Ceramides and sphingomyelins with high proportions of very long-chain polyunsaturated fatty acids in mammalian germ cells

Very long-chain polyunsaturated fatty acids (VLCPUFA) have previously been shown to be components of sphingomyelin (SM) of mammalian testis and spermatozoa. Here we examined the fatty acids of testicular ceramide (Cer) in comparison with those of SM in some mammals with a special focus on the rat testis. In bull, cat, dog, rabbit, mouse, and rat, VLCPUFA were found in both testicular lipids, Cer having a higher percentage of VLCPUFA than SM. Rat testis had the highest percentage of VLCPUFA in both lipids, the major ones being 28:4n-6 and 30:5n-6. VLCPUFA-containing SM and Cer occurred in cells located in the seminiferous tubules, where germ cells had a higher percentage of these species than Sertoli cells. Seminiferous tubule fractionation showed that SM and Cer of mitochondria and lysosomes had mostly saturates and negligible VLCPUFA, the latter being important in the SM and Cer of microsomes and other membrane fractions. VLCPUFA were absent from the SM and Cer of rat prepuberal testis, increased with the onset of spermatogenesis to account for nearly 15 and 40% of the total fatty acids of testicular SM and Cer, respectively, remained at those levels throughout the adult life of fertile rats and tended to decrease at advanced ages. Four conditions that lead to selective death of germ cells in vivo, namely experimental cryptorchidism, post-ischemic reperfusion, focalized x-ray irradiation and treatments with the antineoplasic drug doxorubicin, caused the VLCPUFA to disappear from the testicular SM and Cer of adult fertile rats, showing that these lipids are specific traits of spermatogenic cells.     

Differentiation-related changes in lipid classes with long-chain and very long-chain polyenoic fatty acids in rat spermatogenic cells

In rat seminiferous tubules (ST), cells that contain polar and neutral lipids with long-chain polyenoic fatty acids (PUFA) and sphingomyelins (SM) and ceramides (Cer) with very long chain (VLC) PUFA of the n-6 series coexist. In this study, pachytene spermatocytes and round spermatids were isolated to determine how these lipids change during spermatogenesis. As the amount per cell of PUFA-rich glycerophospholipids (GPL) decreased with cell size, the 22:5/20:4 ratio increased with cell differentiation. The elovl2 and elovl5 genes, required for 22:5 formation, were expressed (mRNA) in both cell types. Residual bodies- particles with compacted organelles and materials discarded from late spermatids-concentrated cholesterol, 22:5-rich triacylglycerols, and GPL, including plasmalogens and phosphatidylserine. Species of SM and Cer with nonhydroxylated (n-) VLCPUFA (28:4, 30:5, and 32:5) predominated in pachytene spermatocytes, whereas species with the corresponding 2-hydroxy (2-OH) VLCPUFA prevailed in round spermatids. Thus, a dramatic increase in the 2-OH/n-VLCPUFA ratio in SM and Cer was a hallmark of differentiation. A substantial decrease of 2-OH SM occurred between spermatids and mature spermatozoa and 2-OH SM species were collected in residual bodies “en route” to Sertoli cells. Notably, spermatids and spermatozoa gained a significant amount of ceramides devoid of n-VLCPUFA but having 2-OH VLCPUFA as their main fatty acids.     

Very long-chain polyunsaturated fatty acids are the major acyl groups of sphingomyelins and ceramides in the head of mammalian spermatozoa

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 ( approximately 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 degrees 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.     

Long-term biopermanence of ceramides,cholesteryl esters,and ether-linked triglycerides with very-long-chain PUFA in the cadmium-damaged testis

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 CdCl₂ doses (1 mg/kg). At day 30 after both treatments, an amorphous material replaced the original seminiferous tubules and the interstitium was populated by macrophages. 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 these cells. The long-term permanence of original VLCPUFA-containing neutral lipids, especially ceramides, indicates that these phagocytes 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.     

