Quantification of seminolipid by LC-ESI-MS/MS-multiple reaction monitoring: compensatory levels in Cgt mice

Abstract

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

Arylsulfatase A deficiency causes seminolipid accumulation and a lysosomal storage disorder in Sertoli cells

“…In Arsa Ϫ / Ϫ mice, an extra glycolipid band chromatographed between SGG and phosphatidylcholine was observed; the area of the HPTLC plate around this band, starting underneath the SGG band, was scraped for lipid extraction by the Bligh/Dyer method ( 24 ). Extracted lipids were subjected to ESI-MS/MS as previously described ( 23,25 ).…”

“…Lipids were extracted from decapsulated testes and PBSwashed epididymal and vas deferens sperm, and then subjected to liquid chromatography ESI-MS/MS and MRM for SGG quantifi cation using deuterated SGG as an internal standard, as previously described ( 23 ).…”

Sperm can act as vectors for HIV‐1 transmission into vaginal and cervical epithelial cells

“…Jurkat cells, vaginal (Vk2/E6E7), ectocervical (Ect1/E6E7), and endocervical (End1/E6E7) epithelial cell lines with culture instructions were obtained from ATCC. SGG was isolated and purified from pig testes in our laboratory. Other lipids (sulfogalactosylceramide [SGC], GC, phosphatidylserine [PS] and phosphatidylcholine [PC] and cholesterol sulfate) were obtained from MilliporeSigma.…”

“…Sperm were separated and washed free from seminal plasma in PBS by low‐speed centrifugation. Lipids were then extracted from sperm by the Bligh‐Dyer method and subjected to high‐performance thin‐layer chromatography (HPTLC) followed by Coomassie Blue staining for all lipid detection or orcinol staining for specific detection of glycolipids . Extracted sperm lipids were then subjected to LC‐ESI‐MS/MS (liquid chromatography‐electrospray ionization tandem mass spectrometry) parent (sulfate m / z 97) ion scanning.…”

“…The effluent from the column was directed at an electrospray ionization source connected to a triple quadrupole mass spectrometer (Agilent 6460, Agilent) scanning in the negative ion parent scan mode (parents of m / z 97) using previously optimized conditions for maximal response from palmitoyl sulfatide standard (fragment or/and collision energy instrumental settings of 200 and 80, respectively). Identification of SGG was from its signature m / z value, whereas identification of cholesterol sulfate was based on its mass as described in database (http://www.lipidmaps.org).…”