Structural analysis of a new glycosphingolipid from the lipopolysaccharide-lacking bacterium Sphingomonas adhaesiva.

A new glycosphingolipid, GSL-4B, was isolated from Sphingomonas adhaesiva and found to share the ceramide moiety with GSL-1 and GSL-3 from Sphingomonas capsulata studied earlier [Kawahara, K.; Moll, H.; Knirel, Y. A.; Seydel, U.; Z?hringer, U. Eur. J. Biochem. 2000, 267, 1837-1846]. It is heterogeneous with respect to the long-chain bases erythro-2-amino-1,3-octadecanediol (sphinganine), (13Z)-erythro-2-amino-13-eicosene-1,3-diol, and (13Z)-erythro-2-amino-13,14-methylene-1,3-eicosanediol which in GSL-4B are present in the ratios of 1.1:1.0:1.1, and all bearing amide-linked (S)-2-hydroxymyristic acid. Methylation analysis and MALDI-TOF-MS along with 1H and 13C NMR spectroscopy showed that the carbohydrate part of GSL-4B has the structure of alpha-D-Glcp-(1-->4)-alpha-D-Galp-(1-->6)-alpha-D-Glcp-(1-->4)-alpha-D-GlcpA-(1-->1)-Cer     

Novel class of glycosphingolipids involved in male fertility

Mice require testicular glycosphingolipids (GSLs) for proper spermatogenesis. Mutant mice strains deficient in specific genes encoding biosynthetic enzymes of the GSL pathway including Galgt1 (encoding GM2 synthase) and Siat9 (encoding GM3 synthase) have been established lacking various overlapping subsets of GSLs. Although male Galgt1-/- mice are infertile, male Siat9-/- mice are fertile. Interestingly, GSLs thought to be essential for male spermatogenesis are not synthesized in either of these mice strains. Hence, these GSLs cannot account for the different phenotypes. A novel class of GSLs was observed composed of eight fucosylated molecules present in fertile but not in infertile mutant mice. These GSLs contain polyunsaturated very long chain fatty acid residues in their ceramide moieties. GSLs of this class are expressed differentially in testicular germ cells. More importantly, the neutral subset of this new GSL class strictly correlates with male fertility. These data implicate polyunsaturated, fucosylated GSLs as essential for spermatogenesis and male mouse fertility.     

Oxidation and base-catalyzed elimination of the saccharide portion of GSLs having very different polarities

Glycosphingolipids (GSLs), present in cell membranes, participate in a variety of biological functions. Although their exact role(s) may not be understood, it has been shown that 1) embryos lacking glucosylceramide synthase activity do not develop normally, 2) GSLs can affect neuritogenesis, and 3) they can function as receptors for some pathogens. To study the role of the saccharide portion of a GSL in any of these functions, it is necessary to either isolate it from the intact GSL or synthesize it. Because syntheses are more complex, modifications were made to the oxidation/elimination procedure previously described for the isolation of the saccharide portion of GM1 and GD1a to enable it to be used with GSLs of varying polarity. The key is to use a mixture of GSLs that differ in polarity. This appears to eliminate problems encountered when purified GSLs such as sulfatide or GT1b are used.     

Isolation and structural characterization of glycosphingolipids of in vitro propagated human umbilical vein endothelial cells

