Cholesterol and phospholipid levels of washed and percoll gradient centrifuged mouse sperm: Presence of lipids possessing inhibitory effects on sperm motility

Abstract

Levels of DNA, cholesterol, and phospholipids of mouse caudal epididymal and vas deferens sperm that were processed through simple washing and Percoll gradient centrifugation were measured. The DNA and cholesterol contents of washed sperm and Percoll gradient centrifuged (PGC) sperm (DNA = 3.6 +/- 0.3 pg/sperm and 3.4 +/- 0.3 pg/sperm, respectively; cholesterol = 0.219 +/- 0.057 nmole/microgram DNA and 0.224 +/- 0.030 nmole/microgram DNA, respectively, for washed and PGC sperm) were not significantly different from each other; however, the phospholipid level of PGC sperm was only one half of that of washed sperm (0.315 +/- 0.071 nmole/microgram DNA versus 0.720 +/- 0.075 nmole/microgram DNA, respectively). The presence of 0.3% bovine serum albumin (BSA) in the culture medium used in sperm washing did not change the cholesterol and phospholipid contents of washed sperm. Similarly, the cholesterol and phospholipid levels of washed sperm and PGC sperm that were further incubated in BSA-containing medium for 30 min remained the same. Interestingly, substantial amounts of lipids, as determined by the cholesterol and phospholipid levels, were released into the supernatants of the sperm washes, and sperm needed to be washed at least twice to ensure their stable levels of cholesterol and phospholipids. The lipid mixture in the first sperm wash supernatant was shown to have inhibitory effects on PGC sperm motility.

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

“…Accumulated evidence reveals that composition and changes of sperm lipids can regulate sperm quality and functions [11]. Contents of major lipids, cholesterol, and phospholipids are much lower in PGC sperm than in washed sperm in both human and mouse species [12][13][14]. These results corroborate the absence of membrane vesicles on the PGC sperm surface.…”

“…Because mouse sperm tend to adhere to the tube wall surface within 10 min in suspension, we used the amount of DNA instead of the sperm number as a denominator of the sperm lipid content. The DNA assay involved treating sperm with DTT, which prevented sperm adherence to the tube wall, thereby allowing the assay to be carried out at a later time [12]. Both PGC capacitated and washed capacitated sperm had the same amount of DNA (4.1 Ϯ 0.2 vs. 4.0 Ϯ 0.4 pg/sperm) ( Table 1).…”

“…Sperm DNA was quantified by a modification of the method outlined by Labarca and Paigen [38] as previously described [12], which was based on the binding of Hoechst 33258 to DNA of live cells treated with a high salt concentration (2 M NaCl) to allow separation of DNA strands and, thus, intercalation of the Hoechst dye. Specifically for sperm cells, 100 mM dithiothreitol (DTT) was added to the sperm suspension to decondense the highly compact sperm chromatin by disrupting protamine disulfide bonds as well as to minimize the adherence of sperm to the surface of the assay tubes.…”

“…Phospholipids were quantified according to the method described by Duck-Chong et al [45] as modified by Tanphaichitr et al [12]. The assay was based on phospholipid digestion by magnesium nitrate at high temperature to inorganic phosphate followed by its color reaction with molybdate and malachite green.…”

PCSK4-null sperm display enhanced protein tyrosine phosphorylation and ADAM2 proteolytic processing during in vitro capacitation

“…Cauda epididymal and vas deferens sperm from both genotypes were collected into Krebs-Ringer bicarbonate (KRB)-HEPES medium (112.4 mmol/L NaCl, 4.8 mmol/L KCl, 1.7 mmol/L CaCl 2 , 1.2 mmol/L KH 2 PO 4 , 1.2 mmol/L Mg 2 SO 4 , 4 mmol/L NaHCO 3 , 21 mmol/L HEPES, 25 mmol/L sodium lactate, 1 mmol/L sodium pyruvate, 5.6 mmol/L glucose, and 28 mmol/L phenol red) containing 0.3% bovine serum albumin (BSA), and subjected to a twostep Percoll gradient centrifugation (PGC) wash as previously described (20). The washed sperm were resuspended in KRB-0.3% BSA to a concentration of 10 7 sperm/mL and incubated at 37 C with 5% CO 2 for up to 3 hours to allow for capacitation.…”