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.     

New insights towards understanding the mechanisms of sperm protection by egg yolk and milk

Mammalian sperm preservation in extenders containing egg yolk (EY) and/or milk has been used for over half a century. However, the mechanism by which EY or milk protects sperm during storage remains elusive. Studies conducted over the past two decades in our laboratory have revealed that a family of lipid-binding proteins (BSP proteins) present in bull seminal plasma is detrimental to sperm preservation since these proteins induce cholesterol and phospholipid removal from the sperm membrane. Interestingly, these detrimental factors of seminal plasma interact with the low-density lipoproteins (LDL) present in EY. This interaction minimizes lipid removal from the sperm membrane, which positively influences sperm storage in liquid or frozen states. Based on several lines of evidence, we suggest that the sequestration of BSP proteins by LDL (BSP proteins: lipoprotein interaction) is the major mechanism of sperm protection by EY. Skimmed milk, which is devoid of lipoproteins, also protects sperm during storage. Several studies indicate that the active components involved in sperm protection by milk are casein micelles. Thus, it appears that the mechanism by which milk protects sperm involves a BSP protein: casein micelle interaction. In view of these new insights, novel strategies have been suggested to improve the efficiency of semen preservation.     

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.     

Seminal plasma choline phospholipid-binding proteins stimulate cellular cholesterol and phospholipid efflux

Bovine seminal plasma (BSP) contains a family of phospholipid-binding proteins (BSP-A1/-A2, BSP-A3 and BSP-30-kDa, collectively called BSP proteins) that potentiate sperm capacitation induced by high-density lipoproteins. We showed recently that BSP proteins stimulate cholesterol efflux from epididymal spermatozoa and play a role in capacitation. Here, we investigated whether or not BSP proteins could stimulate cholesterol and phospholipid efflux from fibroblasts. Cells were radiolabeled ([3H]cholesterol or [3H]choline) and the appearance of radioactivity in the medium was determined in the presence of BSP proteins. Alcohol precipitates of bovine seminal plasma (designated crude BSP, cBSP), purified BSP-A1/-A2, BSP-A3 and BSP-30-kDa proteins stimulated cellular cholesterol and choline phospholipid efflux from fibroblasts. Efflux mechanistic differences were observed between BSP proteins and other cholesterol acceptors. Preincubation of BSP-A1/-A2 proteins with choline prevented cholesterol efflux, an effect not observed with apolipoprotein A-I. Also, the rate of BSP-induced efflux was rapid during the first 20 min, but leveled off thereafter in contrast to a relatively slow, but constant, rate of cholesterol efflux mediated by apolipoprotein A-I, apolipoprotein A-I-containing reconstituted lipoproteins (LpA-I) and high-density lipoproteins. These results indicate that fibroblasts are a good cell model to study the mechanism of lipid efflux mediated by BSP proteins.     

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

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 [(3)H]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 ([(3)H]phospholipid) epididymal sperm were incubated with BSP proteins (20-120 microg/ml) for 8 h, the sperm lost [(3)H]phospholipid in a dose-dependent manner (maximum efflux of approximately 30%). After the incubation with BSP proteins, the efflux particles were fractionated by size-exclusion chromatography. Analysis of the fractions obtained showed that the [(3)H]phospholipid was associated with BSP proteins. BSA (6 mg/ml) stimulated a specific phospholipid efflux of approximately 22%. In contrast, bovine RNase (120 microg/ml) did not stimulate phospholipid efflux. These results indicate that BSP proteins participate in the sperm cholesterol and phospholipid efflux that occurs during capacitation.     

Effect of seminal phospholipid-binding proteins and follicular fluid on bovine sperm capacitation.