To investigate in detail the expression of glycosphingolipids (GSLs) on endothelial cells, 4.85 x 10(9) human umbilical vein endothelial cells (HUVECs) were cultivated in a 2 l bioreactor using microcarriers as a support for anchorage dependent growing cells. Neutral GSLs and gangliosides were isolated and their structures were determined by TLC immunostaining, fast atom bombardment-mass spectrometry (FAB-MS) of the native GSLs, and gas chromatography-electron impact mass spectrometry (GC-EIMS) of partially methylated alditol acetates. GbOse4Cer, GbOse3Cer, and LacCer, all carrying mainly C24- and C16-fatty acid beside C18-sphingosine, were detected as the major neutral GSLs (36%, 23%, and 15% of the total orcinol stain, respectively); GlcCer, nLcOse4Cer, and nLcOse6Cer were expressed to substantial minor amounts (9%, 12%, and 5% of the total orcinol stain, respectively). TLC immunostaining revealed the presence of lipid bound Lewisx antigen, whereas the isomeric Lewisa structure was detectable only in very low quantities. GM3(Neu5Ac) with C18-sphingosine was the major ganglioside constituting about 90% of the whole ganglioside fraction. The fatty acid composition was determined by GC-MS of fatty acid methyl esters, indicating the predominance of C24- and C16-substituted GM3(Neu5Ac), followed by C18- and C22-substituted species. Terminally alpha2-3 sialylated neolacto-series ganglioside IV3Neu5Ac-nLcOse4Cer was the second most abundant ganglioside in HUVECs (8% of the total resorcinol stain), and IV6Neu5Ac-nLcOse4Cer and VI3Neu5Ac-nLcOse6Cer (together less than 2% of total resorcinol stain) were found in minor quantities. Lipid bound sialyl Lewisx antigen with poly-N-acetyllactosaminyl chains, and traces of gangliotetraose-type gangliosides GM1 and GD1a were identified by TLC immunostaining. The expression of dominant neutral GSLs LacCer, GbOse3Cer, and GbOse4Cer, and of ganglioside GM3(Neu5Ac) was assayed by indirect immunofluorescence microscopy of cell layers grown in chamber slides, each showing different plasma membrane and subcellular distribution patterns. The complete structural characterization of GSLs from HUVECs contributes to our understanding about their functional role, not only of the carbohydrate but also of the lipid moiety, as receptors for bacterial toxins, as cell surface antigens of cellular interaction and as receptors for blood components and macromolecules of the extracellular matrix.     

Norwalk virus-like particles bind specifically to A,H and difucosylated Lewis but not to B histo-blood group active glycosphingolipids

Noroviruses and norovirus virus-like particles (VLPs) exhibit strain specific patterns in their binding to ABH and Lewis histo-blood group antigens. In this study we demonstrate for the first time specific binding of Norwalk virus VLPs to type 1 and type 2 chain glycosphingolipids (GSLs) carrying ABH and Lewis antigens. N-succinimidyl-3-tributylstannyl benzoate (ATE) was precursor labeled with ¹²⁵I and then conjugated to VLPs. The ¹²⁵I-VLPs were used in GSL thin-layer chromatogram binding assays and displayed binding to H type 1, Lewis b, A type 1, A Lewis b GSLs but no binding to B type 1 or B Lewis b GSLs. For the type 2 chain GSLs the Norwalk VLPs bound to H type 2, Lewis y, A type 2 and A Lewis y. In addition, the VLPs bound to several complex GSLs from blood group O and A, but not from blood group B red blood cells.     

Chemical structure and function of glycosphingolipids of Sphingomonas spp and their distribution among members of the α-4 subclass of Proteobacteria

Sphingomonas spp are phylogenetically placed in the α-4 subclass of Proteobacteria. They have glycosphingolipids (GSL) in their membranes instead of lipopolysaccharide (LPS) as in other Gram-negative bacteria. S. paucimobilis, the type species of the genus, has GSL-1, which contains only glucuronic acid (GlcA) as a sugar moiety, and GSL-4A, which contains a tetrasaccharide including GlcA. GSL-1 and GSL-4A form the outer membrane of S. paucimobilis with outer membrane proteins and phospholipids. In the outer membrane, GSLs are assumed to locate and function as does the LPS of other Gram-negative bacteria. Sphingomonas spp closely related to the type species contain both GSL-1 and the oligosaccharide-type GSL such as GSL-4A, but other Sphingomonas spp and other genera in the α-4 subclass of Proteobacteria contain only GSL-1. Structural variations of fatty acids and dihydrosphingosines in the GSL-1 are presented. Received 19 April 1999/ Accepted in revised form 18 June 1999     

Newly discovered neutral glycosphingolipids in aureobasidin A-resistant zygomycetes: Identification of a novel family of Gala-series glycolipids with core Gal alpha 1-6Gal beta 1-6Gal beta sequences

