Induction of epididymal boar sperm capacitation by pB1 and BSP-A1/-A2 proteins, members of the BSP protein family.

A family of proteins designated BSP-A1, BSP-A2, BSP-A3, and BSP-30-kDa, collectively called BSP (bovine seminal plasma) proteins, constitute the major protein fraction of bull seminal plasma. BSP proteins can stimulate sperm capacitation by inducing cholesterol and phospholipid efflux from sperm. Boar seminal plasma contains one homologous protein of the BSP family, named pB1; however, its physiological role is still unknown. In the current study, we report a novel method to purify pB1 from boar seminal plasma by chondroitin sulfate B-affinity chromatography and reverse-phase-high performance liquid chromatography. We also studied the effect of pB1, BSP-A1/-A2, and whole boar seminal plasma on boar sperm capacitation. Boar epididymal sperm were washed, preincubated in noncapacitating medium containing pB1 (0, 2.5, 5, 10 or 20 microg/ml), BSP-A1/-A2 (0 or 20 microg/ml) proteins, or whole seminal plasma (0, 250, 500, or 1000 microg/ml), then washed and incubated in capacitating medium. Acrosomal integrity was assessed by chlortetracycline staining. The status of sperm capacitation was evaluated by the capacity of sperm to undergo the acrosome reaction initiated by the addition of the calcium ionophore, A23187. The pB1 and BSP-A1/-A2 proteins increased epididymal sperm capacitation as compared with control (sperm preincubated without proteins). This effect reached a maximum level at 10 microg/ml pB1 and at 20 microg/ml BSP-A1/-A2 (2.3- and 2.2-fold higher than control, respectively). Whole boar seminal plasma did not induce sperm capacitation. In addition, pB1 bound to boar epididymal sperm and was lost during capacitation. These results indicate that BSP proteins and their homologs in other species induce sperm capacitation in a similar way.

Low-density lipoprotein fraction from hen's egg yolk decreases the binding of the major proteins of bovine seminal plasma to sperm and prevents lipid efflux from the sperm membrane.

For sperm preservation, semen is generally diluted with extender containing egg yolk (EY), but the mechanisms of sperm protection by EY are unclear. The major proteins of bull seminal plasma (BSP proteins: BSP-A1/A2, BSP-A3, and BSP-30-kDa) bind to sperm surface at ejaculation and stimulate cholesterol and phospholipid efflux from the sperm membrane. Since EY low-density lipoprotein fraction (LDF) interacts specifically with BSP proteins, it is proposed that the sequestration of BSP proteins in seminal plasma by EY-LDF represents the major mechanism of sperm protection by EY. In order to gain further insight into this mechanism, we investigated the effect of seminal plasma, EY, and EY-LDF on the binding of BSP proteins to sperm and the lipid efflux from the sperm membrane. As shown by immunodetection, radioimmunoassays, and lipid analysis, when semen was incubated undiluted or diluted with control extender (without EY or EY-LDF), BSP proteins bound to sperm in a time-dependent manner, and there is a continuous cholesterol and phospholipid efflux from the sperm membrane. In contrast, when semen was diluted with extender containing EY or EY-LDF, there was 50%-80% fewer BSP proteins associated with sperm and a significant amount of lipid added to sperm membrane during incubation. In addition, sperm function analysis showed that the presence of EY or EY-LDF in the extender preserved sperm motility. These results show that LDF is the constituent of EY that prevents binding of the BSP proteins to sperm and lipid efflux from the sperm membrane and is beneficial to sperm functions during sperm preservation.