Doxorubicin Affects Testicular Lipids with Long-Chain (C18-C22) and Very Long-Chain (C24-C32) Polyunsaturated Fatty Acids

Doxorubicin disrupts spermatogenesis by causing apoptosis of spermatogonia and primary spermatocytes. The aim of this study was to examine the effect of this agent on adult rat testicular lipids and their fatty acids. A single dose (7.5 mg/kg) and a multidose regime (3 mg/kg once a week for 4 weeks) were evaluated. Both treatments resulted in the gradual loss of spermatogenic cells and determined a marked reduction in testicular size and weight 9 weeks after their start. Germ cell loss was accompanied by a decrease in phospholipids, including glycerophospholipids and sphingomyelin. Concomitantly, glycerophospholipids lost selectively their major polyunsaturated fatty acid (PUFA), 22:5n-6, and sphingomyelin lost its major very long-chain PUFA (VLCPUFA), 28:4n-6 and 30:5n-6. The molecular species from which the lost polyenes originated were thus a trait of germ cells. A transient peak of 16:0-ceramide was observed 48 h after the single dose. In both doxorubicin regimes, sphingomyelin and ceramide with reduced amounts of VLCPUFA after about 4 weeks and with no VLCPUFA after 9 weeks resulted. By contrast, triglycerides and especially cholesterol esters (CE) tended to accumulate in the testes undergoing germ cell death, probably in the surviving Sertoli cells, their fatty acid patterns suggesting that initially, these lipids retained part of the PUFA coming from, or no longer used for, the synthesis of germ cell glycerophospholipids. As the latter decreased, CE accumulated massively 9 weeks after starting doxorubicin treatment, 20:4n-6 becoming their major PUFA. Part of these CEs may derive from surviving steroidogenic cells.Fil: Zanetti, Samanta Romina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; ArgentinaFil: Maldonado, Eduardo N.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; ArgentinaFil: Aveldaño, Marta Isabel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentin

Enzymes involved in the biosynthesis of sphingolipids with very-long-chain PUFA concur with male germ cell differentiation

The sphingolipids sphingomyelin (SM) and ceramide (Cer), of male rodent germ cells (spermatocytes, spermatids) and spermatozoa contain very-long-chain (C28-C32) polyenoic fatty acids (VLCPUFA) in nonhydroxy (n-V) and 2-hydroxy forms (h-V) not present in Sertoli cells. During postnatal development, SM and Cer species with n-V appear in testes concomitantly with pachytene spermatocytes and those with h-V with spermatids. Both are able to biosynthesize their own sphingolipids, the former more actively than the latter. The biosynthesis of n-V requires elongation of PUFA (20:4n-6, 22:5n-6) by elongation of very-long-chain fatty acid (Elovl) proteins, while that of h-V requires a fatty acid 2-hydroxylase (Fa2h). Elovl5 and Elovl2, coding for enzymes responsible for PUFA biosynthesis, and Elovl4, in turn responsible for the formation of PUFA longer than C26, are actively expressed in germ cells. The Elovl4 protein is exclusively expressed in germ cells in a stage-dependent manner, spermatocytes displaying the highest Elovl4 protein levels and enzymatic activity. Along with high proportion of h-V Cer and h-V SM species, the Fa2h protein is mainly concentrated in late spermatids, in the step of spermiogenesis in which they elongate and their heads change shape. Consistently, spermatocytes express the highest levels of ceramide synthase 3, required for the N-acylation of sphingosine with a VLCPUFA, while spermatids express the highest levels of SM synthase 2. Irrespective of the maturation stage, the small raftlike domains of the germ cell plasma membrane contain species of SM and Cer with saturated fatty acids, while those containing n-V and h-V abound in the large non-raft areas. These species, with unique physico-chemical properties, could play a structural role facilitating the germ cell shape changes associated with the progress of spermatogenesis. If released from such species, free n-V and h-V could be involved in germ cell differentiation as potential precursors of uncommon elovanoid-like bioactive derivatives.Fil: Oresti, Gerardo Martin. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; ArgentinaFil: Santiago Valtierra, Florencia Ximena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; ArgentinaFil: Reyes, Juan. Pontificia Universidad Católica de Valparaíso; ChileFil: Aveldaño, Marta Isabel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; ArgentinaCongreso Latinoamericano Conjunto de ReproducciónSantiagoChileSociedad Chilena de Reproducción y Desarroll

Unique thermal behavior of sphingomyelin species with nonhydroxy and 2-hydroxy very-long-chain (C28-C32) PUFAs

