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.

Interaction of milk proteins and Binder of Sperm (BSP) proteins from boar, stallion and ram semen

“…The heated skimmed milk was then cooled at room temperature and filtered through metal mesh to remove the coagulum [ 30 ]. The skimmed milk fraction 1 (milk F1) and fraction 2 (milk F2) were prepared by gel filtration on Sepharose CL-4B column as described previously [ 14 ].…”

“…The association of casein micelles with BSP proteins was shown to preclude cholesterol and phospholipid extraction from membranes induced by BSP proteins, while maintaining sperm viability and motility during sperm storage [ 13 ]. Further studies showed that bovine BSP proteins bind to several milk proteins, namely casein micelles, α-lactalbumin and β-lactoglobulin [ 14 ]. These studies led us to propose that the interaction between milk proteins and bovine BSP proteins is the basis for sperm protection during storage using milk-based extenders.…”

Milk, caseinate and lactoferrin addition to equine semen cooling extenders

“…Casein is the major protein component of milk, which represents 75–80% of all milk proteins (Fox & McSweeney, ), and appears to be responsible for the protective effect of milk on the spermatozoon during cooling and cryopreservation (Battelier et al ., , ). It acts through preventing lipid loss from the sperm membrane (Batellier et al ., ; Pagl et al ., ; Bergeron et al ., ; Lusignan et al ., ). Thus, casein appears to be responsible for sperm protection even at low concentrations for cooled (0.6%) and frozen semen (1.35%, Lagares et al ., ).…”

Sodium bicarbonate and HEPES as buffer components for cooling the semen of pony stallions

“…The protection mechanisms involved are not well elucidated. In milk, the protective effect seems to be related to casein micelles (BATELLIER et al, 2001;LUSIGNAN et al, 2011), while in egg yolk, low density lipoproteins are important MANJUNATH, 2006;PUGLIESI et al, 2012).…”