Sequential depletion of rat testicular lipids with long-chain and very long-chain polyenoic fatty acids after X-ray-induced interruption of spermatogenesis

When a single dose of X-rays is applied to the adult rat testis, stem spermatogonia are damaged, and spermatogenesis is interrupted. Supported by Sertoli cells, spermatogenic cells that endure irradiation complete their differentiation and gradually leave the testis as spermatozoa. In this study, the in vivo changes taking place a number of weeks after irradiation revealed cell-specific features of testicular lipid classes. A linear drop, taking about six weeks, in testis weight, nonlipid materials, free cholesterol, and 22:5n-6-rich glycerophospholipids took place with germ cell depletion. Sphingomyelins and ceramides with nonhydroxy very long-chain polyenoic fatty acids (n-VLCPUFA) disappeared in four weeks, together with the last spermatocytes, whereas species with 2-hydroxy VLCPUFA lasted for six weeks, disappearing with the last spermatids and spermatozoa. The amount per testis of 22:5n-6-rich triacylglycerols, unchanged for four weeks, fell between weeks 4 and 6, associating these lipids with spermatids and their residual bodies, detected as small, bright lipid droplets. In contrast, 22:5n-6-rich species of cholesterol esters and large lipid droplets increased in seminiferous tubules up to week 6, revealing they are Sertoli cell products. At week 30, the lipid and fatty acid profiles reflected the resulting permanent testicular involution. Our data highlight the importance of Sertoli cells in maintaining lipid homeostasis during normal spermatogenesis.     

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.     

Changes in lipids containing long- and very long-chain polyunsaturated fatty acids in cryptorchid rat testes

The aim of the present study was to examine the effects of experimental cryptorchidism on rat testicular phospholipids and neutral lipids that contain long-chain (C(18)-C(22)) and very long-chain (VLC) (C(24)-C(32)) polyunsaturated fatty acids (PUFA). The weight of the cryptorchid testis was nearly half that of the contralateral control at postsurgical Days 7-10 owing to the depletion of germ cells. Concomitantly, the amounts of major glycerophospholipids (GPL) and sphingomyelin (SM) per testis decreased. Both these lipids lost their characteristic long-chain and very long-chain PUFA, notably 22:5n-6 and 28:4n-6, respectively, which suggests that these species are linked to the membranes of germ cells. In contrast, the amounts and concentrations of triglycerides (TG; triacylglycerols and 1-O-alkyl-2,3-diacylglycerols) and cholesterol esters (CE) increased several fold in the surviving cells (mainly Sertoli cells) in the cryptorchid testis. All these neutral lipids, but especially CE, accumulated large amounts of the major PUFA of the testis, 22:5n-6, as well as pentaenes with longer carbon chains (i.e., 24:5n-6 in TG and 28:5n-6 in CE). This accretion suggests that neutral lipids may store preformed PUFA coming from dying germ cell GPL and also VLCPUFA no longer needed as a source of PUFA destined to assemble new germ cell GPL. The lipid adjustments observed in cryptorchidism suggest a possible role for Sertoli cell CE in the turnover and conservation of PUFA within seminiferous tubules.     

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.     

Changes of Phosphatidylcholine and Fatty Acids in Germ Cells during Testicular Maturation in Three Developmental Male Morphotypes of Macrobrachium rosenbergii Revealed by Imaging Mass Spectrometry

Testis maturation, germ cell development and function of sperm, are related to lipid composition. Phosphatidylcholines (PCs) play a key role in the structure and function of testes. As well, increases of polyunsaturated fatty acids (PUFA) and highly unsaturated fatty acids (HUFA), especially arachidonic acid (ARA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) are essential for male fertility. This study is the first report to show the composition and distribution of PCs and total fatty acids (FAs) in three groups of seminiferous tubules (STs) classified by cellular associations [i.e., A (STs with mostly early germ cells), B (STs with mostly spermatids), and C (STs with spermatozoa)], in three morphotypes of Macrobrachium rosenbergii, [i.e., small male (SM), orange claw male (OC), and blue claw male (BC)]. Thin layer chromatography exhibited levels of PCs reaching maxima in STs of group B. Imaging mass spectrometry showed remarkably high signals corresponding to PC (16:0/18:1), PC (18:0/18:2), PC (18:2/20:5), and PC (16:0/22:6) in STs of groups A and B. Moreover, most signals were detected in the early developing cells and the intertubular area, but not at the area containing spermatozoa. Finally, gas chromatography-mass spectrometry indicated that the major FAs present in the testes were composed of 14:0, 16:0, 17:0, 18:0, 16:1, 18:1, 18:2, 20:1, 20:2, 20:4, 20:5, and 22:6. The testes of OC contained the greatest amounts of these FAs while the testes of BC contained the least amounts of these FAs, and there was more EPA (20:5) in the testes of SM and OC than those in the BC. The increasing amounts of FAs in the SM and OC indicate that they are important for spermatogenesis and spermiogenesis. This knowledge will be useful in formulating diets containing PUFA and HUFA for prawn broodstocks in order to improve testis development, and lead to increased male fecundity.     

