The major proteins of bovine seminal plasma interact with caseins and whey proteins of milk extender.
ABSTRACT 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.
Article: New insights towards understanding the mechanisms of sperm protection by egg yolk and milk.[show abstract] [hide abstract]
ABSTRACT: 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.Molecular Reproduction and Development 11/2006; 73(10):1338-44. · 2.53 Impact Factor
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ABSTRACT: BSP proteins and their homologs are a family of structurally related proteins characterized by the presence of tandem fibronectin type II domains. In the bovine species, BSP proteins were shown to be involved in sperm capacitation, a posttesticular maturation event necessary for sperm to acquire the ability to fertilize an oocyte. Recently, many new genes from this family have been discovered in numerous mammalian species. However, inconsistency in the nomenclature is creating much confusion. In light of the rapid growth of the BSP superfamily of proteins, we propose a new nomenclature in collaboration with the HUGO Gene Nomenclature Committee.Biology of Reproduction 11/2008; 80(3):394-7. · 4.01 Impact Factor
Article: Purification and biochemical characterization of three major acidic proteins (BSP-A1, BSP-A2 and BSP-A3) from bovine seminal plasma.[show abstract] [hide abstract]
ABSTRACT: Three major acidic proteins of bovine seminal plasma, BSP-A1, BSP-A2 and BSP-A3, were purified to homogeneity, by employing fast protein liquid chromatography, gel filtration and h.p.l.c. The proteins were purified on the basis of their stimulatory effect on the basal release of gonadotropins by rat anterior-pituitary cells in culture. All three proteins migrated as distinct single bands in the presence or absence of 2-mercaptoethanol in SDS/polyacrylamide-gel electrophoresis. Their Mr values were estimated to be between 15,000 and 16,500 by SDS/polyacrylamide-gel electrophoresis. Similar Mr estimates were obtained when they were subjected to gel filtration on a calibrated column of Sephadex G-75 equilibrated in 0.05 M-acetic acid, pH 3.0. However, BSP-A1 and BSP-A2 were eluted as aggregated molecules (Mr 60,000-120,000) during gel filtration on Sephadex G-200 equilibrated in 0.05 M-NH4HCO3, pH 8.5, or phosphate buffer, pH 7.0, containing 0.15 M-NaCl. In the presence of 8 M-urea both BSP-A1 and BSP-A2 were eluted at positions corresponding to Mr values of 17,000-20,000. BSP-A1 and BSP-A2 had an identical amino acid composition, which differed largely from that of BSP-A3. All three proteins contained aspartic acid as the N-terminal residue, and cysteine was identified as the C-terminal residue. BSP-A1 and BSP-A2 are glycoproteins containing galactosamine, sialic acid and neutral sugars, but BSP-A3 did not contain any covalently attached sugars. Whereas BSP-A2 and BSP-A3 were eluted unadsorbed, BSP-A1 bound to wheat-germ lectin-Sepharose 6MB and could be eluted by the competing sugar N-acetyl-D-glucosamine. Treatment of BSP-A1 and BSP-A2 with trypsin resulted in complete loss of gonadotropin-release activity, but BSP-A3 retained full activity. Antibody raised against BSP-A1 did not cross-react with BSP-A3, or vice versa. All these properties indicated marked structural differences between BSP-A3 and BSP-A1 (or BSP-A2). On the basis of amino acid composition it was concluded that BSP-A1, BSP-A2 and BSP-A3 are the same as the gonadostatins [Esch, Ling, Bohlen, Ying & Guillemin (1983) Biochem. Biophys. Res. Commun. 113, 861-867].Biochemical Journal 02/1987; 241(3):685-92. · 4.90 Impact Factor