Bovine seminal plasma (BSP) contains a family of novel phospholipid-binding proteins (BSP-A1/-A2, BSP-A3, and BSP-30-kDa; collectively called BSP proteins) that potentiate sperm capacitation induced by heparin or by serum high-density lipoprotein (HDL). BSP proteins stimulate lipid efflux from sperm that may occur during the early events of capacitation. Here, we investigated the role of BSP proteins, bovine follicular fluid (FF), and bovine follicular fluid HDL (FF-HDL) in sperm capacitation. FF and FF-HDL alone stimulated epididymal sperm capacitation (19.5% +/- 0.8% and 18.2% +/- 2.8%, respectively, control, 9.0% +/- 1.9%) that was increased by preincubation with BSP-A1/-A2 proteins (30.2% +/- 0.4% and 30.9% +/- 1.5%, respectively). In contrast, lipoprotein-depleted follicular fluid (LD-FF) alone was ineffective, and a preincubation with BSP-A1/-A2 proteins was necessary before sperm capacitation was stimulated (up to 22.8% +/- 1.4%). The interaction of BSP proteins with FF components was analyzed using ultracentrifugation, Lipo-Gel electrophoresis, SDS-PAGE, and gel filtration. We established that the BSP proteins interact with factors present in FF including FF-HDL. Additionally, we obtained evidence that BSP proteins, found associated with FF-HDL, were released from the sperm membrane during capacitation. These results confirm that the BSP proteins and the FF-HDL play a role in sperm capacitation.     

Milk caseins decrease the binding of the major bovine seminal plasma proteins to sperm and prevent lipid loss from the sperm membrane during sperm storage

Milk is used as a medium for sperm preservation. Caseins, the major proteins of milk, appear to be responsible for the protective effect of milk on sperm. Recently, we have shown that egg yolk, which is also widely used to preserve semen, protects sperm functions by preventing the binding to sperm of the major proteins of bull seminal plasma (BSP proteins), thereby preventing BSP protein-mediated stimulation of lipid loss from the sperm membrane. In the present study, we investigated whether milk caseins protect sperm in the same manner as egg yolk. Bovine ejaculates were diluted with skimmed milk permeate (skimmed milk devoid of caseins) or permeate that was supplemented with caseins and stored at 4 degrees C for 4 h. In the semen diluted with permeate, sperm viability and motility decreased in a time-dependent manner. However, in semen diluted with milk or permeate supplemented with caseins, sperm functions were maintained. In addition, lower amounts of the BSP proteins were associated with sperm in semen diluted with milk or permeate supplemented with caseins, as compared to semen diluted with permeate. No milk proteins were detected in the sperm protein extracts. Furthermore, sperm diluted with milk or permeate supplemented with caseins showed 3-fold lower losses of cholesterol and choline phospholipids than sperm diluted with permeate during storage. Thus, milk caseins decreased the binding of BSP proteins to sperm and reduced sperm lipid loss, while maintaining sperm motility and viability during storage. These results support our view that milk caseins prevent the detrimental effects of BSP proteins on the sperm membrane during sperm preservation.     

Radioimmunoassays for bull seminal plasma proteins (BSP-A1/-A2, BSP-A3, and BSP-30-Kilodaltons), and their quantification in seminal plasma and sperm

Three proteins, BSP-A1/-A2, BSP-A3, and BSP-30 kilodaltons (collectively called BSP proteins), represent the major proteins of bovine seminal plasma (BSP). At ejaculation, these proteins bind to the sperm surface and induce molecular changes in the plasma membrane that are deemed to be essential for sperm capacitation. The present study was carried out to develop specific radioimmunoassays (RIAs) for the quantification of each of the BSP proteins in BSP and sperm. RIAs were developed using polyclonal antibodies raised in rabbits against each BSP protein. The purified and iodinated BSP proteins were used as standard and tracer, respectively. The RIAs that were developed were shown to be specific for each protein and the cross-reactivity toward various antigens was negligible (<2%). The average sensitivity limit was 5 ng/ml of sample for BSP-A1/-A2 and BSP-A3, and 40 ng/ml of sample for BSP-30-kDa. The concentration of BSP proteins was determined by analyzing the RIA data with spline function. BSP proteins represented 40% to 57% of seminal plasma total protein (25% to 47% of BSP-A1/-A2, 3% to 5% of BSP-A3, and 3% to 7% of BSP-30 kDa) and 4% to 6% of sperm total protein (2.5% to 4% of BSP-A1/-A2, 0.4% to 0.9% of BSP-A3, and 0.5% to 1% of BSP-30-kDa). We also determined the concentration of BSP proteins that were sperm-bound after semen cryopreservation in Tris-egg yolk-glycerol extender. A significant decrease (70%-80%) in sperm-bound BSP proteins was noted after cryopreservation. The availability of reliable RIA procedures should aid in the further understanding of the role of BSP proteins in sperm function as well as their effect on sperm cryopreservation.     