Identification of genes and pathways involved in the synthesis of Mead acid (20:3n − 9), an indicator of essential fatty acid deficiency

In mammals, 5,8,11-eicosatrienoic acid (Mead acid, 20:3n − 9) is synthesized from oleic acid during a state of essential fatty acid deficiency (EFAD). Mead acid is thought to be produced by the same enzymes that synthesize arachidonic acid and eicosapentaenoic acid, but the genes and the pathways involved in the conversion of oleic acid to Mead acid have not been fully elucidated. The levels of polyunsaturated fatty acids in cultured cells are generally very low compared to those in mammalian tissues. In this study, we found that cultured cells, such as NIH3T3 and Hepa1–6 cells, have significant levels of Mead acid, indicating that cells in culture are in an EFAD state under normal culture conditions. We then examined the effect of siRNA-mediated knockdown of fatty acid desaturases and elongases on the level of Mead acid, and found that knockdown of Elovl5, Fads1, or Fads2 decreased the level of Mead acid. This and the measured levels of possible intermediate products for the synthesis of Mead acid such as 18:2n − 9, 20:1n − 9 and 20:2n − 9 in the knocked down cells indicate two pathways for the synthesis of Mead acid: pathway 1) 18:1n − 9 → (Fads2) → 18:2n − 9 → (Elovl5) → 20:2n − 9 → (Fads1) → 20:3n − 9 and pathway 2) 18:1n − 9 → (Elovl5) → 20:1n − 9 → (Fads2) → 20:2n − 9 → (Fads1) → 20:3n − 9.     

Ceramide and sphingomyelin species of fibroblasts and neurons in culture

The ceramide (Cer) and sphingomyelin (SM) species of cultured differentiated rat cerebellar granule cells and human fibroblasts were characterized by electrospray ionization-mass spectrometry. We identified 35 different species of Cer and 18 species of SM in human fibroblasts, and 35 different species of Cer and 9 species of SM were characterized in rat neurons. The main Cer species of rat cerebellar granule cells contained d18:1 sphingosine linked with palmitic, stearic, or nervonic fatty acid, and the two main SM species were d18:1,16:0 and d18:1,18:0. Both sphingolipids were enriched in detergent-resistant membranes (DRMs; or lipid rafts), and significant differences were found in the sphingolipid patterns of DRMs and of detergent-soluble fractions (DSF) from these cells. In human fibroblasts, the main Cer species were d18:1,16:0, d18:2,16:0, d18:1,24:0, d18:2,24:0, d18:1,24:1, and d18:2,24:1; the most represented species of SM were d18:1,16:0, d18:1,24:0, and d18:1,24:1. In these cells, SM was highly enriched in DRMs and Cer was mainly associated with DSF, and the species found in DRMs were markedly different from those found in DSF.     

Lipid metabolism in the testis of the ram

1. Analysis of rams testes revealed that phosphatidylcholine was the major phospholipid and accounted for about 40% of the total. Only small amounts of choline plasmalogen were present. 2. The ratio of phosphatidylcholine to choline plasmalogen in the testis was very different from that occurring in the spermatozoa. This result was in contrast with those for rat testis and rat spermatozoa (obtained from the head of the epididymis), where the ratio of the two lipids was very similar. 3. Infusions of [(32)P]orthophosphate into the testicular artery of rams resulted in incorporation of radioactivity into most phospholipids; phosphatidylinositol labelling accounted for 68% and 39% of the radioactivity after infusions lasting 3hr. and 5hr. respectively. 4. With the exception of phosphatidic acid the specific radioactivity of phosphatidylinositol was higher than that of any other lipid. 5. After the infusion of [U-(14)C]glucose, triglycerides accounted for about 60% of the radioactivity in testicular neutral lipids, whereas diglycerides had only about 15% of the radioactivity. 6. Palmitic acid (16:0) was the major component both in neutral lipids and phospholipids of ram testes. 7. The effects of gonadotrophic hormones (luteinizing hormone and follicle-stimulating hormone) on the incorporation of [(32)P]orthophosphate into total testicular phospholipids in vivo were also examined.     

