, and the SM/Cer ratio was in the order of 2.5 for the spermatozoa whose fatty acids are shown in Table 1, the sum of SM + Cer contributed a total of ∼12–15% of VLCPUFA to the total fatty acid weight of whole bull and ram spermatozoa, a far from negligible amount.The percentage of SM with respect to the total PL was almost twice as large in sperm as in testis (15 and 8% of the total PL, respectively). The percentage of VLCPUFA with respect to total SM fatty acids was 8-fold larger in sperm as in testis (70 and 8%, respectively; Table 1), this concentration obviously being an important feature of epididymal maturation. Spermatozoa isolated from caput and cauda epididymi (not shown) displayed intermediate values between these extremes, indicating that the VLCPUFA-rich SM of sperm originate in the testis and that part of these lipid species are conserved in the cells as they mature in the epididymis. The fact that SM gained importance with respect to other reflects the fact that more of the latter than of the SM was lost in the process of epididymal sperm remodeling, which entails a condensation of the cell (4Williams R.M. Graham J.K. Hammerstedt R.H. Biol. Reprod. 1991; 44: 1080-1091Crossref PubMed Scopus (40) Google Scholar). Similarly, the fact that VLCPUFA concentrated in SM at sperm maturation indicates this lipid lost most of its original saturated and monoenoic fatty acids in the process. Epididymal remodeling of sperm lipid also qualitatively affected the fatty acids of SM, as suggested by the fact that 28:4n-6, from being just one of the VLCPUFA of bull testicular SM, became the major N-acyl group of mature bull spermatozoal SM.The described difference between the fatty acid composition of testicular and spermatozoal SM was also observed in rat. Whereas the SM of rat testis had a series of tetraenoic and pentaenoic VLCPUFA up to 32:5n-6 (3Furland N.E. Zanetti S.R. Oresti M.G. Maldonado E.N. J. Biol. Chem. 2007; 282: 18141-18150Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar), the SM of rat spermatozoa had much less pentaenes, with 28:4n-6 becoming the most prominent component of this group of fatty acids.The spermatozoa of the three mammals examined had in common a significant amount of Cer with a fatty acid pattern similar to that of SM. A contrasting charac" @default.
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• W2044134902 date "2007-06-01" @default.
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• W2044134902 title "Very Long-chain Polyunsaturated Fatty Acids Are the Major Acyl Groups of Sphingomyelins and Ceramides in the Head of Mammalian Spermatozoa" @default.
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• W2044134902 doi "https://doi.org/10.1074/jbc.m700709200" @default.
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