In rat germ cells and spermatozoa, sphingomyelin (SM) contains molecular species with nonhydroxy (n) and 2-hydroxy (h) very-long-chain polyunsaturated fatty acids (V), the most abundant being SMs with (n- and h-) 28:4n-6, 30:5n-6, and 32:5n-6 as acyl chains. The aim of this study was to gain information about their thermotropic behavior and interactions with other lipids. After isolation from rat testis, multilamellar and giant unilamellar vesicles from these SMs were examined using fluorescent probes. Only n-32:5 SM and h-32:5 SM displayed a gel-liquid transition temperature (Tt ≈ 21?22°C), the rest remaining in the liquid state in the 5°C?45°C range. The degree of order was larger in bilayers of any of the h-V SMs than in those of their chain-matched n-V SMs. Both, but n-V SM relatively more than h-V SM, decreased the Tt of dimyristoylphosphatidylcholine as their proportion increased in binary phosphatidylcholine:SM liposomes. In contrast to the established ability of 16:0 SM to form lateral cholesterol/SM-rich ordered domains in ternary dioleoylphosphatidylcholine:cholesterol:SM bilayers, neither n-V SM nor h-V SM showed a tendency to do so. Thus, these SMs are in the fluid state and are not involved in this type of domains in spermatozoa at physiological temperatures. However, this state could be altered at the very low temperatures at which these gametes are usually preserved.Fil: Peñalva, Daniel Alejandro. Consejo Nacional de Investigaciones Cientificas y Técnicas. Centro Científico Tecnológico Bahia Blanca. Instituto de Investigaciones Bioquímicas Bahia Blanca (i); ArgentinaFil: Furland, Natalia Edith. Consejo Nacional de Investigaciones Cientificas y Técnicas. Centro Científico Tecnológico Bahia Blanca. Instituto de Investigaciones Bioquímicas Bahia Blanca (i); ArgentinaFil: Lopez, Gustavo H.. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; ArgentinaFil: Aveldaño, Marta Isabel. Consejo Nacional de Investigaciones Cientificas y Técnicas. Centro Científico Tecnológico Bahia Blanca. Instituto de Investigaciones Bioquímicas Bahia Blanca (i); ArgentinaFil: Antollini, Silvia Susana. Consejo Nacional de Investigaciones Cientificas y Técnicas. Centro Científico Tecnológico Bahia Blanca. Instituto de Investigaciones Bioquímicas Bahia Blanca (i); Argentin

Atypical surface behavior of ceramides with nonhydroxy and 2-hydroxy very long-chain (C28-C32) PUFAs

Unique species of ceramide (Cer) with very-long-chain polyunsaturated fatty acid (VLCPUFA), mainly 28?32 carbon atoms, 4?5 double bonds, in nonhydroxy and 2-hydroxy forms (n-V Cer and h-V Cer, respectively), are generated in rat spermatozoa from the corresponding sphingomyelins during the acrosomal reaction. The aim of this study was to determine the properties of these sperm-distinctive ceramides in Langmuir monolayers. Individual Cer species were isolated by HPLC and subjected to analysis of surface pressure, surface potential, and Brewster angle microscopy (BAM) as a function of molecular packing. In comparison with known species of Cer, n-V Cer and h-V Cer species showed much larger mean molecular areas and increased molecular dipole moments in liquid expanded phases, which suggest bending and partial hydration of the double bonded portion of the VLCPUFA. The presence of the 2-hydoxyl group induced a closer molecular packing in h-V Cer than in their chain-matched n-V Cer. In addition, all these Cer species showed liquid-expanded to liquid-condensed transitions at room temperature. Existence of domain segregation was confirmed by BAM. Additionally, thermodynamic analysis suggests a phase transition close to the physiological temperature for VLCPUFA-Cers if organized as bulk dispersions.Fil: Peñalva, Daniel Alejandro. Consejo Nacional de Investigaciones Cientificas y Técnicas. Centro Científico Tecnológico Bahia Blanca. Instituto de Investigaciones Bioquímicas Bahia Blanca (i); ArgentinaFil: Oresti, Gerardo Martin. Consejo Nacional de Investigaciones Cientificas y Técnicas. Centro Científico Tecnológico Bahia Blanca. Instituto de Investigaciones Bioquímicas Bahia Blanca (i); ArgentinaFil: Dupuy, Fernando Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones En Química Biológica de Córdoba (p); ArgentinaFil: Antollini, Silvia Susana. Consejo Nacional de Investigaciones Cientificas y Técnicas. Centro Científico Tecnológico Bahia Blanca. Instituto de Investigaciones Bioquímicas Bahia Blanca (i); ArgentinaFil: Maggio, Bruno. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones En Química Biológica de Córdoba (p); ArgentinaFil: Aveldaño, Marta Isabel. Consejo Nacional de Investigaciones Cientificas y Técnicas. Centro Científico Tecnológico Bahia Blanca. Instituto de Investigaciones Bioquímicas Bahia Blanca (i); ArgentinaFil: Fanani, Maria Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones En Química Biológica de Córdoba (p); Argentin