Acid sphingomyelinase activity is regulated by membrane lipids and facilitates cholesterol transfer by NPC2

During endocytosis, membrane components move to intraluminal vesicles of the endolysosomal compartment for digestion. At the late endosomes, cholesterol is sorted out mainly by two sterol-binding proteins, Niemann-Pick protein type C (NPC)1 and NPC2. To study the NPC2-mediated intervesicular cholesterol transfer, we developed a liposomal assay system. (Abdul-Hammed, M., B. Breiden, M. A. Adebayo, J. O. Babalola, G. Schwarzmann, and K. Sandhoff. 2010. Role of endosomal membrane lipids and NPC2 in cholesterol transfer and membrane fusion. J. Lipid Res. 51: 1747–1760.) Anionic lipids stimulate cholesterol transfer between liposomes while SM inhibits it, even in the presence of anionic bis(monoacylglycero)phosphate (BMP). Preincubation of vesicles containing SM with acid sphingomyelinase (ASM) (SM phosphodiesterase, EC 3.1.4.12) results in hydrolysis of SM to ceramide (Cer), which enhances cholesterol transfer. Besides SM, ASM also cleaves liposomal phosphatidylcholine. Anionic phospholipids derived from the plasma membrane (phosphatidylglycerol and phosphatidic acid) stimulate SM and phosphatidylcholine hydrolysis by ASM more effectively than BMP, which is generated during endocytosis. ASM-mediated hydrolysis of liposomal SM was also stimulated by incorporation of diacylglycerol (DAG), Cer, and free fatty acids into the liposomal membranes. Conversely, phosphatidylcholine hydrolysis was inhibited by incorporation of cholesterol, Cer, DAG, monoacylglycerol, and fatty acids. Our data suggest that SM degradation by ASM is required for physiological secretion of cholesterol from the late endosomal compartment, and is a key regulator of endolysosomal lipid digestion.     

Sphingomyelin hydrolysis to ceramide during the execution phase of apoptosis results from phospholipid scrambling and alters cell-surface morphology

Apoptosis is generally accompanied by a late phase of ceramide (Cer) production, the significance of which is unknown. This study describes a previously unrecognized link between Cer accumulation and phosphatidylserine (PS) exposure at the cell surface, a characteristic of the execution phase of apoptosis resulting from a loss of plasma membrane phospholipid asymmetry. Using a fluorescent sphingomyelin (SM) analogue, N-(N-[6-[(7-nitrobenz-2-oxa-1, 3-diazol-4-yl)amino]caproyl]-sphingosylphosphorylcholine (C(6)-NBD-SM), we show that Cer is derived from SM, initially located in the outer leaflet of the plasma membrane, which gains access to a cytosolic SMase by flipping to the inner leaflet in a process of lipid scrambling paralleling PS externalization. Lipid scrambling is both necessary and sufficient for SM conversion: Ca(2+) ionophore induces both PS exposure and SM hydrolysis, whereas scrambling-deficient Raji cells do not show PS exposure or Cer formation. Cer is not required for mitochondrial or nuclear apoptotic features since these are still observed in Raji cells. SM hydrolysis facilitates cholesterol efflux to methyl-beta-cyclodextrin, which is indicative of a loss of tight SM-cholesterol interaction in the plasma membrane. We provide evidence that these biophysical alterations in the lipid bilayer are essential for apoptotic membrane blebbing/vesiculation at the cell surface: Raji cells show aberrant apoptotic morphology, whereas replenishment of hydrolyzed SM by C(6)- NBD-SM inhibits blebbing in Jurkat cells. Thus, SM hydrolysis, during the execution phase of apoptosis, results from a loss of phospholipid asymmetry and contributes to structural changes at the plasma membrane.     

