Bovine Seminal Plasma Protein Research Articles

Contrasting effects of molecular crowding on the membrane-perturbing and chaperone-like activities of major bovine seminal plasma protein, PDC-109

Crowded environments inside cells and biological fluids greatly affect protein stability and activity. PDC-109, a polydisperse oligomeric protein of the bovine seminal plasma selectively binds choline phospholipids on the sperm cell surface and causes membrane destabilization and lipid efflux, leading to acrosome reaction. PDC-109 also exhibits chaperone-like activity (CLA) and protects client proteins against various kinds of stress, such as high temperature and low pH. In the present work, we have investigated the effect of molecular crowding on these two different activities of PDC-109 employing Dextran 70 (D70) – a widely used polymeric dextran – as the crowding agent. The results obtained show that presence of D70 markedly increases membrane destabilization by PDC-109. Isothermal titration calorimetric studies revealed that under crowded condition the binding affinity of PDC-109 for choline phospholipids increases approximately 3-fold, which could in turn facilitate membrane destabilization. In contrast, under identical conditions, its CLA was reduced significantly. The decreased CLA could be correlated to reduced surface hydrophobicity, which was due to stabilization of the protein oligomers. These results establish that molecular crowding exhibits contrasting effects on two different functional activities of PDC-109 and highlight the importance of microenvironment of proteins in modulating their functional activities.

Protein signatures of seminal plasma from bulls with contrasting frozen-thawed sperm viability

The present study investigated the seminal plasma proteome of Holstein bulls with low (LF; n = 6) and high (HF; n = 8) sperm freezability. The percentage of viable frozen-thawed sperm (%ViableSperm) determined by flow cytometry varied from -2.2 in LF to + 7.8 in HF bulls, as compared to the average %ViableSperm (54.7%) measured in an 860-sire population. Seminal proteins were analyzed by label free mass spectrometry, with the support of statistical and bioinformatics analyses. This approach identified 1,445 proteins, associated with protein folding, cell–cell adhesion, NADH dehydrogenase activity, ATP-binding, proteasome complex, among other processes. There were 338 seminal proteins differentially expressed (p < 0.05) in LF and HF bulls. Based on multivariate analysis, BSP5 and seminal ribonuclease defined the HF phenotype, while spermadhesin-1, gelsolin, tubulins, glyceraldehyde-3-phosphate dehydrogenase, calmodulin, ATP synthase, sperm equatorial segment protein 1, peroxiredoxin-5, secretoglobin family 1D and glucose-6-phosphate isomerase characterized the LF phenotype. Regression models indicated that %ViableSperm of bulls was related to seminal plasma peroxiredoxin-5, spermadhesin-1 and the spermadhesin-1 × BSP5 interaction (R2 = 0.84 and 0.79; p < 0.05). This report is the largest dataset of bovine seminal plasma proteins. Specific proteins of the non-cellular microenvironment of semen are potential markers of sperm cryotolerance.

Effect of season on bovine seminal plasma proteins in Thailand.

Although season has been shown to affect bull sperm quality and fertility in some studies, the effect of season on seminal plasma proteins has not been examined. In the present study, seminal plasma proteins were analysed by Fast Protein Liquid Chromatography (FPLC), to separate the phosphorylcholine-binding proteins and heparin-binding proteins from the other proteins. Semen samples were collected from bulls in three seasons: winter, summer and the rainy season. Sperm quality was analysed by flow cytometry and computer assisted sperm analysis, and further aliquots of semen were used to prepare the seminal plasma for FPLC. Meteorological data were available from a location close to the bull station. There were slight differences in sperm kinematics between seasons, but other parameters of sperm quality were not different. Minor differences in the phosphorylcholine-binding proteins were detected according to season, being lower in summer than in winter or in the rainy season, although there were no changes in the heparin-binding proteins. Temperature, humidity and rainfall differed between winter and the rainy season, but no differences were observed between summer and the rainy season except in the temperature humidity index (THI). However, the THI was above the threshold indicative of heat stress in all seasons, which could explain why few seasonal differences in protein composition were detected in this study. Alternatively, the bulls could have been well-adapted to heat stress. In conclusion, there were only slight differences in bull sperm quality and seminal plasma proteins between seasons during this study.