We found for the first time that Zygomycetes species showed resistance to Aureobasidin A, an antifungal agent. A novel family of neutral glycosphingolipids (GSLs) was found in these fungi and isolated from Mucor hiemalis, which is a typical Zygomycetes species. Their structures were completely determined by compositional sugar, fatty acid, and sphingoid analyses, methylation analysis, matrix-assisted laser desorption ionization time-of-flight/mass spectrometry, and (1)H NMR spectroscopy. They were as follows: Gal beta 1-6Gal beta 1-1Cer (CDS), Gal alpha 1-6Gal beta 1-6Gal beta 1-1Cer (CTS), Gal alpha 1-6Gal alpha 1-6Gal beta 1-6Gal beta 1-1Cer (CTeS), and Gal alpha 1-6Gal alpha 1-6Gal alpha 1-6Gal beta 1-6Gal beta 1-1Cer (CPS). The ceramide moieties of these GSLs consist of 24:0, 25:0, and 26:0 2-hydroxy acids as major fatty acids and 4-hydroxyoctadecasphinganine (phytosphingosine) as the sole sphingoid. However, the glycosylinositolphosphoceramide families that are the major GSLs components in fungi were not detected in Zygomycetes at all. This seems to be the reason that Aureobasidin A is not effective for Zygomycetes as an antifungal agent. Our results indicate that the biosynthetic pathway for GSLs in Zygomycetes is significantly different from those in other fungi and suggest that any inhibitor of this pathway may be effective for mucormycosis, which is a serious pathogenic disease for humans.     

Glycosphingolipid dynamics in human embryonic stem cell and cancer: their characterization and biomedical implications

Glycosphingolipids (GSLs) are composed of complex glycans linked to sphingosines and various fatty acid chains. Antibodies against several GSLs designated as stage-specific embryonic antigens (SSEAs), have been widely used to characterize differentiation of embryonic stem (ES) cells. In view of the cross-reactivities of these antibodies with multiple glycans, a few laboratories have employed advanced mass spectrometry (MS) technologies to define the dynamic changes of surface GSLs upon ES differentiation. However, the amphiphilic nature and heterogeneity of GSLs make them difficult to decipher. In our studies, systematic survey of GSL expression profiles in human ES cells and differentiated derivatives was conducted, primarily with matrix-assisted laser desorption/ionization MS (MALDI-MS) and MS/MS analyses. In addition to the well-known ES-specific markers, SSEA-3 and SSEA-4, several previously undisclosed globo- and lacto-series GSLs, including Gb4Cer, Lc4Cer, fucosyl Lc4Cer, Globo H, and disialyl Gb5Cer were identified in the undifferentiated human ES and induced pluripotent stem cells. Furthermore, during differentiation to embryoid body outgrowth, the core structures of GSLs switched from globo- and lacto- to ganglio-series. Lineage-specific differentiation was also marked by alterations of specific GSLs. During differentiation into neural progenitors, core structures shifted to primarily ganglio-series dominated by GD3. GSL patterns shifted to prominent expression of Gb4Cer with little SSEA-3 and-?4 or GD3 during endodermal differentiation. Several issues relevant to MS analysis and novel GSLs in ES cells were discussed. Finally, unique GSL signatures in ES and cancer cells are exploited in glycan-targeted anti-cancer immunotherapy and their mechanistic investigations were discussed using anti-GD2 mAb and Globo H as examples.     

The Hypothesis on Function of Glycosphingolipids and ABO Blood Groups Revisited

Twenty-five years ago the author proposed new ideas of glycoprotein (GPs) and glycosphingolipid (GSLs) functions at the cell membrane. The GPs, apart from their glycan carrying capacity, were assumed to have specific, protein associated, functions. In contrast, GSLs such as those of globo and neolacto/lacto series, were considered to be energetically cheap membrane packing substances, filling in membrane spaces not covered with functional GPs. The terminal carbohydrate structures of the neolacto/lacto GSLs, i.e., sialic acid residues and ABH glycotopes, were postulated to have either regulatory or protective functions, respectively. A special active role was ascribed to terminal β-galactosyl residues of GSLs and GPs. Gangliosides were considered to be functional GSLs. In the present review the author discusses these old ideas in context of the contemporary knowledge and comes to the conclusion that they have not aged.     

Characterization of N-glycolyneuraminic acid-containing glycosphingolipids from a Marek's disease lymphoma-derived chicken cell line, MSB1, as tumor-associated heterophile Hanganutziu-Deicher antigens

Heterophile, Hanganutziu-Deicher (HD) antigen-active N-glycolylneuraminic acid-containing glycosphingolipids (GSLs) were detected as tumor-associated foreign antigens of a Marek's disease lymphoma-derived cell line, MSB1, by enzyme-immunoassay with chicken antibody against N-glycolylneuraminyl-lactosylceramide (anti-NeuGc-LacCer). At least three species of HD antigen-active GSLs were detected by two-dimensional thin-layer chromatography (TLC) combined with enzyme-immunoassay. The reactivities of the GSLs with anti-NeuGc-LacCer, their behaviors on two-dimensional TLC and the results of an endo-beta-galactosidase digestion study indicated that these three GSLs were NeuGc-LacCer (NeuGc alpha 2-2Gal beta 1-4Glc-Cer), NeuGc-nLcOse4Cer (NeuGc alpha 2-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc-Cer) and NeuGc-nLcOse6Cer (NeuGc alpha 2-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc-Cer).     