Reversible changes in the testes and epididymides of dog treated with alpha-chlorohydrin.

1. Chronic administration of alpha-chlorohydrin (8 mg/kg body wt for 30 days) caused lesions in the testis of dog. The changes in the germ cells were degenerative. The seminiferous tubule and Leydig cell nuclear diameter were reduced. 2. Epididymal cell height was greatly reduced and the stereocilia had disappeared completely. The lumen was devoid of spermatozoa. 3. Alpha-chlorohydrin administration inhibited the RNA and sialic acid contents in the testes and epididymides of dog. Total cholesterol and lipids/g of testes were increased significantly after alpha-chlorohydrin administration. 4. These effects were reversible. Repopulation of testis tubules occurred following a period of 100 days recovery in dog. Numerous spermatogonia and sperm develop and traverse the epididymides. The RNA, sialic acid, cholesterol and total lipids of testes and epididymides returned to subnormal levels. 5. The possibility of using alpha-chlorohydrin as male contraception is indicated.     

Expression and regulation of delta5-desaturase,delta6-desaturase,stearoyl-coenzyme A (CoA) desaturase 1, and stearoyl-CoA desaturase 2 in rat testis

In mammalian cells, essential polyunsaturated fatty acids (PUFAs) are converted to longer PUFAs by alternating steps of elongation and desaturation. In contrast to other PUFA-rich tissues, the testis is continuously drained of these fatty acids as spermatozoa are transported to the epididymis. Alteration of the germ cell lipid profile from spermatogonia to condensing spermatids and mature spermatozoa has been described, but the male gonadal gene expression of the desaturases, responsible for the PUFA-metabolism, is still not established. The focus of this study was to characterize the expression and regulation of stearoyl-CoA desaturase 1 (SCD1), stearoyl-CoA desaturase 2 (SCD2), and Delta5- and Delta6-desaturase in rat testis. Desaturase gene expression was detected in testis, epididymis, and separated cells from seminiferous tubulus using Northern blot analysis. For the first time, SCD1 and SCD2 expression is demonstrated in rat testis and epididymis, both SCDs are expressed in epididymis, while testis mainly contains SCD2. Examination of the testicular distribution of Delta5- and Delta6-desaturase and SCD1 and SCD2 shows that all four desaturases seem to be localized in the Sertoli cells, with far lower expression in germ cells. In light of earlier published results showing that germ cells are richer in PUFAs than Sertoli cells, this strengthens the hypothesis of a lipid transport from the Sertoli cells to the germ cells. As opposed to what is shown in liver, Delta5- and Delta6-desaturase mRNA levels in Sertoli cells are up-regulated by dexamethasone. Furthermore, dexamethasone induces SCD2 mRNA. Insulin also up-regulates these three genes in the Sertoli cell, while SCD1 mRNA is down-regulated by both insulin and dexamethasone. Delta5- and Delta6-desaturase, SCD1, and SCD2 are all up-regulated by FSH. A similar up-regulation of the desaturases is observed when treating Sertoli cells with (Bu)2cAMP, indicating that the desaturase up-regulation observed with FSH treatment results from elevated levels of cAMP. Finally, testosterone has no influence on the desaturase gene expression. Thus, FSH seems to be a key regulator of the desaturase expression in the Sertoli cell.     

Novel molecular species of sphingomyelin containing 2-hydroxylated polyenoic very-long-chain fatty acids in mammalian testes and spermatozoa.