Characterization of lipid efflux particles generated by seminal phospholipid-binding proteins.

We reported recently that the choline phospholipid-binding proteins (BSP-A1/-A2, BSP-A3 and BSP-30-kDa) of bovine seminal plasma (BSP) stimulate cholesterol and choline phospholipid efflux from fibroblasts. In this study, we characterized the lipid efflux particles generated by BSP proteins. The density gradient ultracentrifugation of the efflux medium from radiolabeled fibroblasts incubated with BSP proteins showed a single peak of [3H]cholesterol between density (d) 1.12 and 1.14 g/ml, which is in the range of high-density lipoproteins. Size-exclusion chromatographic and immunoblot analysis revealed that the efflux particles have a large size equal to or bigger than very low-density lipoproteins and contained BSP proteins. Lipid analysis of density gradient and gel filtration fractions from efflux medium of simultaneously labeled fibroblasts ([3H]cholesterol and [3H]choline) incubated with BSP proteins showed that the efflux particles were homogeneous and composed of cholesterol and choline phospholipids. The lipid particles contained BSP proteins, cholesterol and choline phospholipids in molar ratio of 0.05:1.21:1, respectively. Agarose gel electrophoresis showed that the BSP-generated lipid particles had a gamma migration pattern which is slower than low-density lipoproteins. The sonication of cholesterol and BSP proteins followed by gel filtration chromatographic analysis indicated no direct binding of cholesterol to BSP proteins. These results taken together indicate that BSP proteins induce a concomitant cholesterol and choline phospholipid efflux and generate large protein-lipid particles.     

Effect of progesterone on bovine sperm capacitation and acrosome reaction

Progesterone (P) appears to stimulate sperm capacitation and/or induce the acrosome reaction (AR) in some species. In bovine, it is now well established that the BSP-A1/-A2 proteins (the major proteins of bovine seminal plasma) promote sperm capacitation. In this study, we investigated the effect of P on bovine sperm cholesterol efflux, capacitation, and the AR. Labeled bovine epididymal sperm were incubated (0-6 h) with different concentrations of P (0.01-10 microg/ml) in the presence or absence of BSP-A1/-A2 proteins (capacitating conditions). At different time intervals, aliquots of sperm were taken to determine the sperm cholesterol efflux, sperm capacitation (AR induced by lysophosphatidylcholine, lyso-PC), and sperm AR. The results show that the presence of P in the media did not affect the membrane cholesterol efflux potential of the BSP-A1/-A2 proteins. P alone did not stimulate the AR with or without lyso-PC unless the epididymal sperm were incubated in capacitating conditions (in the presence of BSP-A1/-A2). When washed ejaculated sperm were continuously incubated with P, the P did not stimulate AR. However, when ejaculated sperm were preincubated (6 h) with heparin (capacitation medium) and then incubated 15 min with P (2 microg/ml), the percentage of AR obtained was similar to that obtained with lyso-PC. The effect of P on sperm AR was concentration dependent with a maximum 2.2-fold increase at 2 microg/ml of P. These results demonstrate a potential role of P in bovine sperm AR but not in capacitation.     