Growth in coculture stimulates metabolism of the phenylurea herbicide isoproturon by Sphingomonas sp. strain SRS2

Metabolism of the phenylurea herbicide isoproturon by Sphingomonas sp. strain SRS2 was significantly enhanced when the strain was grown in coculture with a soil bacterium (designated strain SRS1). Both members of this consortium were isolated from a highly enriched isoproturon-degrading culture derived from an agricultural soil previously treated regularly with the herbicide. Based on analysis of the 16S rRNA gene, strain SRS1 was assigned to the beta-subdivision of the proteobacteria and probably represents a new genus. Strain SRS1 was unable to degrade either isoproturon or its known metabolites 3-(4-isopropylphenyl)-1-methylurea, 3-(4-isopropylphenyl)-urea, or 4-isopropyl-aniline. Pure culture studies indicate that Sphingomonas sp. SRS2 is auxotrophic and requires components supplied by association with other soil bacteria. A specific mixture of amino acids appeared to meet these requirements, and it was shown that methionine was essential for Sphingomonas sp. SRS2. This suggests that strain SRS1 supplies amino acids to Sphingomonas sp. SRS2, thereby leading to rapid metabolism of (14)C-labeled isoproturon to (14)CO(2) and corresponding growth of strain SRS2. Proliferation of strain SRS1 suggests that isoproturon metabolism by Sphingomonas sp. SRS2 provides unknown metabolites or cell debris that supports growth of strain SRS1. The role of strain SRS1 in the consortium was not ubiquitous among soil bacteria; however, the indigenous soil microflora and some strains from culture collections also stimulate isoproturon metabolism by Sphingomonas sp. strain SRS2 to a similar extent.     

Stimulatory effect of gangliosides on phagocytosis, phagosome–lysosome fusion, and intracellular signal transduction system by human polymorphonuclear leukocytes

Gangliosides are known to be differentiation-inducing molecules in mammalian stem cells. We studied the interaction between the molecular structure of glycosphingolipids (GSLs) and their promoting mechanisms of the phagocytic processes in human polymorphonuclear leukocytes (PMN). The effect of various gangliosides from mammalian tissues on adhesion, phagocytosis, phagosome–lysosome (P–L) fusion and superoxide anion production was examined by human PMN using heat-killed cells of Staphylococcus aureus coated with GSLs. Gangliosides GM3, GD1a, GD3 and GT1b showed a marked stimulatory effect on the phagocytosis and P–L fusion in a dose-dependent manner, while ganglioside GM1, asialo GM1 and neutral GSLs did not. The relative phagocytic rate of ganglioside GM3-coated S. aureus was the highest among the tested GSLs. Both P–L fusion rate and phagocytosis of S. aureus were elevated significantly when coated with ganglioside GD1a, GD3 or GT1b, and GT1b gave a five times higher rate than that of the non-coated control. These results suggest that the terminal sialic acid moiety is essential for the enhancement of phagocytosis and that the number of sialic acid molecules in the ganglioside is related to the enhancement of the P–L fusion process. On the other hand, the superoxide anion release from PMN was not affected by ganglioside GM2, GM3, GD1a or GT1b. Furthermore, to clarify the trigger or the signal transduction mechanism of phagocytic processes, we examined the effect of protein kinase inhibitors such as H-7, staurosporine (protein kinase C inhibitor), H-89 (protein kinase A inhibitor), genistein (tyrosine kinase inhibitor), ML-7 (myosin light chain kinase inhibitor), and KN-62 (Ca2+/calmodulin-dependent protein kinase II inhibitor) on ganglioside-induced phagocytosis. H-7, staurosporine and KN-62 inhibited ganglioside-induced phagocytosis in the range of concentration without cell damage, while H-89, genistein and ML-7 did not. Moreover, H-7 and KN-62 inhibited ganglioside-induced P–L fusion. These results suggest that protein kinase C and Ca2+/calmodulin-dependent protein kinase II may be involved in the induction of phagocytosis and P–L fusion stimulated by gangliosides. This revised version was published online in November 2006 with corrections to the Cover Date.     