Characterisation of ADAMs (protein)of bovine seminal plasma by Mass Spectrometry

This study has been designed to analyse the secreted and soluble proteins in bovine seminal plasma by mass spectrometry. The majority of these proteins are produced by accessory glands, and partially by testis, epididymis, ductus deferens and vas deferens of male reproductive tract.&#x0D; Seminal plasma of bovine was collected freshly and isolated after centrifuged and removed the sperm. Non boiled and boiled seminal plasma lysate were run to identify and detect the total proteins, as well as the individual single member of ADAMs protein is determined.&#x0D; The non-boiled lysate of seminal plasma was displayed diversity of proteins more than boiled sample lysate of seminal plasma.&#x0D; However, boiled and non-boiled seminal plasma have distinguished several types of ADAMs protein which are included: ADAM10, ADAM9, ADAM7, ADAM15 in non-boiled samples, while boiled lysate of bovine seminal plasma was displayed ADAM10, ADAM9, ADAM28, and ADAM22.&#x0D; Our finding concluded that bovine seminal plasma is very rich in different types of soluble, cleaved and shed proteins such ADAMs protein which could potentially have a biological and physiological role in protection and interactions of sperm during motility inside female reproductive tract, as it might support it to fertilise the ovum. This protection might be via immunosuppression behaviour or by block specific receptors in female reproductive tract for enhancing sperm motility, and avoid sperm the singling response of immunity.

Conserved core tryptophans of FnII domains are crucial for the membranolytic and chaperone-like activities of bovine seminal plasma protein PDC-109.

The fibronectin type II (FnII) domain, present in diverse vertebrate proteins, plays crucial roles in several fundamental biological processes. PDC-109, the major bovine seminal plasma protein, contains two FnII domains that bind to choline phospholipids on sperm plasma membrane and induce lipid efflux crucial for successful fertilization. PDC-109 also exhibits chaperone-like activity and protects other proteins against various types of stress. Here, we show that a core tryptophan residue is highly conserved across species in the FnII domains. Mutation of conserved tryptophan residues W47, W93, and W106 in the FnII domains of PDC-109 to alanine leads to drastic decrease or complete abolition of membrane-binding and chaperone-like activities. These observations suggest that conserved tryptophans are important for the function of FnII proteins.

Inhibition of influenza virus activity by the bovine seminal plasma protein PDC-109.

A number of viruses causing sexually transmissible diseases are transmitted via mammalian seminal plasma. Several components of seminal plasma have been shown to influence those viruses and their physiological impact. To unravel whether components of seminal plasma could affect viruses transmitted via other pathways, it was investigated here whether the bovine seminal plasma protein PDC-109, belonging to the Fn-type 2 protein family, influences the activity of influenza A viruses, used as a model for enveloped viruses. We found that PDC-109 inhibits the fusion of influenza virus with human erythrocyte membranes and leads to a decreased viral infection in MDCK cells. In the presence of the head group of the phospholipid phosphatidylcholine, phosphorylcholine, the inhibitory effect of PDC-109 was attenuated. This indicates that the impact of the protein is mainly caused by its binding to viral and to erythrocyte membranes thereby interfering with virus-cell binding. Our study underlines that Fn-type 2 proteins have to be considered as new antiviral components present in mammalian seminal plasma.

Glycosylation differentially modulates membranolytic and chaperone-like activities of PDC-109, the major protein of bovine seminal plasma

The major bovine seminal plasma protein, PDC-109, is a mixture of glycosylated (BSP-A1) and non-glycosylated (BSP-A2) isoforms of a 109-residue long polypeptide. It binds to spermatozoa by specifically recognizing choline phospholipids on the plasma membrane and destabilizes it by penetrating the hydrophobic interior, resulting in lipid efflux, which is necessary for sperm capacitation and successful fertilization. PDC-109 also acts as a molecular chaperone and protects target proteins from denaturation and aggregation induced by various types of stress. In order to investigate the role of glycosylation in these activities, we have separated BSP-A1 and BSP-A2 from PDC-109, and also cloned and expressed BSP-A2 in E. coli and purified the recombinant BSP-A2 (rBSP-A2) to homogeneity. Employing biophysical and biochemical approaches we have investigated the membrane-perturbing and chaperone-like activities (CLA) of PDC-109, BSP-A1, BSP-A2 and recombinant BSP-A2 (rBSP-A2). The results obtained demonstrate that glycan-lacking wild-type BSP-A2 and rBSP-A2 exhibit higher membrane-perturbing activity but decreased CLA as compared to PDC-109. In contrast, BSP-A1 exhibits significantly higher CLA than PDC-109, but its ability to destabilize membranes is considerably lower. This differential modulation of the membrane-perturbing and chaperone-like activities has been explained on the basis of higher membrane-penetrating ability and lower solubility of glycan-lacking BSP-A2 as compared to the glycosylated BSP-A1.