Characterization of lipid efflux particles generated by seminal phospholipid-binding proteins

We reported recently that the choline phospholipid-binding proteins (BSP-A1/-A2, BSP-A3 and BSP-30-kDa) of bovine seminal plasma (BSP) stimulate cholesterol and choline phospholipid efflux from fibroblasts. In this study, we characterized the lipid efflux particles generated by BSP proteins. The density gradient ultracentrifugation of the efflux medium from radiolabeled fibroblasts incubated with BSP proteins showed a single peak of [³H]cholesterol between density (d) 1.12 and 1.14 g/ml, which is in the range of high-density lipoproteins. Size-exclusion chromatographic and immunoblot analysis revealed that the efflux particles have a large size equal to or bigger than very low-density lipoproteins and contained BSP proteins. Lipid analysis of density gradient and gel filtration fractions from efflux medium of simultaneously labeled fibroblasts ([³H]cholesterol and [³H]choline) incubated with BSP proteins showed that the efflux particles were homogeneous and composed of cholesterol and choline phospholipids. The lipid particles contained BSP proteins, cholesterol and choline phospholipids in molar ratio of 0.05:1.21:1, respectively. Agarose gel electrophoresis showed that the BSP-generated lipid particles had a γ migration pattern which is slower than low-density lipoproteins. The sonication of cholesterol and BSP proteins followed by gel filtration chromatographic analysis indicated no direct binding of cholesterol to BSP proteins. These results taken together indicate that BSP proteins induce a concomitant cholesterol and choline phospholipid efflux and generate large protein–lipid particles.     

Isolation and characterization of gelatin-binding bison seminal vesicle secretory proteins

Bovine seminal plasma (BSP) contains a family of major proteins designated BSP-A1/A2, BSP-A3, and BSP-30kDa (collectively called BSP proteins) that bind to sperm at ejaculation and potentiate sperm capacitation. Homologous proteins have been identified in stallion, boar, goat, and ram seminal plasma. We report here the isolation and characterization of homologous proteins from bison seminal vesicle secretions. Seminal vesicle secretory proteins were precipitated by adding cold ethanol and recovered by centrifugation. The precipitates were resuspended in ammonium bicarbonate, dialyzed, and lyophilized. Lyophilized proteins were dissolved in 0.05 M phosphate buffer (PB) and loaded onto a gelatin-agarose column. The unadsorbed proteins and adsorbed proteins were eluted with PB and 5 M urea in PB, respectively. The gelatin-adsorbed fraction was analyzed by SDS-PAGE and revealed the presence of four major proteins designated BiSV-16kDa, BiSV-17kDa, BiSV-18kDa, and BiSV-28kDa (BiSV: bison seminal vesicle proteins). Heparin-Sepharose chromatography allowed the separation of BiSV-16kDa, which did not bind heparin from other BiSV proteins, which bound heparin. Immunoblotting revealed that BiSV-16kDa cross-reacted with BSP-A3 antibodies, BiSV-17kDa and BiSV-18kDa cross-reacted with BSP-A1/-A2 antibodies, and BiSV-28kDa cross-reacted with BSP-30kDa antibodies. Radioimmunoassays indicated that approximately 25% of bison seminal vesicle total proteins are related to BSP proteins. The amino-terminal sequencing indicated that BiSV proteins share almost 100% sequence identity with BSP proteins. In addition, BiSV proteins bind to low-density lipoproteins isolated from hen's egg yolk. These results confirm that BSP protein homologs are present in mammalian seminal plasma and they may share the same biological role.     

Phosphorylcholine-binding proteins from the seminal fluids of different species share antigenic determinants with the major proteins of bovine seminal plasma