Surface topography of sulfatide and gangliosides in unilamellar vesicles of dipalmitoylphosphatidylcholine

The property of the dyes, acridine orange and methylene blue, to exhibit metachromatic changes upon binding to negatively charged groups that are within a defined spatial separation was employed to study the lateral and transverse topography of sulfatide and gangliosides GM1 and GD1a mixed with dipalmitoylphosphatidylcholine (DPPC) in unilamellar vesicles. The spectral changes of the dyes in the presence of liposomes containing anionic glycosphingolipids (GSLs) (hypochromism and frequency shift) are typical of polyanionic lattices while minor changes are found for neutral lipids. The metachromatic changes are abolished by the presence of Ca2+ in the external medium. The proportion of anionic GSLs accessible to the dyes on the external surface of the liposomes is greater as the GSLs are more complex (sulfatide less than GM1 less than GD1a) and as its proportion in the mixture decreases. The number of molecules of anionic GSLs that are laterally distributed on the external surface in a position favorable for the formation of dye dimers (at intermolecular distances not exceeding 1 nm) is greater for sulfatide than for ganglioside. This is correlated to the greater intermolecular distances and delocalization in ganglioside-, compared to sulfatide-containing interfaces. The experimental values indicate that the mixture with DPPC of any of the anionic GSLs studied behaves as if it was more enriched in the GSLs compared to the proportions of the whole mixture.     

Glycosphingolipids of the globo-series are associated with the monocytic lineage of human myeloid cells

Neutral glycosphingolipids (neutral GSLs) of the human myeloid leukemia cell lines ML-2, ML-3, HL-60 and THP-1-0 were metabolically labeled with [3H]galactose and [3H]glucosamine, and analyzed by high-performance liquid chromatography. They were compared with unlabeled neutral GSLs from purified human granulocytes and monocytes. Neutral GSLs were identified by retention times and the structures were further confirmed by degradation with specific exoglycosidases. Two neutral GSLs of the globoseries, globotetraosylceramide and globotriaosylceramide were found in monocytes and the monoblastic leukemia line THP-1-0. The leukemia-derived cell-lines, ML-3 and HL-60, representing successively earlier stages of myeloid differentiation, contained respectively less neutral GSLs of the globoseries and an increasing proportion of (neo)lacto neutral GSLs. Granulocytes and the cell line ML-2 contained almost exclusively neutral GSLs of the (neo)lacto series.     

Isolation and characterization of Sphingomonas sp. GTIN11 capable of carbazole metabolism in petroleum

A bacterial culture was isolated from a manufactured gas plant (MGP) soil based on its ability to metabolize the nitrogen-containing heterocycle carbazole. The culture was identified as a Sphingomonas sp. and was given the designation GTIN11. A cloned 4.2kb DNA fragment was confirmed to contain genes responsible for carbazole degradation. DNA sequence analysis revealed that the fragment contained five open reading frames (ORFs) with the deduced amino acid sequence showing homology to; carbazole terminal dioxygenase (ORF1), 2,3-dihydroxybiphenyl dioxygenase subunits (ORF2 and ORF3), meta-cleavage compound hydrolases (ORF4), and ferrodoxin component of bacterial multicomponent dioxygenases (ORF5). The percent similarity was 61% of these proteins or less to known proteins. The specific activity of Sphingomonas sp. GTIN11 for the degradation of carbazole at 37 degrees C was determined to be 8.0 micromol carbazole degraded/min/g dry cell. This strain is unique in expressing the carbazole degradation trait constitutively. Resting cells of Sphingomonas sp. GTIN11 removed 95% of carbazole and 50% of C1-carbazoles from petroleum in a 16-h treatment time.     

