Marta Aveldaño | Universidad Nacional del Sur (original) (raw)
Papers by Marta Aveldaño
Biochemical Journal, 1988
The synthesis of very long chain (C24 to C36) polyunsaturated (four, five and six double bonds) f... more The synthesis of very long chain (C24 to C36) polyunsaturated (four, five and six double bonds) fatty acids (VLCPUFA) is investigated in bovine retina using [14C]acetate. Saturates on the one hand (mainly palmitate), and polyenes on the other (mainly VLCPUFA), incorporate most of the label found in lipids. Phosphatidylcholine (PC) is the most highly labelled lipid class, since both types of '4C-labelled fatty acids, but especially this novel series of VLCPUFA, are concentrated in this phospholipid. Radioactivity from [14C]acetate is found in very long chain tetra, penta and hexaenoic fatty acids of PC. The labelling of 20: 4(n-6), 20: 5(n-3), 22: 5(n-6) and 22: 6(n-3) is much lower than that of longer polyenes of each of these series, indicating that VLCPUFA are synthesized in situ by successive elongations of the above polyenes, pre-existing in retina lipids. In various subcellular fractions isolated from retinas after incubations with [14C]acetate (including cytosol, microsomes, mitochondria and photoreceptor membranes), the labelling of the VLCPUFA of PC is very high, even at relatively short intervals of incubation. The results suggest that not only the synthesis but also the intracellular traffic among membranes of VLCPUFAcontaining species of PC are very active processes in the retina. Recently it was shown that the levels of VLCPUFA in retina PC decrease with aging in rats (Rotstein et al., 1987). The information about important aspects of the biochemistry and biophysics of the novel VLCPUFAcontaining lipids is quite limited at present. This paper is concerned with the synthesis of these fatty acids in the bovine retina. It is shown that the VLCPUFA of PC are very actively synthesized from ["4C]acetate in entire retinas in vitro, and that in the PC of various subcellular fractions, including photoreceptor membranes, these fatty acids are labelled to a considerable extent. MATERIALS AND METHODS Materials Bovine eyes were obtained from a local abattoir and transported to the laboratory in crushed ice. They were dark-adapted for 2 h and the retinas were excised on ice, under dim red light. [1-'4C]Acetate (sodium salt, specific activity 58.3 mCi/mmol, ethanolic solution) was obtained from New England Nuclear (Boston, MA, U.S.A.). Incubation of retinas Retinas were incubated in a C02-saturated Krebs-Ringer bicarbonate buffer (118 mM-NaCl, 5 mM-KCl, 2.5 mM-CaCl2, 1 mM-KH2PO4, lmM-MgSO4 and 25 mm-NaHCO3), pH 7.0 (7 ml/retina). Glucose (2 mg per ml of medium) was added at the beginning of incubation and (dissolved in 500 p1 of medium) after every 1 h of incubation. The ['4C]acetate was added to the media and thoroughly mixed before adding the retinas. Three retinas per sample (each weighing 300-500 mg) were used in the Vol. 249 Abbreviations used: PC, phosphatidylcholine; VLCPUFA, very long chain polyunsaturated fatty acids; FAME, fatty acid methyl esters; ROS, rod outer segments; fatty acids are abbreviated by the convention, number of carbon atoms:number of double bonds.
Lipids, Feb 13, 2011
Spermatogenesis is known to be vulnerable to temperature. The aim of this study was to investigat... more Spermatogenesis is known to be vulnerable to temperature. The aim of this study was to investigate the effects on testicular lipids of the transient germ cell loss that is induced by mild testicular hyperthermia. Adult rat testes were exposed once a day to 43 °C for 15 min for 5 days and the effects were followed for several weeks. Two week after the last heat exposure, spermatocytes and early spermatids had virtually disappeared and the seminiferous tubules were populated mostly by mature spermatids and spermatozoa. One week later, the latter were also absent and mostly Sertoli cells populated the tubules. During these 3 weeks, glycerophospholipids (Gpl) and triacylglycerols with long‐chain polyunsaturated fatty acids (PUFA) (e.g., 22:5n‐6) and species of sphingomyelin and ceramide with nonhydroxy and 2‐hydroxy very long‐chain (VLC) PUFA (e.g., 28:4n‐6, 2‐OH 30:5n‐6) decreased alongside the germ cells. Concomitantly, the amounts of cholesteryl esters and ether‐linked triglycerides increased, both lipids accumulating long‐chain and very‐long‐chain polyenes. This concurred with a considerable buildup of lipid droplets in Sertoli cells, evidently containing these neutral lipids, apparently formed during germ cell‐derived membrane lipid catabolism. Between week 4 and week 6, new cohorts of spermatocytes appeared, and by week 12 most cell changes were reversed. Accordingly, as germ cell differentiation proceeded, 22:5n‐6‐rich Gpl augmented and spermatocyte‐associated sphingolipids with nonhydroxy VLCPUFA appeared before their 2‐hydroxy counterparts. The unique fatty acids of rat testicular lipids after mild hyperthermia reveal lipid catabolic and biosynthetic reactions that occur in normal spermatogenesis.
Biochemical Journal, Apr 1, 1992
In their transit from the caput to the cauda segments of the epididymis, rat spermatozoa undergo ... more In their transit from the caput to the cauda segments of the epididymis, rat spermatozoa undergo significant modifications in lipid content and composition. The amount of lipid phosphorus per cell decreases, and most lipid classes show specific changes in their constituent fatty acids. A depletion of phosphatidylcholine and phosphatidylethanolamine, concomitant with a virtually unchanged amount of the corresponding plasmalogens, are the major alterations, plasmenylcholine thereby becoming the major phospholipid. Diphosphatidylglycerol, sphingomyelin and the phosphoinositides decrease to a lesser extent or do not change at all, also resulting in relative increases with sperm maturation. Concerning the fatty acids, the proportions of oleate (C18:1 n-9) and linoleate (C18:2 n-6) in most lipids decrease on movement of sperm from caput to cauda, augmenting in turn the proportions of longer-chain (C20 to C24) and more unsaturated fatty acids. Docosapentaenoate (C22:5 n-6) is a major acyl chain present in all lipids at both stages, but uncommon long-chain polyenoic fatty acids of the n-9 series are also present, being almost exclusively found in the choline glycerophospholipids. These fatty acids are found to undergo the most significant changes during sperm maturation. They are minor components of plasmenylcholine in immature spermatozoa, but increase severalfold on maturation, representing more than half of the acyl chains of this major lipid in cells from the cauda. The high concentration of n-9 polyenes in mature sperm plasmenylcholine raises intriguing questions on the possible role epididymal cells may play in providing spermatozoa with such an unusual phospholipid. These plasmenylcholines could contribute to the characteristic lipid domain organization of the mature spermatozoa plasma membrane. Abbreviations used: CGP and EGP are used to denote the (diradyl)-choline and-ethanolamine glycerophospholipids respectively; the terms phosphatidyl-, plasmenyland plasmanylare reserved respectively for the diacyl-, alk-1-enyl-2-acyl-and alkyl-2-acylsubclasses of the corresponding glycerophospholipid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PA, phosphatidic acid; PS, phosphatidylserine; DPG, diphosphatidylglycerol; FAME, fatty acid methyl esters.
