Percoll Gradient-Centrifuged Capacitated Mouse Sperm Have Increased Fertilizing Ability and Higher Contents of Sulfogalactosylglycerolipid and Docosahexaenoic Acid-Containing Phosphatidylcholine Compared to Washed Capacitated Mouse Sperm1

Furimsky, Anna; Vuong, Ngoc Q.; Xu, Hongbin; Kumarathasan, Premkumari; Xu, Min; Weerachatyanukul, Wattana; Khalil, Maroun Bou; Kates, M.; Tanphaichitr, Nongnuj

doi:10.1095/biolreprod.104.036095

Cited by 42 publications

(33 citation statements)

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“…Whereas most phospholipids are nonreactive, polyphosphate phospholipids such as cardiolipin, react with the dye ( 15,16 ). Cardiolipin is present in male germ cells at a level comparable to SGG, and it migrates on the HPTLC plate in close proximity to SGG ( 14 ). Preparative HPTLC, involving scraping chromatographed lipids around the SGG band, as used for partially purifying the mouse testis lipid samples, was likely ineffi cient in removing cardiolipin.…”

Section: Discussionmentioning

confidence: 99%

“…used ( 13,14 ). Azure A is a cationic dye that reacts with anionic lipids, including sulfolipids ( 15,16 ).…”

Section: Methodsmentioning

confidence: 99%

“…Testes were removed, decapsulated, weighed ( ‫ف‬ 100-120 mg/testis) and individually hand-homogenized in 1 ml of phosphate buffered saline (PBS), which was then used for lipid extraction. Sperm were retrieved from the caudal epididymis and vas deferens from each animal into 1 ml of Hepes-buffered Krebs Ringer solution (KRBHepes; 119.4 mM NaCl, 4.8 mM KCl, 1.7 mM CaCl 2 , 1.2 mM KH 2 PO 4 , 1.2 mM Mg 2 SO 4 , 4 mM NaHCO 3 , 1 mM sodium pyruvate, 25 mM sodium lactate, 5.6 mM glucose, 1 U/ml of penicillin G, 1 µg/ml of streptomycin sulfate, 21 mM Hepes, and 28 µM phenol red at pH 7.4) ( 14 ). For lipid extraction, the sperm suspension was centrifuged (430 g , 10 min, 25°C), then pellet was washed twice with PBS and fi nally resuspended in 0.5 ml PBS.…”

Section: Mouse Testis and Sperm Preparationmentioning

confidence: 99%

“…Following a modifi ed Bligh-Dyer method ( 14,(28)(29)(30), methanol (2.5 ml) and chloroform (1.25 ml) was added to the 1 ml PBS testis homogenate. After thorough mixing and 1 h incubation at room temperature, the sample was diluted further with chloroform and water (1.25 ml each), then vigorously mixed and centrifuged (800 g , 5 min, room temperature).…”

Section: Lipid Extraction From Mouse Testes and Spermmentioning

confidence: 99%

“…After plate development, lipids were detected by various dyes. These included Coomassie Brilliant Blue G-250 (0.03% in 30% methanol/100 mM NaCl at room temperature), the excess of which was removed with 30% methanol in 100 mM NaCl ( 14,32 ); and Azure A (0.016% Azure in 1 mM H 2 SO 4 ), the excess of which was removed with 50 mM H 2 SO 4 /methanol (3/1, v/v) ( 7,14 ). Coomassie Blue stained all lipids blue, whereas Azure A stained sulfolipids blue ( 29 ).…”

Section: High-performance Thin-layer Chromatographymentioning

confidence: 99%

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

Kongmanas

Yaghoubian

et al. 2010

Journal of Lipid Research

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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

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Section: Discussionmentioning

confidence: 99%

“…used ( 13,14 ). Azure A is a cationic dye that reacts with anionic lipids, including sulfolipids ( 15,16 ).…”

Section: Methodsmentioning

confidence: 99%

Section: Mouse Testis and Sperm Preparationmentioning

confidence: 99%

Section: Lipid Extraction From Mouse Testes and Spermmentioning

confidence: 99%

Section: High-performance Thin-layer Chromatographymentioning

confidence: 99%

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

Kongmanas

Yaghoubian

et al. 2010

Journal of Lipid Research

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Glycosphingolipids, Chemistry of

Lingwood

Abram

Mylvaganum

2008

Wiley Encyclopedia of Chemical Biology

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Eukaryotic cell glycosphingolipids (GSLs) are central components of membrane lipid microdomains that function as trans plasmamembrane signaling foci. GSLs serve as important receptors and coreceptors, primarily to mediate host/microbial pathogen interactions, and undergo unique intracellular trafficking pathways. As such, their chemical modification can generate interventive therapeutic strategies and biologic probes. The process of achieving such goals via chemistry of the carbohydrate is in its infancy, but substitution within the lipid moiety has been used extensively. A consideration of the importance of the lipid moiety in GSL function has led to novel chemical approaches that attempt to retain this property.

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Proteomic Characterization of Pig Sperm Anterior Head Plasma Membrane Reveals Roles of Acrosomal Proteins in ZP3 Binding

Kongmanas

Kruevaisayawan

Saewu

et al. 2014

Journal Cellular Physiology

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The sperm anterior head plasma membrane (APM) is the site where sperm first bind to the zona pellucida (ZP). This binding reaches the maximum following the sperm capacitation process. To gain a better understanding of the sperm-ZP binding mechanisms, we compared protein profiles obtained from mass spectrometry of APM vesicles isolated from non-capacitated and capacitated sperm. The results revealed that ZP-binding proteins were the most abundant group of proteins, with a number of them showing increased levels in capacitated sperm. Blue native gel electrophoresis and far-western blotting revealed presence of high molecular weight (HMW) protein complexes in APM vesicles of both non-capacitated and capacitated sperm, but the complexes (∼750-1300 kDa) from capacitated sperm possessed much higher binding capacity to pig ZP3 glycoprotein. Proteomic analyses indicated that a number of proteins known for their acrosome localization, including zonadhesin, proacrosin/acrosin and ACRBP, were components of capacitated APM HMW complexes, with zonadhesin being the most enriched protein. Our immunofluorescence results further demonstrated that a fraction of these acrosomal proteins was transported to the surface of live acrosome-intact sperm during capacitation. Co-immunoprecipitation indicated that zonadhesin, proacrosin/acrosin and ACRBP interacted with each other and they may traffic as a complex from the acrosome to the sperm surface. Finally, the significance of zonadhesin in the binding of APM HMW complexes to pig ZP3 was demonstrated; the binding ability was decreased following treatment of the complexes with anti-zonadhesin antibody. Our results suggested that acrosomal proteins, especially zonadhesin, played roles in the initial sperm-ZP binding during capacitation.

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Percoll Gradient-Centrifuged Capacitated Mouse Sperm Have Increased Fertilizing Ability and Higher Contents of Sulfogalactosylglycerolipid and Docosahexaenoic Acid-Containing Phosphatidylcholine Compared to Washed Capacitated Mouse Sperm1

Cited by 42 publications

References 64 publications

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

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

Glycosphingolipids, Chemistry of

Proteomic Characterization of Pig Sperm Anterior Head Plasma Membrane Reveals Roles of Acrosomal Proteins in ZP3 Binding

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