This article is available online at http://www.jlr.org Seminolipid, 1-O -alkyl-2-O -acyl[ ␤ -D-(3 ′ -sulfatoxy)galactopyranosyl(1'-3)] sn -glycerol, also known as sulfogalactosylglycerolipid (SGG) and SM4g ( Fig. 1A ), is present selectively and substantially in mammalian male germ cells, comprising about 10 mol% of total lipids ( 1 ). SGG consists of glycerol with a sn -1 alkyl and sn -2 acyl chain as the lipid backbone, and a (3 ′ -sulfo)-galactopyranose ␤ -linked to the sn -3 position. Consistently across mammalian species studied so far, C16:0/C16:0 SGG is the main molecular species ( 1, 2 ), although minor molecular species, such as C16:0/C14:0, C14:0/C16:0, C15:0/C16:0, C17:0/C16:0, C16:0/C18:0, C18:0/C16:0, and C17:0/C18:0 SGG, representing < 10% of the main species, have also been described ( 3, 4 ). Accumulated evidence has pointed to the signifi cance of SGG for mammalian male reproduction ( 1 ). SGG on the sperm surface is involved in sperm-zona pellucida (ZP) binding and sperm-egg plasma membrane binding ( 5, 6 ). As a structurally ordered lipid, SGG participates in the formation of lipid rafts in the sperm head, which act as platforms for ZP binding ( 7-10 ). Genetic deletion of Cgt and Cst , coding for two enzymes (ceramide galactosyltransferase and cerebroside sulfotransferase, respectively) in the SGG biosynthetic pathway, leads to spermatogenesis arrest in the primary spermatocyte stage and, thus, male infertility ( 11, 12 ).Due to its physiological signifi cance, an accurate, sensitive, specifi c method for SGG quantifi cation is needed. A simple colorimetric Azure A assay has been conventionally

A mass spectrometric method is described for monitoring cerebrosides in the presence of excess concentrations of alkali metal salts. This method has been adapted for use in the assay of arylsulfatase A (ASA) and the cerebroside sulfate activator protein (CSAct or saposin B). Detection of the neutral glycosphingolipid cerebroside product was achieved via enhancement of ionization efficiency in the presence of lithium ions. Assay samples were extracted into the chloroform phase as for the existing assays, dried, and diluted in methanol-chloroform-containing lithium chloride. Samples were analyzed by electrospray ionization mass spectrometry with a triple quadrupole mass spectrometer in the multiple reaction monitoring tandem mass spectrometric mode. The assay has been used to demonstrate several previously unknown or ambiguous aspects of the coupled ASA/CSAct reaction, including an absolute in vitro preference for CSAct over the other saposins (A, C, and D) and a preference for the nonhydroxylated species of the sulfatide substrate over the corresponding hydroxylated species. The modified assay for the coupled ASA/CSAct reaction could find applicability in settings in which the assay could not be performed previously because of the need for radiolabeled substrate, which is now not required.

Lipid rafts are ordered membrane microdomains e n r i c h e d i n c h o l e s t e r o l , g l y c o l i p i d s a n d s a t u r a t e d phospholipids. Lipid rafts also assemble molecules involved in cell adhesion and signaling, and they are thus considered platforms for these phenomena. This has made the lipid raft topic a very active research field. However, a number of issues remain controversial in this research line. Advanced imaging studies have recently indicated that individual lipid/membrane rafts are of nanometer sizes. This suggests that rafts isolated from cells as low-density membrane fractions may rather represent "macrorafts" due to coalescence of individual rafts. Further controversy lies in the use of Triton X-100 in raft isolation, as the detergent may induce lipid microdomain formation. Investigators in the field are moving towards using milder non-ionic detergents or physical forces in raft isolation. Sperm-egg interaction is an ideal system to attest the implication of lipid rafts in cell adhesion/signaling. Recently, we have shown that Triton X-100 resistant membrane rafts of capacitated pig sperm have direct binding to pig zona pellucida (ZP) with a K d value much lower than that of noncapacitated sperm rafts-ZP binding. These results may partially explain the enhanced ZP binding ability of capacitated sperm, relative to non-capacitated sperm. Another possible contribution to the greater ZP binding ability of capacitated sperm is their possession of higher lipid raft levels despite a cholesterol efflux occurring during capacitation. Significantly, GM1 ganglioside, normally used as a raft marker of somatic cells, does not exist in capacitated sperm rafts. Rather, 70% of male germ cell specific sulfogalacotosylglycerolipid (SGG) is present in isolated capacitated sperm rafts, making it an attractive candidate as a sperm raft marker. Nonetheless, since sperm lipid rafts used in these studies were isolated by the Triton X-100 treatment method, work should be repeated using lipid rafts prepared from sperm by a physical force (e.g., nitrogen cavitation). Finally, advanced imaging studies

Arylsulfatase A (AS-A) is a lysosomal enzyme, which catalyzes the desulfation of certain sulfogalactolipids, including sulfogalactosylglycerolipid (SGG), a molecule implicated in cell adhesion. In this report, immunocytochemistry revealed the selective presence of AS-A in the corpus luteum of mouse ovaries. Immunoblotting indicated that mouse corpus luteum AS-A had a molecular mass of 66 kDa, similar to AS-A of other tissues. Corpus luteum AS-A was active, capable of desulfating the artificial substrate, p-nitrocatechol sulfate, at the optimum pH of five. To understand further the role of AS-A in female reproduction, levels of AS-A were determined during corpus luteum development in pseudopregnant mice and during luteolysis after cessation of pseudopregnancy. Immunocytochemistry, immunoblotting and desulfation activity showed that AS-A expression was evident at the onset of pseudopregnancy in the newly formed corpora lutea, and its level increased steadily during gland development. The increase in the expression and activity of AS-A continued throughout luteolysis after the decrease in serum progesterone levels. We also observed the selective presence of SGG on the luteal cell surface in developed corpora lutea, as shown by immunofluorescence of mouse ovary sections as well as high-performance thin-layer chromatography of lipids isolated from mouse and pig corpora lutea. The identity of the "SGG" band on the thin layer silica plate was further validated by electrospray ionization mass spectrometry. Significantly, SGG disappeared in regressing corpora lutea. Therefore, lysosomal AS-A may be involved in cell-surface remodeling during luteolysis by desulfating SGG after its endocytosis and targeting to the lysosome.