International Journal of Biochemistry, 1990
1. The distribution of phospholipids between the two leaflets of the lipid bilayer in acetylcholi... more 1. The distribution of phospholipids between the two leaflets of the lipid bilayer in acetylcholine receptor (AChR)-rich membranes from T. marmorata has been examined with two complementary techniques: chemical derivatization with the membrane-impermeable reagent trinitrobenzenesulphonate (TNBS) and B.cereus phospholipase C hydrolysis. 2. AChR-membranes were reacted with TNBS at 0-4 and 37 degrees C and the accessibility of their aminophospholipids was compared to that of rod outer segment and erythrocyte membranes. The results indicate that more of the total ethanolamine glycerophospholipid (EGP) than of the total phosphatidylserine (PS) is located in the outer monolayer. 3. Nearly half the phospholipid content of AChR membranes is hydrolyzed by phospholipase C with a half-time of ca. 1.6 min at 25 degrees C. Consistent with the TNBS results, more of the total EGP than of the total PS is degraded. Beyond 3 min the reaction slows down, relatively smaller additional amounts of lipids are hydrolyzed, and all phospholipid classes are attacked to a similar extent, indicating that after half the lipid is removed all phospholipids become accessible to the enzyme. 4. The results indicate that the outer leaflet of the bilayer is richer in ethanolamine and choline glycerophospholipids, whereas phosphatidylinositol, most of the sphingomyelin, and ca 65% of the PS are located on the inner leaflet.
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Biochimica et biophysica acta, Sep 1, 1987
The labeling of molecular species of phosphatidylcholine (PC) has been studied in bovine retinas ... more The labeling of molecular species of phosphatidylcholine (PC) has been studied in bovine retinas incubated for 2 h with (1-14C)-labeled (n-6) eicosatetraenoate (n-3) docosapentaenoate and (n-3) docosahexaenoate (20: 4, 22: 5 and 22: 6, respectively) and in four subcellular fractions isolated after such incubations. Of the total radioactivity incorporated in PC, the following percentages of the above fatty acids, respectively, are found in its dipolyunsaturated species: 58, 56 and 53% in rod outer segments; 29, 41 and 49% in mitochondria; 24,28 and 39% in microsomes; 12, 14 and 16% in postmicrosomal supernatants; 28,36 and 58% in entire retinas. The remainder percentages are in tetra-, penta-and hexaenoic species of PC, respectively. The levels of pentaenoic species in the PCs of all fractions are similar, while tetraenes are lowest and hexaenes highest in photoreceptor membranes. Dipolyunsaturated species are highly concentrated in photoreceptor membranes, but are minor components of mitochondrial, microsomal and cytosolic PC. The specific radioactivities of tetraenoic, pentaenoic and hexaenoic PCs are decreasingly lower in the following order: postmicrosomal supematants, microsomes, mitochondria, photoreceptor membranes. In contrast, the specific radioactivities of dipolyunsaturated PCs are higher in mitochondria and microsomes than in the other fractions, especially with 22 : 5 and 22 : 6. It is suggested that mitochondria as well as the endoplasmic reticulum could play a role in the synthesis and further modifications of dipolyunsaturated PCs before being supplied to photoreceptor membranes.
Plant Physiology and Biochemistry, Apr 1, 2013
Phosphatidic acid (PA) is the common lipid product in abscisic acid (ABA) and gibberellic acid (G... more Phosphatidic acid (PA) is the common lipid product in abscisic acid (ABA) and gibberellic acid (GA) response. In this work we investigated the lipid metabolism in response to both hormones. We could detect an in vivo phospholipase D activity (PLD, EC 3.1.4.4). This PLD produced [ 32 P]PA (phosphatidic acid) rapidly (minutes) in the presence of ABA, confirming PA involvement in signal transduction, and transiently, indicating rapid PA removal after generation. The presence of PA removal by phosphatidate phosphatase 1 and 2 isoforms (E.C. 3.1.3.4) was verified in isolated aleurone membranes in vitro, the former but not the latter being specifically responsive to the presence of GA or ABA. The in vitro DGPP phosphatase activity was not modified by short time incubation with GA or ABA while the in vitro PA kinase e that allows the production of 18:2-DGPP from 18:2-PA e is stimulated by ABA. The long term effects (24 h) of ABA or GA on lipid and fatty acid composition of aleurone layer cells were then investigated. An increase in PC and, to a lesser extent, in PE levels is the consequence of both hormone treatments. ABA, in aleurone layer cells, specifically activates a PLD whose product, PA, could be the substrate of PAP1 and/or PAK activities. Neither PLD nor PAK activation can be monitored by GA treatment. The increase in PAP1 activity monitored after ABA or GA treatment might participate in the increase in PC level observed after 24 h hormone incubation.
![Research paper thumbnail of Labeling of lipids of retina subcellular fractions by [1-14C]eicosatetraenoate (20:4(n − 6)) docosapentaenoate (22:5(n − 3)) and docosahexaenoate (22:6(n − 3))](https://attachments.academia-assets.com/111692808/thumbnails/1.jpg)
](Biochimica et biophysica acta, Sep 1, 1987
(l-l4 C)-labeled (n-6) eicosatetraenoate, (n-3) docosapentaenoate and (n-3) docosahexaenoate (20 ... more (l-l4 C)-labeled (n-6) eicosatetraenoate, (n-3) docosapentaenoate and (n-3) docosahexaenoate (20 : 4, 22: 5 and 22: 6, respectively) are efficiently taken up and actively esterified into the lipids of bovine retina after 2 h incubation. Photoreceptor membranes, mitochondria, microsomes and postmicrosomal supernatants, which display significant differences in phospholipid and fatty acid compositions, are isolated after such incubations to study the labeling of lipids. The lipid classes preferentially labeled with the acids (1) largely differ among and within subcellular fractions, while (2) some common features in the treatment of the three polyenes are observed in each fraction. In all of them, the three acids are actively incorporated in phosphatidylcholine; ethanolamine glycerophospholipid, phosphatidylserine (PS) and phosphatidylinositol (PI) are highly labeled with 22 : 6, 22 : 5 and 20 : 4 respectively; within ethanolamine glycerophospholipid, the three label phosphatidylethanolamine in preference to plasmenylethanolamine. Most of the 14C esterified in mitochondria is in phospholipids. The endoplasmic reticulum produces in addition highly labeled triacylglycerols, also found in cytosol. High levels of l4 C-labeled diacylglycerols are observed exclusively in photoreceptor membranes, where the specific radioactivity of PI is very high. The total amounts of 14C incorporated (1) are in general similar within a given fraction for the three polyenes, but (2) largely differ among fractions. The labeling of the highly unsaturated phospholipids of photoreceptor membranes is the lowest, while the postmicrosomal supematant (whose lipids are relatively the poorest in polycnoic fatty acids) contains most of the labeled lipids isolated from retinas under these conditions. The results indicate that polyunsaturated species of retina phospholipids undergo an active synthesis and turnover, as well as an intense intracellular traffic among membranes.