The major proteins of bovine seminal plasma, BSP-A1, BSP-A2, BSP-A3, and BSP-30kDa (collectively named BSP proteins) bind to phospholipids containing the phosphorylcholine moiety. An affinity purification method using a p-aminophenyl phosphorylcholine-Agarose (PPC-Agarose) affinity matrix was developed for their purification. In this study, we investigated the distribution of BSP-like analogues in seminal fluid of the human, porcine, hamster, mouse, and rat using this affinity matrix. Alcohol precipitates of the seminal plasma/seminal vesicle secretions (SP/SVS) were further delipidated using isopropyl ether:n-butanol (60:40). The protein preparations obtained were solubilized in a minimal volume of buffer A (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 0.02% NaN₃), dialyzed against the same buffer, and applied to a PPC-Agarose column connected to a FPLC system. The unbound material was washed out and the adsorbed proteins eluted with buffer A containing 10 mM phosphorylcholine (PrC) and 10 M urea. The fractions were separated by SDS-PAGE, stained or transferred onto a nitrocellulose membrane, and probed with rabbit polyclonal anti-BSP antibodies. Anti-BSP cross-reacting proteins were detected in the seminal fluids of all the species investigated. Moreover, many of these proteins bound to the affinity matrix. The BSP proteins and their immunoreacting analogues appear to be ubiquitous in mammals and may possibly be involved in a common function such as in the modification of the lipid content of the sperm plasma membrane. © 1993 Wiley-Liss, Inc.     

Bovine seminal plasma proteins PDC-109, BSP-A3, and BSP-30-kDa share functional roles in storing sperm in the oviduct

On ejaculation, sperm become coated with proteins secreted by the male accessory sex glands. In the bull, these proteins consist predominantly of the bovine seminal plasma family of proteins (BSPs): PDC-109 (BSP-A1/-A2), BSP-A3, and BSP-30-kDa. PDC-109 plays a role in forming an oviductal sperm reservoir by enabling sperm to bind to oviductal epithelium. Because PDC-109 has high sequence identity with the other BSPs, we tested BSP-A3 and BSP-30-kDa for the capacity to bind sperm to oviductal epithelium. BSP-A3 and BSP-30-kDa each increased binding of epididymal sperm to epithelium and were as effective as PDC-109 in competitively inhibiting binding of ejaculated sperm. Because binding extends the motile life of sperm, BSPs were tested for the ability to maintain sperm motility. BSP-treated epididymal sperm incubated with plasma membrane vesicles from bovine oviductal epithelium maintained progressive motility longer than untreated sperm. To our knowledge, this is the first report of this protective effect of BSPs. Similarities in function among the BSPs were reflected in their three-dimensional structure, whereas surface maps of electrostatic potential indicated differences in binding affinities and kinetics. Such differences may provide sperm with greater adaptability to variations among females. Altogether, these results indicate that BSPs play a crucial role in fertilization by maintaining sperm motility during storage.     

Isolation and characterization of gelatin-binding proteins from goat seminal plasma

A family of proteins designated BSP-A1, BSP-A2, BSP-A3 and BSP-30 kDa (collectively called BSP proteins for Bovine Seminal Plasma proteins) constitute the major protein fraction in the bull seminal plasma. These proteins interact with choline phospholipids on the sperm surface and play a role in the membrane stabilization (decapacitation) and destabilization (capacitation) process. Homologous proteins have been isolated from boar and stallion seminal plasma. In the current study we report the isolation and preliminary characterization of homologous proteins from goat seminal plasma. Frozen semen (-80°C) was thawed and centrifuged to remove sperm. The proteins in the supernatant were precipitated by the addition of cold ethanol. The precipitates were dissolved in ammonium bicarbonate and lyophilised. The lyophilised proteins were dissolved in phosphate buffer and loaded onto a gelatin-agarose column, which was previously equilibrated with the same buffer. The column was successively washed with phosphate buffer, with phosphate buffer saline and with 0.5 M urea in phosphate buffer saline to remove unadsorbed proteins, and the adsorbed proteins were eluted with 5 M urea in phosphate buffer saline. Analysis of pooled, dialysed and lyophilised gelatin-agarose adsorbed protein fraction by SDS-PAGE indicated the presence of four protein bands that were designated GSP-14 kDa, GSP-15 kDa, GSP-20 kDa and GSP-22 kDa (GSP, Goat Seminal Plasma proteins). Heparin-affinity chromatography was then used for the separation of GSP-20 and -22 kDa from GSP-14 and -15 kDa. Finally, HPLC separation permitted further isolation of each one from the other. Amino acid sequence analysis of these proteins indicated that they are homologous to BSP proteins. In addition, these BSP homologs bind to hen's egg-yolk low-density lipoproteins. These results together with our previous data indicate that BSP family proteins are ubiquitous in mammalian seminal plasma, exist in several forms in each species and possibly play a common biological role.     

