Bovine seminal plasma phospholipid-binding proteins stimulate phospholipid efflux from epididymal sperm

Abstract

Several studies have shown that sperm capacitation was accompanied by a change in the lipid composition of the sperm membrane. In cattle, the major proteins of (bovine)seminal plasma (BSP proteins: BSP-A1/A2, BSP-A3, and BSP- 30-kDa) potentiate sperm capacitation induced by high-density lipoprotein (HDL). Our recent studies indicate that these proteins and HDL stimulate sperm cholesterol efflux during capacitation. In order to gain more insight into the mechanisms of BSP-mediated sperm capacitation, we studied whether or not BSP proteins induce phospholipid efflux from epididymal sperm membrane. By direct determination of choline phospholipids on unlabeled epididymal sperm, the results show that sperm incubated in the presence of BSP-A1/A2 protein lost 34.4% of their choline phospholipids compared with the control (11.5%). Similar results were obtained using labeled epididymal sperm. Labeling was carried out by incubating washed epididymal sperm for 1 h with medium containing [3H]palmitic acid. The majority of the label was incorporated into sperm phosphatidylcholine. Studies of sperm phospholipid efflux were done by incubating the labeled sperm with purified BSP proteins, delipidated BSA, or bovine seminal ribonuclease (RNase, control protein). When labeled ([3H]phospholipid) epididymal sperm were incubated with BSP proteins (20-120 μg/ml) for 8 h, the sperm lost [3H]phospholipid in a dose- dependent manner (maximum efflux of ~30%). After the incubation with BSP proteins, the efflux particles were fractionated by size-exclusion chromatography. Analysis of the fractions obtained showed that the [3H]phospholipid was associated with BSP proteins. BSA (6 mg/ml) stimulated a specific phospholipid efflux of ~22%. In contrast, bovine RNase (120 μg/ml) did not stimulate phospholipid efflux. These results indicate that BSP proteins participate in the sperm cholesterol and phospholipid efflux that occurs during capacitation.