Journal of Biological Chemistry, Jun 1, 1993
The formation of long and very long chain (VLC) n-6 polyunsaturated fatty acids (PUFA) in isolate... more The formation of long and very long chain (VLC) n-6 polyunsaturated fatty acids (PUFA) in isolated rat seminiferous tubules was investigated by following the metabolism of three l-14C-labeled n-6 tetraenoic fatty acids (20:4, 24:4, and 3 2 4) and [U-"Clacetate. In contrast to [I4C]32:4, which was poorly incorporated and altered, ['4C]20:4 and [14C]24:4 were efficiently taken up by the tubules, esterified into lipids, elongated to VLCPUFA, and desaturated to pentaenoic fatty acids; the rate of [14C]24:4 desaturation to [14C]24:5 was notably high. The main products with [I4C]acetate as precursor were labeled saturates and VLCPUFA, most of the label in tetraenoic and pentaenoic acids appearing in 24:4 and 24:5, respectively. These two Car polyenes, connected by a A 6 desaturation, may play a central role in n-6 PUFA metabolism, in their capacity as potential precursors of longer polyenes via elongation and of shorter ones, such as 22:5n-6, via retroconversion. Triacylglycerols, rich in Czz and Cz4 polyenes, incorporated the greatest amounts of both ["C] acetate-derived and exogenous "C-PUFA, suggesting that this lipid class is involved in the traffic and metabolism of testicular PUFA. The detection of a series of unusual odd-chain tetraenoic and pentaenoic acids, also labeled with [U-14C]acetate, suggests that a PUFA chain shortening mechanism occurs in testis involving a-in addition to &oxidation. We speculate that aoxidation plays a role in the retroconversion of PUFA. Docosapentaenoic (22:5n-6)' and arachidonic (204n-6) acids are known to be abundant polyunsaturated fatty acids
Biochimica Et Biophysica Acta - Molecular And Cell Biology Of Lipids, 2014
Cadmium is known to harm rat testis by causing the dose-dependent apoptotic or necrotic death of ... more Cadmium is known to harm rat testis by causing the dose-dependent apoptotic or necrotic death of seminiferous epithelium cells. Here we investigated how this affects the lipids with long-chain (C18-C22) and very-long-chain (C24-C32) polyunsaturated fatty acids (VLCPUFA) typical of spermatogenic and Sertoli cells. A severe acute inflammatory reaction resulted from the massive necrotic death of these cells two days after a single high (4 mg/kg) dose of CdCl2. This led to the conversion of most testicular glycerophospholipids to diradylglycerols (DRG) and free fatty acids (FFA) and of most sphingomyelins to ceramides (Cer). By day 30 the testis weight had decreased three fold. The DRG and FFA had been metabolized but, unexpectedly, ceramides persisted. Also slow to disappear were VLCPUFA-containing triacylglycerols from former germ cells and ether-linked triglycerides and cholesteryl esters (CE) from former Sertoli cells. Similar results were observed 30 and 45 days after administering repeated small non pro-inflammatory CdCl2doses (1 mg/kg). At day 30 after both treatments, an amorphous material replaced the original seminiferous tubules and testicular macrophages populated the interstitium. Species of CE and ether-linked triglycerides containing fatty acids other than VLCPUFA steadily accumulated in the irreversibly damaged testis, a manifestation of the activity of phagocytic cells. The long-term permanence of original VLCPUFAcontaining neutral lipids, especially ceramides, indicates that these cells were slow to clear out the acellular material contained in seminiferous tubules, pointing to a form of silent chronic inflammation as an additional outcome of the multifactorial commotion caused in the testis by experimentally administered cadmium.
Reproduction, Nov 1, 2013
Male germ cell differentiation entails the synthesis and remodeling of membrane polar lipids and ... more Male germ cell differentiation entails the synthesis and remodeling of membrane polar lipids and the formation of triacylglycerols (TAGs). This requires fatty acid-binding proteins (FABPs) for intracellular fatty acid traffic, a diacylglycerol acyltransferase (DGAT) to catalyze the final step of TAG biosynthesis, and a TAG storage mode. We examined the expression of genes encoding five members of the FABP family and two DGAT proteins, as well as the lipid droplet protein perilipin 2 (PLIN2), during mouse testis development and in specific cells from seminiferous epithelium. Fabp5 expression was distinctive of Sertoli cells and consequently was higher in prepubertal than in adult testis. The expression of Fabp3 increased in testis during postnatal development, associated with the functional differentiation of interstitial cells, but was low in germ cells. Fabp9, together with Fabp12, was prominently expressed in the latter. Their transcripts increased from spermatocytes to spermatids and, interestingly, were highest in spermatid-derived residual bodies (RB). Both Sertoli and germ cells, which produce neutral lipids and store them in lipid droplets, expressed Plin2. Yet, while Dgat1 was detected in Sertoli cells, Dgat2 accumulated in germ cells with a similar pattern of expression as Fabp9. These results correlated with polyunsaturated fatty acid-rich TAG levels also increasing with mouse germ cell differentiation highest in RB, connecting DGAT2 with the biosynthesis of such TAGs. The age-and germ cell type-associated increases in Fabp9, Dgat2, and Plin2 levels are thus functionally related in the last stages of germ cell differentiation.
Se realizaron dos experimentos en el invernaculo para estudiar la influencia de la deficiencia de... more Se realizaron dos experimentos en el invernaculo para estudiar la influencia de la deficiencia de P en la nutricion con nitrogeno de plantas de soja no nodulada. Hubo una reduccion en el contenido de N y P en las plantas deficientes en P (P-) de casi el 50% y del 33% en la materia seca, luego de 50 dias. La actividad de la nitrato reductasa y el contenido de nitratos en tallos fueron menores en plantas P- que en aquellas P+. Del total de nitratos en las plantas P-, 75% estuvo en las raices. La acumulacion de nitratos en las raices seria debido a la menor actividad de la nitrato reductasa en las mismas, y a una disminucion en el flujo de agua hacia el vastago. El incremento en la concentracion de nitratos en la raiz causaria una retroalimentacion negativa que reduciria su absorcion por la planta
Journal of Lipid Research, 1983
Glycerolipids and sphingolipids are hydrolyzed with 0.5 M HCI in acetonitrile-water 9:l (by vol) ... more Glycerolipids and sphingolipids are hydrolyzed with 0.5 M HCI in acetonitrile-water 9:l (by vol) for 45 min at 100°C or 4 hr at 70°C. After hydrolysis, free fatty acids (FFA) are recovered in chloroform and separated by thin-layer chromatography. More than 95% of the radioactivity from labeled phospholipids is recovered as FFA, and more than 97% of the lipid phosphorus is recovered as water-soluble phosphate. The yields of FFA, including polyunsaturated acids, after hydrolysis are as good as or better than those obtained for methyl esters using methanolysis catalyzed by acid, alkali, or BFS. High recoveries of FFA from glycerophospholipids, sphingomyelin, and neutral glycerides are attained. The procedure is quantitative, simple, inexpensive, and produces no methyl esters as secondary products.-Aveldaiio, M. I., and L. A. Horrocks. Quantitative release of fatty acids from lipids by a simple hydrolysis procedure.
Journal of Lipid Research, 1983
Reverse phase high pressure liquid chromatography (HPLC) on octadecylsilyl columns separates mixt... more Reverse phase high pressure liquid chromatography (HPLC) on octadecylsilyl columns separates mixtures of either free fatty acids or fatty acid methyl esters prepared from mammalian tissue phospholipids. Acetonitrile-water mixtures are used for the elution of esters. Aqueous phosphoric acid is substituted for water for the separation of the free acids. Unsaturated compounds are detected and quantitated by their absorption at 192 nm. Saturates are detected better at 205 nm. T h e order of elution of fatty acids in complex mixtures varies as a function of acetonitrile concentration. At any given concentration, some compounds overlap. However, by varying the solvent strength, any fatty acid of interest can be resolved including many geometrical and positional isomers. Methyl esters prefractionated according to unsaturation by argentation thin-layer chromatography (TLC) are rapidly and completely separated by elution with CH'CN alone. Argentation TLC-reverse phase HPLC can be used as an analytical as well as a preparative procedure. Octylsilyl columns are used for rapid resolution and improved detection of minor or low ultraviolet-absorbing components in the fractions. For example, monoenoic fatty acids with up to 32 carbons have been detected in bovine brain glycerophospholipids. Specific radioactivities of 'H-and ''C-labeled fatty acids and the distribution of radioactivity among acyl groups from complex lipids are measured. T h e method is not recommended for complete compositional analysis, but is useful for determinations of specific radioactivities during studies on turnover and metabolic Abbreviations: GLC, gas-liquid chromatography; HPLC, high pressure liquid chromatography: TLC, thin-layer chromatography; FFA, free fatty acids; FAME, fatty acid methyl esters. Fatty acids are abbreviated by the convention, number of carbon atomsmumber of double bonds.
