Major Proteins of Bovine Seminal Plasma and High-Density Lipoprotein Induce Cholesterol Efflux from Epididymal Sperm

Abstract

One of the hypotheses to explain the mechanism of capacitation involves the loss of sperm membrane cholesterol. Here, we studied whether or not the major proteins of bovine seminal plasma designated as BSP-A1, -A2, -A3, and -30-kDa (collectively called BSP proteins), which are implicated in sperm capacitation, induce cholesterol efflux. When epididymal sperm were labeled with [³H]cholesterol and incubated with bovine seminal plasma (0.05–2%) or BSP proteins (20–120 μg/ml) for 8 h, the sperm lost [³H]cholesterol (3.6-fold and 3-fold, respectively). The same results in the presence of BSP-A1/-A2 were obtained (3.5–fold) by direct determination of cholesterol on unlabeled epididymal sperm. Analysis of efflux particles by ultracentrifugation on a sucrose gradient revealed a single symmetrical peak of radioactivity at 1.14 g/ml. Immunoblotting of the fractions obtained from size-exclusion chromatography of the efflux particles showed that a portion of the BSP proteins were associated with [³H]cholesterol. Heparin (12 μg/ml) alone did not stimulate cholesterol efflux. In contrast, high-density lipoprotein (HDL, 100 μg/ml) alone stimulated cholesterol efflux up to 3.1–fold after 8 h. When labeled epididymal sperm were preincubated for 20 min with BSP-A1/-A2 (120 μg/ml), washed, and incubated with HDL (100 μg/ml) for 8 h, the total cholesterol efflux of the sperm suspension was 51.8 ± 5.0% compared to 39.3 ± 1.2% when HDL alone was used. These results indicate that BSP proteins and HDL play an important role in the sperm sterol efflux that occurs during capacitation. Furthermore, the heparin-induced sperm capacitation did not involve the efflux of sperm membrane cholesterol.