The major proteins of bovine seminal plasma interact with caseins and whey proteins of milk extender

Milk has been used routinely as an extender for sperm preservation. Caseins, the major proteins in milk, are proposed to be the protective constituents of milk during sperm preservation. It is unclear whether the whey proteins in milk are also implicated in the protection of sperm. Our previous studies have shown that the major proteins of bovine seminal plasma (recently named as binder of sperm or BSP, which comprises BSP1, BSP3, and BSP5 proteins) mediate a continuous phospholipid and cholesterol efflux from the sperm plasma membrane that is detrimental for sperm preservation. In this study, we investigated whether the protective effect of milk could be due to an interaction between BSP proteins and milk proteins. The binding of BSP proteins to milk proteins was demonstrated by gel filtration chromatography. Milk was fractionated into three fractions: the first containing whey protein aggregates and kappa-casein, the second containing all milk proteins, and the third containing small peptides, salts, and sugars. BSP1 has a higher affinity for the milk proteins in the milk fractions as compared to BSP3 and BSP5. The binding of BSP proteins to milk proteins was further characterized by isothermal titration calorimetry. We demonstrated that BSP1 binds to caseins and the titration could be simulated with a Scatchard approach, leading to an affinity constant (K(a)) of 350 mM(-1) and a stoichiometric parameter for the association (n) of 4.5 BSP1 per casein. The association between BSP1 and alpha-lactalbumin was characterized by a K(a) of 240 mM(-1) and an n value of 0.8. These results indicate the existence of an interaction between BSP proteins and milk proteins that could be the origin of the protection of sperm during preservation in milk.     

Apolipoprotein A-I binds to a family of bovine seminal plasma proteins

Bovine seminal plasma contains four similar acidic proteins, previously designated as BSP (bovine seminal plasma)-A1, BSP-A2, BSP-A3, and BSP-30-kDa, that when added to pituitary cell cultures result in the immediate secretion of gonadotropins (follitropin and lutropin). However, when calf or horse serum was included in the culture medium the secretion of gonadotropins was completely prevented. This effect was seen at levels up to 200 micrograms of BSP protein/ml while the presence of more than 200 micrograms of BSP protein/ml in the serum medium continued to release gonadotropins. This could be explained by the presence in the sera of a binding factor to the BSP proteins which prevents their action. This binding factor has been detected in all the sera tested, including human serum, in dot-blot experiments using 125I-labeled BSP-A1, -A2, -A3, or -30-kDa protein. Thus, it was of interest to isolate this binding factor from human serum by affinity chromatography on a column of BSP-A1/-A2-agarose. The purified binding factor was then identified as apolipoprotein A-I (apoA-I) by the following criteria: (a) it has a molecular mass of 27,000 daltons, (b) the amino acid composition is similar to apoA-I, (c) the first 25 residues at the amino-terminal end of this binding factor are identical to apoA-I, and (d) the binding factor cross-reacts in the radioimmunoassay of apoA-I. Furthermore, BSP proteins also bind to purified plasma apoA-I and apoA-I associated with high density lipoprotein. ApoA-I is the major protein of plasma high density lipoprotein and plays an important role in lipid transport and metabolism. Thus, the binding of bovine seminal plasma proteins to apoA-I suggests some physiological significance in lipoprotein function or vice versa.     