Clinical Chemistry, 1996
Blood cell and plasma lipid classes and their fatty acids were analyzed in a child with X-linked ... more Blood cell and plasma lipid classes and their fatty acids were analyzed in a child with X-linked adrenoleukodystrophy. The increase in saturated fatty acids with very long chains typical of this disease occurred almost exclusively in sphingomyelin. In this lipid, the proportion of lignoceric (24:0) and hexacosanoic (26:0) acids increased while that of 18:0, 20:0, and 24:1 decreased. In the rest of the lipid classes, but especially in cholesteryl esters and triacylglycerols, the proportion of linoleate (18:2) decreased while that of oleate (18:1) increased. In glycerophospholipids, polyunsaturated fatty acids such as 20:4n-6, 22:5n-6, and 22:6n-3 were reduced while their immediate precursors, 20:3n-6, 22:4n-6, and 22:5n-3, respectively, were relatively increased, suggesting a defect in fatty acid desaturation mechanisms. Although less pronounced, a similar trend of changes was seen in the patient's mother; in both, all alterations were more marked in serum than in blood cells.
![Research paper thumbnail of {"__content__"=>" and expression during rat spermatogenesis: a link to the very-long-chain PUFA typical of germ cell sphingolipids.", "i"=>[{"__content__"=>"Elovl4"}, {"__content__"=>"Fa2h"}]}](https://attachments.academia-assets.com/111692792/thumbnails/1.jpg)
](Journal of lipid research, Jan 3, 2018
The sphingolipids (SL) of rodent spermatogenic cells (spermatocytes, spermatids) and spermatozoa ... more The sphingolipids (SL) of rodent spermatogenic cells (spermatocytes, spermatids) and spermatozoa contain nonhydroxylated and 2-hydroxylated versions of very-long-chain (C26 to C32) PUFA (n-V and h-V, respectively), not present in Sertoli cells. Here we investigated the expression of selected fatty acid elongases (), 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, though the protein was absent from, immature testes and Sertoli cells. 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 to C32 PUFA. 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...
Journal of Lipid Research, 2017
of the n-6 series. In spermatogenic cells from the adult rat, major ester-bound PUFAs of GPLs are... more of the n-6 series. In spermatogenic cells from the adult rat, major ester-bound PUFAs of GPLs are docosapentaenoic acid (22:5n-6) and arachidonic acid (20:4n-6) (1); whereas major amide-bound fatty acids of sphingomyelins (SMs) and ceramides (Cers) are 28:4n-6, 30:5n-6, and 32:5n-6 (2). Previous work has shown that in rat and boar testes, such fatty acids occur in SM in nonhydroxylated (n-V) and 2-hydroxylated (h-V) versions (3). Focus on the fatty acids of rat testis Cer showed it to contain even higher percentages of such fatty acids than SM (4, 5). The atypical 2-hydroxy-VLCPUFAs (e.g., h-30:5n-6) were also found by Sandhoff et al. (6) to be components of a novel series of glycosphingolipids (GSLs) that contain fucose in their head groups (FGSLs), which they discovered and thoroughly characterized in the rodent testis (mouse, rat). In the mouse, these complex sphingolipids were shown to be required in spermatogenic cells for a proper completion of meiosis (7). More recently, by conditionally knocking out (endoplasmic reticulum-located) Cer synthase 3 (CerS3) and glucosyl-Cer synthase specifically in mouse germ cells, the essentiality of both simple and complex sphingolipids that contain VLCPUFAs has been demonstrated, not only for meiosis, but also for the stability of spermatids (8). Neither Cer with VLCPUFA, nor the species of SM or FGSL that contain such fatty acids are produced in the testis in the absence of CerS3, resulting in spermatogenic arrest, increased apoptosis, and formation of multinuclear giant cells.
Biology of Reproduction, 2015
In spermatozoa isolated from rat epididymis, lipids are differentially modified after in vitro in... more In spermatozoa isolated from rat epididymis, lipids are differentially modified after in vitro induction of capacitation (Cap) and the acrosomal reaction (AR). This study uses Laurdan fluorescence generalized polarization values (GPv) to evaluate the effect of lipid changes occurring after isolation and functional activation on sperm membrane biophysical properties. In gametes isolated in the presence of a divalent cation chelator, no lipid changes occurred and the GPv were the lowest recorded, indicating maximal membrane lipid mobility. In sperm isolated as rapidly and gently as possible in the absence of chelator, part of the sphingomyelins (SM) were converted into ceramides (Cer), giving rise to higher GPv. In samples incubated as controls for Cap and AR, unchanged cholesterol and reduced glycerophospholipid levels were accompanied by the accumulation of free fatty acids (FFA), leading to even higher GPv. After completion of Cap, the GPv returned to lower levels as a result of the spermatozoa losing part of their cholesterol and FFA. Cap samples became relatively enriched in polyunsaturated fatty acids-containing plasmalogens because hydrolysis affected phosphatidyl rather than plasmenyl glycerophospholipid subclasses. The highest Cer:SM ratio and the highest GPv were found after completion of AR induced by A23187. The degree of SM ! Cer conversion among the samples, including controls, correlated with the extent of AR. FFA and Cer augmented GPv when added to liposomes prepared from the membrane lipid of intact sperm. Our results underscore the importance of hydrolytic changes that affect sperm lipids, especially the decisive lipid SM and Cer pair, not only after inducing sperm functional changes such as Cap and AR, but also under control conditions.
Journal of Biological Chemistry
ABSTRACT
Neurobiology of Essential Fatty Acids, 1992
Photoreceptor cells and spermatozoa have long been known to be the richest sources of polyunsatur... more Photoreceptor cells and spermatozoa have long been known to be the richest sources of polyunsaturated fatty acids (PUFA) in vertebrates. Docosahexaenoate (22:6n-3) and docosapentaenoate (22:5n-6), according to species and in diverse proportions, are common major acyl chains of the glycerophospholipids of these highly specialized cells. Both 22:6n-3 and 22:5n-6 are the products of a reaction that involves the introduction of a double bond in theΔ4 position of the chain in 22:5n-3 and 22:4n-6 respectively. Even though this reaction has yet to be fully characterized, the most highly unsaturated fatty acids that occur in vertebrate membranes are known to result from such desaturation. The four fatty acids just mentioned and their predecessors are grouped into two“lineages,”defined by the position of the first double bond counting from the methyl end, as the n-3 and the n-6 families. However, each of the four can be elongated, thus producing four lines of descendants, each line comprising various n-3 hexaenoic, n-6 pentaenoic, n-3 pentaenoic, and n-6 tetraenoic PUFA respectively. This chapter summarizes our findings on retina, joining them with more recent ones on spermatozoa, PUFA components, and the peculiar glycerophospholipids in which they occur. It is shown that, just as the glycerophospholipids of retina contribute to extend our view of existing PUFA by disclosing a whole variety of chain lengths within each of the known PUFA“lineages,”the glycerophospholipids of spermatozoa do so by disclosing new lineages for each of the known PUFA lengths. Intricate as this may sound, this overview expands, but at the same time simplifies, our picture of PUFA structural and metabolic relationships.
