Mammalian spermatogenesis is an extraordinary cell transformation process. It includes asymmetric division of spermatogonia, rapid proliferation and differentiation into primary spermatocytes, a meiotic phase in which the genetic material in spermatocytes is recombined and segregated to produce haploid cells, a postmeiotic phase in which secondary spermatocytes develop into round spermatids, and differentiation of the latter into late spermatids and spermatozoa ( 1 ). During this last stage, known as spermiogenesis, spermatids undergo relevant cytological transformations, with changes in nuclear shape, chromatin condensation, formation of acrosome and fl agellum, and disposal of surplus organelles and materials by production and release of membrane-enclosed remnants known as "residual bodies" ( 2, 3 ). These minute, densely packed particles are then engulfed by Sertoli cells ( 4 ).The sphingomyelin (SM) ( 5 ) and ceramide (Cer) ( 6 ) of cells located in mammalian seminiferous tubules are rich in very long chain (C24 to C34) polyunsaturated fatty acids (VLCPUFA) of the n-6 or the n-3 series, the main ones being 28:4n-6 and 30:5n-6, followed by 32:5n-6 in the rat. These lipids belong to spermatogenic cells, as indicated by the facts that i) in the prepubertal rat testis, VLCPUFAcontaining species of SM and Cer are not detected; ii) in

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 (ϳ70%) of SM and Cer. In rat epididymal spermatozoa the SM/Cer ratio was low in the absence of and could be maintained high in the presence of the cation chelator EDTA, added to the medium used for sperm isolation. This fact points to the occurrence of an active divalent cation-dependent sphingomyelinase. Bull and rat sperm had an uneven head-tail distribution of phospholipid, with virtually all the VLCPUFA-rich SM located at the head, the lower SM content in the rat being determined by the lower sperm head/tail size ratio. Most of the SM from bull sperm heads was readily solubilized with 1% Triton X-100 at 4°C. The detergent-soluble SM fraction was richer in VLCPUFA than the nonsoluble fraction and richer in saturated fatty acids. Cer was produced at the expense of SM, thus decreasing severalfold the SM/Cer ratio in rat spermatozoa incubated for 2 h in presence of the spermcapacitating agents, calcium, bicarbonate, and albumin. The generation of Cer from SM in the sperm head surface may be an early step among the biochemical and biophysical changes known to take place in the spermatozoon in the physiological events preceding fertilization.In a number of mammals including humans a series of very long-chain polyunsaturated fatty acids (VLCPUFA), 2 i.e. n-6 and n-3 tetraenoic, pentaenoic and hexaenoic fatty acids with up to 32 or 34 carbon atoms, depending on the species, was characterized in the sphingomyelin (SM) from testis and spermatozoa (1, 2). In the testis of various mammals, we focused on the fatty acids of the ceramide (Cer), a lipid molecule with which SM bears a close precursor-product relationship, showing that SM and Cer species containing VLCPUFA are a specific feature of cells of the spermatogenic lineage (3). Because these testicular cells are predecessors of spermatozoa, the question arose as to the quantitative importance of these molecules in spermatozoa, where they could play a role in sperm functions related to fertilization. Transit through the epididymis is a crucial phase in sperm maturation. Spermatozoa exiting the testis are immotile, unable to bind to eggs and to undergo the acrosomal reaction in vitro in response to commonly used stimuli. By the time they reach the region of cauda epididymis, sperm cells have acquired their progressive motility and their ability to bind, penetrate, and fertilize eggs (4). One of the questions we addressed was whether epididymal maturation gives rise to spermatozoa with a larger or a smaller proportion of SM and Cer containing these VLCPUFA as opposed to other fatty acids.Spermatozoa are functionally regionalized cells. Sperm-oocyte interactions are head-relat...

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.

This article is available online at http://www.jlr.org motherapeutic drugs and X-ray irradiation. Precisely because of its effi cacy to kill rapidly dividing cells, exposure of the testis to these agents, whether incidental or therapeutic, selectively targets the population of spermatogenic cells and is a potential cause of male infertility. While somatic cells, including Sertoli cells, are apparently unaffected, spermatogonia that are mitotically dividing and spermatocytes during the early phases of meiosis are the testicular cells most vulnerable to X-rays ( 1 ). Relatively less susceptible are nondividing spermatogonial stem cells on the one hand and postmeiotic cells on the other. Spermatids and spermatozoa, with their much more compact nuclei, are notably resistant. Germ cell death induced by X-ray irradiation mostly occurs via apoptosis ( 2, 3 ), which results from radiation-induced free radical generation damaging DNA. The protein P53, the known intracellular sensor of DNA damage, is required for this response ( 1, 4 ). P53 upregulates in germ cells the production of Fas, the surface receptor that, on binding the Fas ligand produced by Sertoli cells, activates apoptosis via a caspase-mediated cascade ( 5, 6 ).A few weeks after having locally irradiated the testis with X-rays, spermatogenesis recommences from type A spermatogonial stem cells that had not been affected at irradiation time in some mammalian species like mice ( 1 ) but not in others like LBNF1 rats, which are in this regard a good model of the human testicular sensitivity to X-rays ( 7 ). In these rats, despite the presence and normal proliferation of apparently undamaged type A spermatogonia, it is their further differentiation that is after some time impeded, The mammalian testis is known to be highly susceptible to a variety of anticancer agents, including a number of che- This work was supported by funding from Consejo Nacional de Investigaciones Científi cas y Técnicas (CONICET), Agencia Nacional de Promoción de la Ciencia y la Tecnología (ANPCyT), and Universidad Nacional del Sur (UNS).

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.

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.

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Previous work showed that rat germ cells and spermatozoa contain ceramides and sphingomyelins with high proportions of nonhydroxy and 2-hydroxy (2-OH) polyunsaturated fatty acids (PUFA) with very long chains (VLCPUFA). The aim of this study was to assess how these lipids are distributed between the heads and tails of mature spermatozoa in comparison with other membrane lipid classes. In addition to quantitative differences due to the fact that these gametes have a long, voluminous tail and a minute head, several compositional dissimilarities emerged between these two regions. The total cholesterol/total phospholipid ratio, the choline/ethanolamine glycerophospholipid (ChoGpl/EtnGpl) ratio, and the proportion of plasmalogens within these two classes, were much larger in the head than in the tail. Whereas EtnGpl was rich in 22:5n-6 in both regions, ChoGpl had plenty of 22:4n-9, especially in the heads. An important proportion of the head EtnGpl- 22:5n-6 and ChoGpl 22:4n-9 was in plasmenyl- (rather than in phosphatidyl-) subclasses. The heads concentrated all of the sphingomyelin species with nonhydroxy- and 2-OH VLCPUFA, and the tails most of the saturated fatty acids that are present in total sperm sphingomyelin. Unexpectedly, virtually all of the abundant spermatozoal ceramides, predominantly made up by species with 2-OH VLCPUFA, was located in the tail. The fact that intact rat spermatozoa constitutively have much more VLCPUFA-containing ceramide than sphingomyelin is explained by the present findings, since the former are mostly lipids of the large tail while the latter mostly collect in the small head.