Identification of PDC-109-like protein(s) in buffalo seminal plasma

The FN-2 family of seminal plasma proteins represents the major protein fraction of bovine seminal plasma. These proteins also constitute the major seminal plasma proteins fraction in horse, goat and bison seminal plasma and are present in pig, rat, mouse, hamster and human seminal plasma. BSP-A1 and BSP-A2, the predominant proteins of the FN-2 family, are collectively termed as PDC-109. Fn-2 proteins play an important role in fertilization, including sperm capacitation and formation of oviductal sperm reservoirs. Significantly, BSP proteins were also shown to have negative effects in the context of sperm storage. No conclusive evidence for the presence of buffalo seminal plasma protein(s) similar to PDC-109 exists. Studies with buffalo seminal plasma indicated that isolation and identification of PDC-109-like protein(s) from buffalo seminal plasma by conventional methods might be difficult. Thus, antibodies raised against PDC-109 isolated, and purified from cattle seminal plasma, were used for investigating the presence of PDC-109-like protein(s) in buffalo seminal plasma. Buffalo seminal plasma proteins were resolved on SDS-PAGE, blotted to nitro cellulose membranes and probed for the presence of PDC-109-like protein(s) using the PDC-109 antisera raised in rabbits. A distinct immunoreactive band well below the 20-kDa regions indicated the presence of PDC-109-like protein(s) in buffalo seminal plasma.     

Major proteins of bovine seminal plasma exhibit novel interactions with phospholipid.

A group of similar proteins, namely BSP-A1, BSP-A2, BSP-A3, and BSP-30-kDa (collectively called BSP proteins), are the major proteins found in bovine seminal fluid. These proteins are secretory products of seminal vesicles, and they bind to spermatozoa upon ejaculation, suggesting that there are binding sites for these proteins on the spermatozoa. It was of interest to characterize these binding sites on spermatozoa which may help in the elucidation of the biological function of BSP proteins. The binding sites on spermatozoa are resistant to protease or acid treatment and are heat-stable but extractable with organic solvents. The solvent-extractable material, when coated on plastic microtitration wells, binds radiolabeled BSP proteins thus indicating the lipid nature of the BSP binding sites on spermatozoa. We investigated the specificity of interaction of BSP proteins with lipids using liposomes of phospholipids, solid-phase, and thin-layer chromatography-overlay techniques. Results showed that BSP-A1, -A2, and -A3 proteins bound specifically to those phospholipids which contain the phosphorylcholine group. In contrast, BSP-30-kDa protein preferentially bound to phospholipids containing the phosphorylcholine moiety but also interacted with phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, and cardiolipin. Furthermore, of those lipids that were extracted from spermatozoa, only phospholipids which contain the phosphorylcholine moiety bound radiolabeled BSP proteins. These data suggest that the BSP protein binding sites on spermatozoa are phospholipids. We propose that this specific interaction plays an important role in the membrane modification of spermatozoa that occurs during capacitation and/or acrosome reaction.     

Binding of bovine seminal plasma protein BSP-A1/-A2 to model membranes: lipid specificity and effect of the temperature

Bovine seminal plasma (BSP) contains a family of phospholipid-binding proteins. The affinity of the protein BSP-A1/-A2 for lipid membranes composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), and POPC containing 30% (mol/mol) 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) or cholesterol, has been investigated by the isothermal titration calorimetry (ITC). This study confirms the association of these proteins to lipid bilayers, and provides a direct characterization of this exothermic process, at 37 degrees C. The measurements indicate that the protein affinity for lipid bilayers is modulated by the lipid composition, the lipid/protein ratio, and the temperature. The saturation lipid/protein ratio was increased in the presence of cholesterol and, to a lesser extent, of phosphatidylethanolamine, suggesting that it is modulated by the lipid acyl chain order. For all the investigated systems, the binding of BSP-A1/-A2 could not be modeled using a simple partitioning of the proteins between the aqueous and lipid phases. The existence of "binding sites", and lipid phase separations is discussed. The decrease of temperature, from 37 to 10 degrees C, converts the exothermic association of the proteins to the POPC bilayers to an endothermic process. A complementary 1-D and 2-D infrared spectroscopy study excludes the thermal denaturation of BSP-A1/-A2 as a contributor in the temperature dependence of the protein affinity for lipid bilayers. The reported findings suggest that changes in the affinity of BSP-A1/-A2 for lipid bilayers could be involved in modulating the association of these proteins to sperm membranes as a function of space and time; this would consequently modulate the extent of lipid extraction, including cholesterol, at a given place and given time.