Biochemical Journal, 1988
The synthesis of very long chain (C24 to C36) polyunsaturated (four, five and six double bonds) f... more The synthesis of very long chain (C24 to C36) polyunsaturated (four, five and six double bonds) fatty acids (VLCPUFA) is investigated in bovine retina using [14C]acetate. Saturates on the one hand (mainly palmitate), and polyenes on the other (mainly VLCPUFA), incorporate most of the label found in lipids. Phosphatidylcholine (PC) is the most highly labelled lipid class, since both types of '4C-labelled fatty acids, but especially this novel series of VLCPUFA, are concentrated in this phospholipid. Radioactivity from [14C]acetate is found in very long chain tetra, penta and hexaenoic fatty acids of PC. The labelling of 20: 4(n-6), 20: 5(n-3), 22: 5(n-6) and 22: 6(n-3) is much lower than that of longer polyenes of each of these series, indicating that VLCPUFA are synthesized in situ by successive elongations of the above polyenes, pre-existing in retina lipids. In various subcellular fractions isolated from retinas after incubations with [14C]acetate (including cytosol, microsomes, mitochondria and photoreceptor membranes), the labelling of the VLCPUFA of PC is very high, even at relatively short intervals of incubation. The results suggest that not only the synthesis but also the intracellular traffic among membranes of VLCPUFAcontaining species of PC are very active processes in the retina. Recently it was shown that the levels of VLCPUFA in retina PC decrease with aging in rats (Rotstein et al., 1987). The information about important aspects of the biochemistry and biophysics of the novel VLCPUFAcontaining lipids is quite limited at present. This paper is concerned with the synthesis of these fatty acids in the bovine retina. It is shown that the VLCPUFA of PC are very actively synthesized from ["4C]acetate in entire retinas in vitro, and that in the PC of various subcellular fractions, including photoreceptor membranes, these fatty acids are labelled to a considerable extent. MATERIALS AND METHODS Materials Bovine eyes were obtained from a local abattoir and transported to the laboratory in crushed ice. They were dark-adapted for 2 h and the retinas were excised on ice, under dim red light. [1-'4C]Acetate (sodium salt, specific activity 58.3 mCi/mmol, ethanolic solution) was obtained from New England Nuclear (Boston, MA, U.S.A.). Incubation of retinas Retinas were incubated in a C02-saturated Krebs-Ringer bicarbonate buffer (118 mM-NaCl, 5 mM-KCl, 2.5 mM-CaCl2, 1 mM-KH2PO4, lmM-MgSO4 and 25 mm-NaHCO3), pH 7.0 (7 ml/retina). Glucose (2 mg per ml of medium) was added at the beginning of incubation and (dissolved in 500 p1 of medium) after every 1 h of incubation. The ['4C]acetate was added to the media and thoroughly mixed before adding the retinas. Three retinas per sample (each weighing 300-500 mg) were used in the Vol. 249 Abbreviations used: PC, phosphatidylcholine; VLCPUFA, very long chain polyunsaturated fatty acids; FAME, fatty acid methyl esters; ROS, rod outer segments; fatty acids are abbreviated by the convention, number of carbon atoms:number of double bonds.
Lipids, Feb 13, 2011
Spermatogenesis is known to be vulnerable to temperature. The aim of this study was to investigat... more Spermatogenesis is known to be vulnerable to temperature. The aim of this study was to investigate the effects on testicular lipids of the transient germ cell loss that is induced by mild testicular hyperthermia. Adult rat testes were exposed once a day to 43 °C for 15 min for 5 days and the effects were followed for several weeks. Two week after the last heat exposure, spermatocytes and early spermatids had virtually disappeared and the seminiferous tubules were populated mostly by mature spermatids and spermatozoa. One week later, the latter were also absent and mostly Sertoli cells populated the tubules. During these 3 weeks, glycerophospholipids (Gpl) and triacylglycerols with long‐chain polyunsaturated fatty acids (PUFA) (e.g., 22:5n‐6) and species of sphingomyelin and ceramide with nonhydroxy and 2‐hydroxy very long‐chain (VLC) PUFA (e.g., 28:4n‐6, 2‐OH 30:5n‐6) decreased alongside the germ cells. Concomitantly, the amounts of cholesteryl esters and ether‐linked triglycerides increased, both lipids accumulating long‐chain and very‐long‐chain polyenes. This concurred with a considerable buildup of lipid droplets in Sertoli cells, evidently containing these neutral lipids, apparently formed during germ cell‐derived membrane lipid catabolism. Between week 4 and week 6, new cohorts of spermatocytes appeared, and by week 12 most cell changes were reversed. Accordingly, as germ cell differentiation proceeded, 22:5n‐6‐rich Gpl augmented and spermatocyte‐associated sphingolipids with nonhydroxy VLCPUFA appeared before their 2‐hydroxy counterparts. The unique fatty acids of rat testicular lipids after mild hyperthermia reveal lipid catabolic and biosynthetic reactions that occur in normal spermatogenesis.
Biochemical Journal, Apr 1, 1992
In their transit from the caput to the cauda segments of the epididymis, rat spermatozoa undergo ... more In their transit from the caput to the cauda segments of the epididymis, rat spermatozoa undergo significant modifications in lipid content and composition. The amount of lipid phosphorus per cell decreases, and most lipid classes show specific changes in their constituent fatty acids. A depletion of phosphatidylcholine and phosphatidylethanolamine, concomitant with a virtually unchanged amount of the corresponding plasmalogens, are the major alterations, plasmenylcholine thereby becoming the major phospholipid. Diphosphatidylglycerol, sphingomyelin and the phosphoinositides decrease to a lesser extent or do not change at all, also resulting in relative increases with sperm maturation. Concerning the fatty acids, the proportions of oleate (C18:1 n-9) and linoleate (C18:2 n-6) in most lipids decrease on movement of sperm from caput to cauda, augmenting in turn the proportions of longer-chain (C20 to C24) and more unsaturated fatty acids. Docosapentaenoate (C22:5 n-6) is a major acyl chain present in all lipids at both stages, but uncommon long-chain polyenoic fatty acids of the n-9 series are also present, being almost exclusively found in the choline glycerophospholipids. These fatty acids are found to undergo the most significant changes during sperm maturation. They are minor components of plasmenylcholine in immature spermatozoa, but increase severalfold on maturation, representing more than half of the acyl chains of this major lipid in cells from the cauda. The high concentration of n-9 polyenes in mature sperm plasmenylcholine raises intriguing questions on the possible role epididymal cells may play in providing spermatozoa with such an unusual phospholipid. These plasmenylcholines could contribute to the characteristic lipid domain organization of the mature spermatozoa plasma membrane. Abbreviations used: CGP and EGP are used to denote the (diradyl)-choline and-ethanolamine glycerophospholipids respectively; the terms phosphatidyl-, plasmenyland plasmanylare reserved respectively for the diacyl-, alk-1-enyl-2-acyl-and alkyl-2-acylsubclasses of the corresponding glycerophospholipid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PA, phosphatidic acid; PS, phosphatidylserine; DPG, diphosphatidylglycerol; FAME, fatty acid methyl esters.