Phosphorus nutrition on nitrogen metabolism of non-nodulated soybean

Se realizaron dos experimentos en el invernáculo para estudiar la influencia de la deficiencia de P en la nutrición con nitrógeno de plantas de soja no nodulada. Hubo una reducción 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 días. 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 raíces. La acumulación de nitratos en las raíces sería debido a la menor actividad de la nitrato reductasa en las mismas, y a una disminución en el flujo de agua hacia el vástago. El incremento en la concentración de nitratos en la raíz causaría una retroalimentación negativa que reduciría su absorción por la planta.Two experiments were conducted under greenhouse conditions to study the influence of P deficiency on nitrogen nutrition of non-nodulated soybean. There was a reduction of N and P content in P deficient (P-) plants of almost 50% after 50 days. During this period, dry matter of these plants was reduced by 33%. Nitrate reductase activity and shoot nitrate content were lower in Pthan in P+ plants. Seventy five percent of nitrates in P- plants were in the roots. Nitrate accumulation in roots would be due to the lower nitrate reductase activity in this organ and a reduced water flux to shoots. Root nitrate concentration increases would cause a negative feedback which would reduce plant nitrate uptake.Fil: Brevedan, Roberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Centro de Recursos Naturales Renovables de la Zona Semiárida. Universidad Nacional del Sur. Centro de Recursos Naturales Renovables de la Zona Semiárida; ArgentinaFil: Aveldaño, Marta Isabel. Universidad Nacional del Sur; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Fioretti, Maria Nelida. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Centro de Recursos Naturales Renovables de la Zona Semiárida. Universidad Nacional del Sur. Centro de Recursos Naturales Renovables de la Zona Semiárida; ArgentinaFil: Varillas, María Alejandra. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia. Cátedra de Control de Calidad de Medicamentos; ArgentinaFil: Baioni, Sandra Sonia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Centro de Recursos Naturales Renovables de la Zona Semiárida. Universidad Nacional del Sur. Centro de Recursos Naturales Renovables de la Zona Semiárida; ArgentinaFil: Mirasson, H. R.. Universidad Nacional de la Pampa. Facultad de Agronomia. Cátedra de Fisiología Vegetal; ArgentinaFil: Palomo, Iris Rosana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Centro de Recursos Naturales Renovables de la Zona Semiárida. Universidad Nacional del Sur. Centro de Recursos Naturales Renovables de la Zona Semiárida; Argentin

Very long chain PUFA in murine testicular triglycerides and cholesterol esters

Very long chain (VLC) PUFA of the n-6 and n-3 series are known to occur in mammalian testis. The aim of this work was to characterize further two testicular lipid classes with VLCPUFA, cholesterol esters (CE) and total triglycerides (TG) in rat and mouse testis. The VLCPUFA predominating in these lipids were a series of n-6 pentaenes and tetraenes with 24 to 32 carbons, including small amounts of odd-chain PUFA, 28:5n-6 and 24:5n-6 prevailing in CE and TG, respectively. Most of the VLCPUFA of TG were concentrated in a small fraction of TG, made up by 1-O-alkyl-2,3-DAG. This TG subclass was absent altogether from the TG of sexually immature testis. The TG and the CE with VLCPUFA only occurred in testis of adult fertile animals. The proportion of VLCPUFA in total TG and CE was higher in rodents than in other mammals. In the n-6 PUFA-rich adult mouse testis, the amounts of testicular triacylglycerols decreased significantly after consumption of fish oil for 2 wk. Whereas 18:2n-6 was significantly reduced, the amounts of 22:5n-6 and longer n-6 PUFA were less affected in all major testicular lipids including PC and PE, where they were unchanged. The 1-O-alkyl-2,3-DAG and their n-6 VLCPUFA were virtually unaffected by the diet. The VLCPUFA-containing molecular species of CE and TG may represent a form of storage of cholesterol and polyenoic FA required to sustain spermatogenesis. Via chain-shortening, VLCPUFA stored in the neutral lipids may serve as precursors of the major C22 PUFA typical of cell membrane glycerophospholipids, protecting testicular cells against shifts in FA composition induced by dietary changes.Fil: Furland, Natalia Edith. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; ArgentinaFil: Maldonado, Eduardo Néstor. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; ArgentinaFil: Aveldaño, Marta Isabel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentin

Major clofibrate effects on liver and plasma lipids are independent of changes in polyunsaturated fatty acid composition induced by dietary fat