Direct analysis of sialylated or sulfated glycosphingolipids and other polar and neutral lipids using TLC-MS interfaces

Gangliosides and sulfatides (STs) are acidic glycosphingolipids (GSLs) that have one or more sialic acids or sulfate substituents, in addition to neutral sugars, attached to the C-1 hydroxyl group of the ceramide long chain base. TLC is a widely employed and convenient technique for separation and characterization of GSLs. When TLC is directly coupled to MS, it provides both the molecular mass and structural information without further purification. Here, after development of the TLC plates, the structural analyses of acidic GSLs, including gangliosides and STs, were investigated using the liquid extraction surface analysis (LESA™) and CAMAG TLC-MS interfaces coupled to an ESI QSTAR Pulsar i quadrupole orthogonal TOF mass spectrometer. Coupling TLC with ESI-MS allowed the acquisition of high resolution mass spectra of the acidic GSLs with high sensitivity and mass accuracy, without the loss of sialic acid residues that frequently occurs during low-pressure MALDI MS. These systems were then applied to the analysis of total lipid extracts from bovine brain. This allowed profiling of many different lipid classes, not only gangliosides and STs, but also SMs, neutral GSLs, and phospholipids.     

A novel sphingoglycolipid containing galacturonic acid and 2-hydroxy fatty acid in cellular lipids of Sphingomonas yanoikuyae

A novel sphingoglycolipid was isolated from Sphingomonas yanoikuyae, and its structure was identified as a galacturonosyl-beta (1-->1)-ceramide. This was a characteristic sphingoglycolipid present in S. yanoikuyae and certain other species of Sphingomonas, such as Sphingomonas mali, Sphingomonas terrae, and Sphingomonas macrogoltabidus, but not in the type species of Sphingomonas, Sphingomonas paucimobilis.     

Structural analysis of two glycosphingolipids from the lipopolysaccharide-lacking bacterium Sphingomonas capsulata.

Two glycosphingolipids, GSL-1 and GSL-3, were isolated from Sphingomonas capsulata and studied by methylation analysis, laser desorption mass spectrometry, and 1H and 13C NMR spectroscopy, including two-dimensional 1H,1H COSY and heteronuclear 13C,1H COSY experiments. GSL-1 and GSL-3 differ in their carbohydrate part, their structures being alpha-D-GlcpA-(1-->1)-Cer and alpha-D-Galp-(1-->6)-alpha-D-GlcpN-(1-->4)-alpha-D-GlcpA(1-- >1)Cer, respectively. Variations occur in the ceramide of GSL-1 and GSL-3, both having the same long-chain bases, erythro-2-amino-1, 3-octadecanediol (sphinganine), (13Z)-erythro-2-amino-13-eicosene-1, 3-diol and (13Z)-erythro-2-amino-13,14-methylene-1,3-eicosanediol, in the ratios 2.6 : 1 : 3.5 in GSL-1 and 1 : 1.2 : 1.5 in GSL-3. All bases are quantitatively substituted by amide-linked (S)-2-hydroxymyristic acid.     

Male germ cells require polyenoic sphingolipids with complex glycosylation for completion of meiosis: a link to ceramide synthase-3

Previously, it was found that a novel class of neutral fucosylated glycosphingolipids (GSLs) is required for male fertility. These lipids contain very long-chain (C26-C32) polyunsaturated (4-6 double bonds) fatty acid residues (VLC-PUFAs). To assess the role of these complex GSLs in spermatogenesis, we have now investigated with which of the testicular cell types these lipids are associated. During postnatal development, complex glycosylated and simple VLC-PUFA sphingolipids were first detectable at day 15, when the most advanced germ cells are pachytene spermatocytes. Their synthesis is most likely driven by ceramide synthase-3. This enzyme is encoded by the Cers3/Lass3 gene (longevity assurance genes), and out of six members of this gene family, only Cers3 mRNA expression was limited to germ cells, where it was up-regulated more than 700-fold during postnatal testicular maturation. Increasing levels of neutral complex VLC-PUFA GSLs also correlated with the progression of spermatogenesis in a series of male sterile mutants with arrests at different stages of spermatogenesis. Remarkably, fucosylation of the complex VLC-PUFA GSLs was not essential for spermatogenesis, as fucosylation-deficient mice produced nonfucosylated versions of the complex testicular VLC-PUFA GSLs, had complete spermatogenesis, and were fertile. Nevertheless, sterile Galgt1(-/-) mice, with a defective meiotic cytokinesis and a subsequent block in spermiogenesis, lacked complex but contained simple VLC-PUFA GSLs, as well as VLC-PUFA ceramides and sphingomyelins, indicating that the latter lipids are not sufficient for completion of spermatogenesis. Thus, our data imply that both glycans and the particular acyl chains of germinal sphingolipids are relevant for proper completion of meiosis.