International Journal of Biochemistry, 1990
1. The distribution of phospholipids between the two leaflets of the lipid bilayer in acetylcholi... more 1. The distribution of phospholipids between the two leaflets of the lipid bilayer in acetylcholine receptor (AChR)-rich membranes from T. marmorata has been examined with two complementary techniques: chemical derivatization with the membrane-impermeable reagent trinitrobenzenesulphonate (TNBS) and B.cereus phospholipase C hydrolysis. 2. AChR-membranes were reacted with TNBS at 0-4 and 37 degrees C and the accessibility of their aminophospholipids was compared to that of rod outer segment and erythrocyte membranes. The results indicate that more of the total ethanolamine glycerophospholipid (EGP) than of the total phosphatidylserine (PS) is located in the outer monolayer. 3. Nearly half the phospholipid content of AChR membranes is hydrolyzed by phospholipase C with a half-time of ca. 1.6 min at 25 degrees C. Consistent with the TNBS results, more of the total EGP than of the total PS is degraded. Beyond 3 min the reaction slows down, relatively smaller additional amounts of lipids are hydrolyzed, and all phospholipid classes are attacked to a similar extent, indicating that after half the lipid is removed all phospholipids become accessible to the enzyme. 4. The results indicate that the outer leaflet of the bilayer is richer in ethanolamine and choline glycerophospholipids, whereas phosphatidylinositol, most of the sphingomyelin, and ca 65% of the PS are located on the inner leaflet.
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Biochimica et biophysica acta, Sep 1, 1987
The labeling of molecular species of phosphatidylcholine (PC) has been studied in bovine retinas ... more The labeling of molecular species of phosphatidylcholine (PC) has been studied in bovine retinas incubated for 2 h with (1-14C)-labeled (n-6) eicosatetraenoate (n-3) docosapentaenoate and (n-3) docosahexaenoate (20: 4, 22: 5 and 22: 6, respectively) and in four subcellular fractions isolated after such incubations. Of the total radioactivity incorporated in PC, the following percentages of the above fatty acids, respectively, are found in its dipolyunsaturated species: 58, 56 and 53% in rod outer segments; 29, 41 and 49% in mitochondria; 24,28 and 39% in microsomes; 12, 14 and 16% in postmicrosomal supernatants; 28,36 and 58% in entire retinas. The remainder percentages are in tetra-, penta-and hexaenoic species of PC, respectively. The levels of pentaenoic species in the PCs of all fractions are similar, while tetraenes are lowest and hexaenes highest in photoreceptor membranes. Dipolyunsaturated species are highly concentrated in photoreceptor membranes, but are minor components of mitochondrial, microsomal and cytosolic PC. The specific radioactivities of tetraenoic, pentaenoic and hexaenoic PCs are decreasingly lower in the following order: postmicrosomal supematants, microsomes, mitochondria, photoreceptor membranes. In contrast, the specific radioactivities of dipolyunsaturated PCs are higher in mitochondria and microsomes than in the other fractions, especially with 22 : 5 and 22 : 6. It is suggested that mitochondria as well as the endoplasmic reticulum could play a role in the synthesis and further modifications of dipolyunsaturated PCs before being supplied to photoreceptor membranes.
Plant Physiology and Biochemistry, Apr 1, 2013
Phosphatidic acid (PA) is the common lipid product in abscisic acid (ABA) and gibberellic acid (G... more Phosphatidic acid (PA) is the common lipid product in abscisic acid (ABA) and gibberellic acid (GA) response. In this work we investigated the lipid metabolism in response to both hormones. We could detect an in vivo phospholipase D activity (PLD, EC 3.1.4.4). This PLD produced [ 32 P]PA (phosphatidic acid) rapidly (minutes) in the presence of ABA, confirming PA involvement in signal transduction, and transiently, indicating rapid PA removal after generation. The presence of PA removal by phosphatidate phosphatase 1 and 2 isoforms (E.C. 3.1.3.4) was verified in isolated aleurone membranes in vitro, the former but not the latter being specifically responsive to the presence of GA or ABA. The in vitro DGPP phosphatase activity was not modified by short time incubation with GA or ABA while the in vitro PA kinase e that allows the production of 18:2-DGPP from 18:2-PA e is stimulated by ABA. The long term effects (24 h) of ABA or GA on lipid and fatty acid composition of aleurone layer cells were then investigated. An increase in PC and, to a lesser extent, in PE levels is the consequence of both hormone treatments. ABA, in aleurone layer cells, specifically activates a PLD whose product, PA, could be the substrate of PAP1 and/or PAK activities. Neither PLD nor PAK activation can be monitored by GA treatment. The increase in PAP1 activity monitored after ABA or GA treatment might participate in the increase in PC level observed after 24 h hormone incubation.
Biochimica et biophysica acta, Sep 1, 1987
(l-l4 C)-labeled (n-6) eicosatetraenoate, (n-3) docosapentaenoate and (n-3) docosahexaenoate (20 ... more (l-l4 C)-labeled (n-6) eicosatetraenoate, (n-3) docosapentaenoate and (n-3) docosahexaenoate (20 : 4, 22: 5 and 22: 6, respectively) are efficiently taken up and actively esterified into the lipids of bovine retina after 2 h incubation. Photoreceptor membranes, mitochondria, microsomes and postmicrosomal supernatants, which display significant differences in phospholipid and fatty acid compositions, are isolated after such incubations to study the labeling of lipids. The lipid classes preferentially labeled with the acids (1) largely differ among and within subcellular fractions, while (2) some common features in the treatment of the three polyenes are observed in each fraction. In all of them, the three acids are actively incorporated in phosphatidylcholine; ethanolamine glycerophospholipid, phosphatidylserine (PS) and phosphatidylinositol (PI) are highly labeled with 22 : 6, 22 : 5 and 20 : 4 respectively; within ethanolamine glycerophospholipid, the three label phosphatidylethanolamine in preference to plasmenylethanolamine. Most of the 14C esterified in mitochondria is in phospholipids. The endoplasmic reticulum produces in addition highly labeled triacylglycerols, also found in cytosol. High levels of l4 C-labeled diacylglycerols are observed exclusively in photoreceptor membranes, where the specific radioactivity of PI is very high. The total amounts of 14C incorporated (1) are in general similar within a given fraction for the three polyenes, but (2) largely differ among fractions. The labeling of the highly unsaturated phospholipids of photoreceptor membranes is the lowest, while the postmicrosomal supematant (whose lipids are relatively the poorest in polycnoic fatty acids) contains most of the labeled lipids isolated from retinas under these conditions. The results indicate that polyunsaturated species of retina phospholipids undergo an active synthesis and turnover, as well as an intense intracellular traffic among membranes.
