No 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 microg/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 microg/ml) alone did not stimulate cholesterol efflux. In contrast, high-density lipoprotein (HDL, 100 microg/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 microg/ml), washed, and incubated with HDL (100 microg/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.

Bovine seminal plasma (BSP) contains four similar acidic proteins, previously designated as BSP-A1, BSP-A2, BSP-A3, and BSP-30-kDa. These proteins are secreted by the seminal vesicles and coat the surface of the spermatozoa after ejaculation. The binding site of BSP proteins on the sperm surface has been identified as choline phospholipids on the plasma membrane. This study was undertaken to determine whether BSP proteins modulate capacitation of bovine spermatozoa induced by heparin. Bovine epididymal spermatozoa were washed and incubated in buffer containing BSP proteins and then washed and incubated with heparin. The percentage of capacitated spermatozoa was determined under the microscope after the acrosome reaction has been initiated with the addition of lysophosphatidylcholine. The results demonstrated that epididymal sperm undergo the acrosome reaction only in the presence of BSP proteins. This effect was concentration-dependent and reached a maximum level of a 3-5-fold increase at 20-40 micrograms/ml BSP protein concentrations. In contrast, ribonuclease (purified from bovine seminal fluid) or seminal fluid proteins depleted of BSP proteins (by sequential absorption of BSP proteins on gelatin-Agarose and DEAE-Sephadex columns) showed no significant potentiating activity. The purified BSP proteins were more active than crude alcohol precipitates of bovine seminal plasma. These results indicate that BSP proteins are regulatory factors of capacitation.