The effects of clofibrate on the content and composition of liver and plasma lipids was studied in mice fed for 4 wk on diets enriched in n-6 or n-3 polyunsaturated fatty acids (PUFA) from sunflower oil (SO) or fish oil (FO), respectively; both oils were fed at 9% of the diet (dry weight basis). Only FO was hypolipidemic. Both oil regimes led to slightly increased concentrations of phospholipids (PL) and triacylglycerols (TG) in liver as compared with a standard chow diet containing 2% fat. Clofibrate promoted hypolipidemia only in animals fed SO. Its main effect was to enlarge the liver, such growth increasing the amounts of major glycerophospholipids while depleting the TG. SO and FO consumption changed the proportion of n-6 or n-3 PUFA in liver and plasma lipids in opposite ways. After clofibrate action, the PUFA of liver PL were preserved better than in the absence of oil supplementation. However, most of the drug-induced changes (e.g., increased 18:1n-9 and 20:3n-6, decreased 22:6/20:5 ratios) occurred irrespective of lipids being rich in n-6 or n-3 PUFA. The concentration of sphingomyelin (SM), a minor liver lipid that virtually lacks PUFA, increased with the dietary oils, decreased with clofibrate, and changed its fatty acid composition in both situations. Thus, oil-increased SM had more 22:0 and 24:0 than clofibrate-decreased SM, which was significantly richer in 22:1 and 24:1.Fil: Pennacchiotti, Graciela Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; ArgentinaFil: Maldonado, Eduardo Néstor. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; ArgentinaFil: Aveldaño, Marta Isabel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentin

Growth inhibition by gamma rays affects lipids and fatty acids in garlic sprouts during storage

Bulbs of cv. Colorado garlic were irradiated at dormancy with a dose of 60 Gy of gamma rays and stored for 8 months, during which period the content and fatty acid composition of phospholipids (PL), glycolipids (GL) and neutral lipids were analyzed on three occasions. No significant changes were observed a few hours after irradiation, but the treatment resulted in a considerable reduction in lipid and fatty acid content 150 and 240 days post-harvest, with a concomitant reduction in the process of sprout growth. In total lipid, all fatty acids including the major linoleic acid (18:2) decreased, the largest decrease being in linolenic acid (18:3). The latter was a relatively minor component of PL (phosphatidylcholine and -ethanolamine) and a major acyl group of GL (monogalactosyl- and digalactosylglycerol). Radioinhibition had the opposite effect on polyunsaturated fatty acids of PL and GL, the 18:3/18:2 ratio decreasing in the former and increasing in the latter. Accretion of lipids and fatty acids is a normal biosynthetic process accompanying sprout growth, and the long-term effects of irradiation are interpreted to reflect a delay or slowing down of such process.Fil: Perez, Monica Beatriz. Universidad Nacional del Sur; ArgentinaFil: Aveldaño, Marta Isabel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Universidad Nacional del Sur; ArgentinaFil: Croci, Clara Ana. Universidad Nacional del Sur; Argentin

Biosynthesis of sphingolipids with very-long-chain pufa a hallmark of differentiating male germ cells

The sphingomyelins (SM) and ceramides (Cer) of rodent spermatogenic cells contain very-long-chain (C28-C32) polyenoic fatty acids (VLCPUFA), in non-hydroxy (n-V) and 2-hydroxy (h-V) forms. The SM and Cer species with n-V, present in meiotic spermatocytes, become in part h-V species in post-meiotic spermatids. In each of these cells, the mentioned species are located in the non-raft fraction of the plasma membrane. The enzymes required for PUFA elongation to n-V, Elovl5, Elovl2 and Elovl4, and the fatty acid 2-hydroxylase (Fa2h) that converts n-V to h-V, are expressed in germ cells, with Elovl4 and Fa2h protein levels being highest in spermatocytes and spermatids, respectively. The Cer and SM species with n-V and h-V are biosynthesized de novo in a germ cell type-specific and steroid hormone-dependent manner. CerS3, which specifically N-acylates VLCPUFA to sphinganine, is highly expressed in meiotic cells. The Elovl4 and CerS3 protein expression prevails in spermatocytes, is seminiferous stage-specific, and is mostly concomitant. In spermatids, the Fa2h protein appears concentrated in late stages, especially when they elongate and their heads change shape. The unique sphingolipid species with n-V and h-V, as well as the enzymes involved in their biosynthesis, are useful biomarkers for investigating normal and pathological aspects of germ and sperm cell functions. Supported by SGCyT UNS-PGI-UNS (24/B272 to GMO and 24/B218 to MIA), FONCyT (PICT2017-2535 to GMO).Fil: Oresti, Gerardo Martin. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; ArgentinaFil: Santiago Valtierra, Florencia Ximena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; ArgentinaFil: Aveldaño, Marta Isabel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; ArgentinaThe LV Annual SAIB Meeting and XIV PABMB CongressArgentinaSociedad Argentina de Investigación Bioquímica y Biología MolecularPanamerican Association of Biochemestry and Molecular Biolog