Journal of Biological Chemistry, Jun 1, 1993
The formation of long and very long chain (VLC) n-6 polyunsaturated fatty acids (PUFA) in isolate... more The formation of long and very long chain (VLC) n-6 polyunsaturated fatty acids (PUFA) in isolated rat seminiferous tubules was investigated by following the metabolism of three l-14C-labeled n-6 tetraenoic fatty acids (20:4, 24:4, and 3 2 4) and [U-"Clacetate. In contrast to [I4C]32:4, which was poorly incorporated and altered, ['4C]20:4 and [14C]24:4 were efficiently taken up by the tubules, esterified into lipids, elongated to VLCPUFA, and desaturated to pentaenoic fatty acids; the rate of [14C]24:4 desaturation to [14C]24:5 was notably high. The main products with [I4C]acetate as precursor were labeled saturates and VLCPUFA, most of the label in tetraenoic and pentaenoic acids appearing in 24:4 and 24:5, respectively. These two Car polyenes, connected by a A 6 desaturation, may play a central role in n-6 PUFA metabolism, in their capacity as potential precursors of longer polyenes via elongation and of shorter ones, such as 22:5n-6, via retroconversion. Triacylglycerols, rich in Czz and Cz4 polyenes, incorporated the greatest amounts of both ["C] acetate-derived and exogenous "C-PUFA, suggesting that this lipid class is involved in the traffic and metabolism of testicular PUFA. The detection of a series of unusual odd-chain tetraenoic and pentaenoic acids, also labeled with [U-14C]acetate, suggests that a PUFA chain shortening mechanism occurs in testis involving a-in addition to &oxidation. We speculate that aoxidation plays a role in the retroconversion of PUFA. Docosapentaenoic (22:5n-6)' and arachidonic (204n-6) acids are known to be abundant polyunsaturated fatty acids
Biochimica Et Biophysica Acta - Molecular And Cell Biology Of Lipids, 2014
Cadmium is known to harm rat testis by causing the dose-dependent apoptotic or necrotic death of ... more Cadmium is known to harm rat testis by causing the dose-dependent apoptotic or necrotic death of seminiferous epithelium cells. Here we investigated how this affects the lipids with long-chain (C18-C22) and very-long-chain (C24-C32) polyunsaturated fatty acids (VLCPUFA) typical of spermatogenic and Sertoli cells. A severe acute inflammatory reaction resulted from the massive necrotic death of these cells two days after a single high (4 mg/kg) dose of CdCl2. This led to the conversion of most testicular glycerophospholipids to diradylglycerols (DRG) and free fatty acids (FFA) and of most sphingomyelins to ceramides (Cer). By day 30 the testis weight had decreased three fold. The DRG and FFA had been metabolized but, unexpectedly, ceramides persisted. Also slow to disappear were VLCPUFA-containing triacylglycerols from former germ cells and ether-linked triglycerides and cholesteryl esters (CE) from former Sertoli cells. Similar results were observed 30 and 45 days after administering repeated small non pro-inflammatory CdCl2doses (1 mg/kg). At day 30 after both treatments, an amorphous material replaced the original seminiferous tubules and testicular macrophages populated the interstitium. Species of CE and ether-linked triglycerides containing fatty acids other than VLCPUFA steadily accumulated in the irreversibly damaged testis, a manifestation of the activity of phagocytic cells. The long-term permanence of original VLCPUFAcontaining neutral lipids, especially ceramides, indicates that these cells were slow to clear out the acellular material contained in seminiferous tubules, pointing to a form of silent chronic inflammation as an additional outcome of the multifactorial commotion caused in the testis by experimentally administered cadmium.
Reproduction, Nov 1, 2013
Male germ cell differentiation entails the synthesis and remodeling of membrane polar lipids and ... more Male germ cell differentiation entails the synthesis and remodeling of membrane polar lipids and the formation of triacylglycerols (TAGs). This requires fatty acid-binding proteins (FABPs) for intracellular fatty acid traffic, a diacylglycerol acyltransferase (DGAT) to catalyze the final step of TAG biosynthesis, and a TAG storage mode. We examined the expression of genes encoding five members of the FABP family and two DGAT proteins, as well as the lipid droplet protein perilipin 2 (PLIN2), during mouse testis development and in specific cells from seminiferous epithelium. Fabp5 expression was distinctive of Sertoli cells and consequently was higher in prepubertal than in adult testis. The expression of Fabp3 increased in testis during postnatal development, associated with the functional differentiation of interstitial cells, but was low in germ cells. Fabp9, together with Fabp12, was prominently expressed in the latter. Their transcripts increased from spermatocytes to spermatids and, interestingly, were highest in spermatid-derived residual bodies (RB). Both Sertoli and germ cells, which produce neutral lipids and store them in lipid droplets, expressed Plin2. Yet, while Dgat1 was detected in Sertoli cells, Dgat2 accumulated in germ cells with a similar pattern of expression as Fabp9. These results correlated with polyunsaturated fatty acid-rich TAG levels also increasing with mouse germ cell differentiation highest in RB, connecting DGAT2 with the biosynthesis of such TAGs. The age-and germ cell type-associated increases in Fabp9, Dgat2, and Plin2 levels are thus functionally related in the last stages of germ cell differentiation.
Se realizaron dos experimentos en el invernaculo para estudiar la influencia de la deficiencia de... more Se realizaron dos experimentos en el invernaculo para estudiar la influencia de la deficiencia de P en la nutricion con nitrogeno de plantas de soja no nodulada. Hubo una reduccion en el contenido de N y P en las plantas deficientes en P (P-) de casi el 50% y del 33% en la materia seca, luego de 50 dias. La actividad de la nitrato reductasa y el contenido de nitratos en tallos fueron menores en plantas P- que en aquellas P+. Del total de nitratos en las plantas P-, 75% estuvo en las raices. La acumulacion de nitratos en las raices seria debido a la menor actividad de la nitrato reductasa en las mismas, y a una disminucion en el flujo de agua hacia el vastago. El incremento en la concentracion de nitratos en la raiz causaria una retroalimentacion negativa que reduciria su absorcion por la planta
Journal of Lipid Research, 1983
Glycerolipids and sphingolipids are hydrolyzed with 0.5 M HCI in acetonitrile-water 9:l (by vol) ... more Glycerolipids and sphingolipids are hydrolyzed with 0.5 M HCI in acetonitrile-water 9:l (by vol) for 45 min at 100°C or 4 hr at 70°C. After hydrolysis, free fatty acids (FFA) are recovered in chloroform and separated by thin-layer chromatography. More than 95% of the radioactivity from labeled phospholipids is recovered as FFA, and more than 97% of the lipid phosphorus is recovered as water-soluble phosphate. The yields of FFA, including polyunsaturated acids, after hydrolysis are as good as or better than those obtained for methyl esters using methanolysis catalyzed by acid, alkali, or BFS. High recoveries of FFA from glycerophospholipids, sphingomyelin, and neutral glycerides are attained. The procedure is quantitative, simple, inexpensive, and produces no methyl esters as secondary products.-Aveldaiio, M. I., and L. A. Horrocks. Quantitative release of fatty acids from lipids by a simple hydrolysis procedure.
Journal of Lipid Research, 1983
Reverse phase high pressure liquid chromatography (HPLC) on octadecylsilyl columns separates mixt... more Reverse phase high pressure liquid chromatography (HPLC) on octadecylsilyl columns separates mixtures of either free fatty acids or fatty acid methyl esters prepared from mammalian tissue phospholipids. Acetonitrile-water mixtures are used for the elution of esters. Aqueous phosphoric acid is substituted for water for the separation of the free acids. Unsaturated compounds are detected and quantitated by their absorption at 192 nm. Saturates are detected better at 205 nm. T h e order of elution of fatty acids in complex mixtures varies as a function of acetonitrile concentration. At any given concentration, some compounds overlap. However, by varying the solvent strength, any fatty acid of interest can be resolved including many geometrical and positional isomers. Methyl esters prefractionated according to unsaturation by argentation thin-layer chromatography (TLC) are rapidly and completely separated by elution with CH'CN alone. Argentation TLC-reverse phase HPLC can be used as an analytical as well as a preparative procedure. Octylsilyl columns are used for rapid resolution and improved detection of minor or low ultraviolet-absorbing components in the fractions. For example, monoenoic fatty acids with up to 32 carbons have been detected in bovine brain glycerophospholipids. Specific radioactivities of 'H-and ''C-labeled fatty acids and the distribution of radioactivity among acyl groups from complex lipids are measured. T h e method is not recommended for complete compositional analysis, but is useful for determinations of specific radioactivities during studies on turnover and metabolic Abbreviations: GLC, gas-liquid chromatography; HPLC, high pressure liquid chromatography: TLC, thin-layer chromatography; FFA, free fatty acids; FAME, fatty acid methyl esters. Fatty acids are abbreviated by the convention, number of carbon atomsmumber of double bonds.