Polyenoic very-long-chain fatty acids (VLCFA) have been shown to be localized in unusual molecular species of sphingomyelin in the testes and spermatozoa of the ram, bull, rat, and boar and in the spermatozoa of man. The composition of polyenoic VLCFA-sphingomyelin was comparable in the testes and spermatozoa of each mammalian species; however, the sphingolipid was more concentrated in spermatozoa. The composition of testicular and spermatozoan polyenoic VLCFA-sphingomyelin differed considerably between animal types. Human spermatozoa mainly contained n-6 polyenoic VLCFA with two to four double bonds and even-carbon chain lengths up to 32. In ram and bull testes and spermatozoa, n-3 and n-6, tetra-, penta-, and hexaenoic VLCFA with even-carbon chain lengths up to 34 predominated. In rat and boar testes and spermatozoa, the polyenoic VLCFA were mainly n-6 derivatives with three to five double bonds and even- and odd-carbon chain lengths up to 34. The testes and spermatozoa of the latter two animal species contained 2-hydroxylated, in addition to non-hydroxylated, polyenoic VLCFA in sphingomyelin. This is the first time that 2-hydroxylated polyenoic VLCFA have been recognized in biological systems. Non-hydroxylated polyenoic VLCFA were initially observed in the sphingomyelin of rat testes 25 days after birth, followed by 2-hydroxylated derivatives at 30 days. The total amount of polyenoic VLCFA associated with rat testicular sphingomyelin increased dramatically from 25 to 40 days of postnatal life and then remained constant to 60 days (sexual maturity). The ratio of 2-hydroxylated to non-hydroxylated polyenoic VLCFA increased during this period. Polyenoic VLCFA-sphingomyelin seems to occur exclusively in the testes and spermatozoa of mammals, and it is postulated that this lipid plays a role in reproduction.     

Fatty acid composition in matrix vesicles and in microvilli from femurs of chicken embryos revealed selective recruitment of fatty acids

Hypertrophic chondrocytes participate in matrix mineralization by releasing matrix vesicles (MVs). These MVs, by accumulating Ca²⁺ and phosphate initiate the formation of hydroxyapatite. To determine the types of lipids essential for mineralization, we analyzed fatty acids (FAs) in MVs, microvilli and in membrane fractions of chondrocytes isolated from femurs of chicken embryos. The FA composition in the MVs was almost identical to that in microvilli, indicating that the MVs originated from microvilli. These fractions contained more monounsaturated FAs especially oleic acid than in membrane homogenates of chondrocytes. They were enriched in 5,8,11-eicosatrienoic acid (20:3n−9), in eicosadienoic acid (20:2n−6), and in arachidonic acid (20:4n−6). In contrast, membrane homogenates from chondrocytes were enriched in 20:1n−9, 18:3n−3, 22:5n−3 and 22:5n−6. Due to their relatively high content in MVs and to their selective recruitment within microvilli from where MV originate, we concluded that 20:2n−6 and 20:3n−9 (pooled values), 18:1n−9 and 20:4n−6 are essential for the biogenesis of MVs and for bone mineralization.     

Synthesis of very long chain (up to 36 carbon) tetra, penta and hexaenoic fatty acids in retina.

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 14C-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 VLCPUFA-containing species of PC are very active processes in the retina.     

Assessment of lipid and essential fatty acids requirements of black seabream (Spondyliosoma cantharus) by comparison of lipid composition in muscle and liver of wild and captive adult fish

The primary aim of the present study was to compare the contents of total lipid, lipid classes and their associated fatty acids in muscle and liver of wild and one-year captive black seabream (Spondyliosoma cantharus) adults, in order to elucidate the lipid and fatty acids requirements of this fish species of potential interest for aquaculture. The total lipid contents (TL) of muscle and liver of the captive fish were 2.5-fold greater than those of the wild fish. In consequence, contents of triacylglycerols were much higher in tissues of the captive fish. Distribution of fatty acids in total lipids and lipid classes of muscle and liver was also different between both groups of fish. For instance, percentages of 20:4n−6, 20:5n−6 and 22:6n−3 were considerably higher in the wild fish, whereas 18:1, 20:1, and 22:1n−9 as well as 18:2n−6 and 20:5n−3 were more abundant in the captive fish. These results suggest that the lipid composition of the commercial diet supplied to the captive black seabream differed greatly from that of the diet consumed by the fish in the wild, which hypothetically contains the desirable composition for the lipid nutrition of this fish species. Despite the good growth and survival achieved by the black seabream after one year in captivity, the significant accumulation of lipids and the imbalance of essential fatty acids in their muscle and livers, together with the absence of spawning, suggest that future research on the lipid requirements of this omnivorous species is necessary in order to establish whether the administration of currently available aquaculture formulated feeds is adequate for good black seabream performance and reproduction.