Clinical Chemistry, 1996
Blood cell and plasma lipid classes and their fatty acids were analyzed in a child with X-linked ... more Blood cell and plasma lipid classes and their fatty acids were analyzed in a child with X-linked adrenoleukodystrophy. The increase in saturated fatty acids with very long chains typical of this disease occurred almost exclusively in sphingomyelin. In this lipid, the proportion of lignoceric (24:0) and hexacosanoic (26:0) acids increased while that of 18:0, 20:0, and 24:1 decreased. In the rest of the lipid classes, but especially in cholesteryl esters and triacylglycerols, the proportion of linoleate (18:2) decreased while that of oleate (18:1) increased. In glycerophospholipids, polyunsaturated fatty acids such as 20:4n-6, 22:5n-6, and 22:6n-3 were reduced while their immediate precursors, 20:3n-6, 22:4n-6, and 22:5n-3, respectively, were relatively increased, suggesting a defect in fatty acid desaturation mechanisms. Although less pronounced, a similar trend of changes was seen in the patient's mother; in both, all alterations were more marked in serum than in blood cells.
Journal of lipid research, Jan 3, 2018
The sphingolipids (SL) of rodent spermatogenic cells (spermatocytes, spermatids) and spermatozoa ... more The sphingolipids (SL) of rodent spermatogenic cells (spermatocytes, spermatids) and spermatozoa contain nonhydroxylated and 2-hydroxylated versions of very-long-chain (C26 to C32) PUFA (n-V and h-V, respectively), not present in Sertoli cells. Here we investigated the expression of selected fatty acid elongases (), 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, though the protein was absent from, immature testes and Sertoli cells. 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 to C32 PUFA. 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...
Journal of Lipid Research, 2017
of the n-6 series. In spermatogenic cells from the adult rat, major ester-bound PUFAs of GPLs are... more of the n-6 series. In spermatogenic cells from the adult rat, major ester-bound PUFAs of GPLs are docosapentaenoic acid (22:5n-6) and arachidonic acid (20:4n-6) (1); whereas major amide-bound fatty acids of sphingomyelins (SMs) and ceramides (Cers) are 28:4n-6, 30:5n-6, and 32:5n-6 (2). Previous work has shown that in rat and boar testes, such fatty acids occur in SM in nonhydroxylated (n-V) and 2-hydroxylated (h-V) versions (3). Focus on the fatty acids of rat testis Cer showed it to contain even higher percentages of such fatty acids than SM (4, 5). The atypical 2-hydroxy-VLCPUFAs (e.g., h-30:5n-6) were also found by Sandhoff et al. (6) to be components of a novel series of glycosphingolipids (GSLs) that contain fucose in their head groups (FGSLs), which they discovered and thoroughly characterized in the rodent testis (mouse, rat). In the mouse, these complex sphingolipids were shown to be required in spermatogenic cells for a proper completion of meiosis (7). More recently, by conditionally knocking out (endoplasmic reticulum-located) Cer synthase 3 (CerS3) and glucosyl-Cer synthase specifically in mouse germ cells, the essentiality of both simple and complex sphingolipids that contain VLCPUFAs has been demonstrated, not only for meiosis, but also for the stability of spermatids (8). Neither Cer with VLCPUFA, nor the species of SM or FGSL that contain such fatty acids are produced in the testis in the absence of CerS3, resulting in spermatogenic arrest, increased apoptosis, and formation of multinuclear giant cells.
Biology of Reproduction, 2015
In spermatozoa isolated from rat epididymis, lipids are differentially modified after in vitro in... more In spermatozoa isolated from rat epididymis, lipids are differentially modified after in vitro induction of capacitation (Cap) and the acrosomal reaction (AR). This study uses Laurdan fluorescence generalized polarization values (GPv) to evaluate the effect of lipid changes occurring after isolation and functional activation on sperm membrane biophysical properties. In gametes isolated in the presence of a divalent cation chelator, no lipid changes occurred and the GPv were the lowest recorded, indicating maximal membrane lipid mobility. In sperm isolated as rapidly and gently as possible in the absence of chelator, part of the sphingomyelins (SM) were converted into ceramides (Cer), giving rise to higher GPv. In samples incubated as controls for Cap and AR, unchanged cholesterol and reduced glycerophospholipid levels were accompanied by the accumulation of free fatty acids (FFA), leading to even higher GPv. After completion of Cap, the GPv returned to lower levels as a result of the spermatozoa losing part of their cholesterol and FFA. Cap samples became relatively enriched in polyunsaturated fatty acids-containing plasmalogens because hydrolysis affected phosphatidyl rather than plasmenyl glycerophospholipid subclasses. The highest Cer:SM ratio and the highest GPv were found after completion of AR induced by A23187. The degree of SM ! Cer conversion among the samples, including controls, correlated with the extent of AR. FFA and Cer augmented GPv when added to liposomes prepared from the membrane lipid of intact sperm. Our results underscore the importance of hydrolytic changes that affect sperm lipids, especially the decisive lipid SM and Cer pair, not only after inducing sperm functional changes such as Cap and AR, but also under control conditions.
Journal of Biological Chemistry
ABSTRACT
Neurobiology of Essential Fatty Acids, 1992
Photoreceptor cells and spermatozoa have long been known to be the richest sources of polyunsatur... more Photoreceptor cells and spermatozoa have long been known to be the richest sources of polyunsaturated fatty acids (PUFA) in vertebrates. Docosahexaenoate (22:6n-3) and docosapentaenoate (22:5n-6), according to species and in diverse proportions, are common major acyl chains of the glycerophospholipids of these highly specialized cells. Both 22:6n-3 and 22:5n-6 are the products of a reaction that involves the introduction of a double bond in theΔ4 position of the chain in 22:5n-3 and 22:4n-6 respectively. Even though this reaction has yet to be fully characterized, the most highly unsaturated fatty acids that occur in vertebrate membranes are known to result from such desaturation. The four fatty acids just mentioned and their predecessors are grouped into two“lineages,”defined by the position of the first double bond counting from the methyl end, as the n-3 and the n-6 families. However, each of the four can be elongated, thus producing four lines of descendants, each line comprising various n-3 hexaenoic, n-6 pentaenoic, n-3 pentaenoic, and n-6 tetraenoic PUFA respectively. This chapter summarizes our findings on retina, joining them with more recent ones on spermatozoa, PUFA components, and the peculiar glycerophospholipids in which they occur. It is shown that, just as the glycerophospholipids of retina contribute to extend our view of existing PUFA by disclosing a whole variety of chain lengths within each of the known PUFA“lineages,”the glycerophospholipids of spermatozoa do so by disclosing new lineages for each of the known PUFA lengths. Intricate as this may sound, this overview expands, but at the same time simplifies, our picture of PUFA structural and metabolic relationships.