Lectin-Binding Specificity of the Fertilization-Relevant Protein PDC-109 by Means of Surface Plasmon Resonance and Carbohydrate REcognition Domain EXcision-Mass Spectrometry

Seminal plasma proteins are relevant for sperm functionality and some appear responsible for establishing sperm interactions with the various environments along the female genital tract towards the oocyte. In recent years, research has focused on characterizing the role of these proteins in the context of reproductive biology, fertility diagnostics and treatment of related problems. Herein, we focus on the main protein of bovine seminal plasma, PDC-109 (BSP-A1/-A2), which by virtue of its lectin properties is involved in fertilization. By means of surface plasmon resonance, the interaction of PDC-109 with a panel of the most relevant glycosidic epitopes of mammals has been qualitatively and quantitatively characterized, and a higher affinity for carbohydrates containing fucose has been observed, in line with previous studies. Additionally, using the orthogonal technique of Carbohydrate REcognition Domain EXcision-Mass Spectrometry (CREDEX-MS), the recognition domain of the interaction complexes between PDC-109 and all fucosylated disaccharides [(Fuc-α1,(3,4,6)-GlcNAc)] has been defined, revealing the specific glycotope and the peptide domain likely to act as the PDC-109 carbohydrate binding site.

Spermine and spermidine act as chemical chaperones and enhance chaperone-like and membranolytic activities of major bovine seminal plasma protein, PDC-109

The major bovine seminal plasma protein, PDC-109, binds to choline phospholipids of the sperm plasma membrane and induces an efflux of cholesterol and choline phospholipids (cholesterol efflux), which is crucial for sperm capacitation. PDC-109 also exhibits chaperone-like activity and protects target proteins against various kinds of stress. Here we show that the polyamines spermine and spermidine, present in high concentration in the seminal plasma of various mammals, increase the ability of PDC-109 to perturb membrane structure as well as its chaperone-like activity. Interestingly, spermine/spermidine alone did not perturb membrane structure but exhibited chaperone-like activity by protecting target proteins against thermal and oxidative stress. When spermine/spermidine was used along with PDC-109, the observed chaperone-like activity was considerably higher than that expected for a simple additive effect, suggesting that PDC-109 and the polyamines act in a synergistic fashion. These results indicate that at the high concentrations present in the seminal plasma spermine/spermidine exhibit a positive modulatory effect on the chaperone-like activity of PDC-109 and may also function as chemical chaperones and protect other seminal plasma proteins from various kinds of stress.

Sequestration of bovine seminal plasma proteins by different assemblies of phosphatidylcholine: A new technical approach

Binder of SPerm (BSP) proteins, the main proteins from bovine seminal plasma, are known to partially intercalate into the outer leaflet of the spermatozoa membrane and bind to choline-containing lipids being present therein. This insertion generates a negative effect on semen quality after cryopreservation by inducing an early-stage capacitation of spermatozoa. The assumption of surface properties exhibited by BSP proteins was checked by tensiometry measurements: BSP proteins are highly surface active. This suggests that BSP proteins can reach the interface covered by phospholipids not only by interactions between one and each other but also due to their own surface activity. The insertion of BSP proteins into the lipid domains outer leaflet of spermatozoa was reproduced on a biomimetic system such as Langmuir monolayers. The insertion of BSP proteins can be performed in the compressible fluid domains which contain choline-bearing lipids. Monolayer films were used as well to study the complexation of BSP proteins by two phospholipid assemblies: low density lipoprotein (LDLs) from egg yolk or liposomes produced from egg phospholipids. Irrespective of the phospholipid structure (lipoprotein or liposome), BSP was hindered to alter the structure of the membrane. Only the overall ratio BSP proteins:phosphatidylcholine was important. The difference between the two sequestering agents lies on their surface properties: LDL have a strong tendency to merge with the outer layer whereas liposomes mainly remain in the bulk on the same time scale.

Altered Expression Pattern of Major Seminal Plasma Protein Genes in Seminal Vesicles of Water Buffalo (Bubalus bubalis)

The binder of sperm proteins (BSP) are secretary products of seminal vesicles. Secretions from the seminal vesicles contribute more to seminal plasma which determine the quality of the semen. The concentration of BSPs in seminal plasma has been negatively correlated with cryopreservation. An analysis of expression profile of seminal plasma proteins could be critical for better freezability of semen. Present study was carried out to assess the expression levels of major seminal plasma protein genes viz., BSP1, BSP3 and BSP5 from seminal vesicles of water buffalo using Real Time PCR. The study consisted of identification of a suitable reference gene in seminal vesicles followed by the expression analysis of major bovine seminal plasma protein (BSP) genes. The most stable reference gene among the three (glyceraldehyde 3-phosphate dehydrogenase, ribosomal protein S15a (RPS15a) and beta-actin), was found to be RPS15a and therefore, was used for normalization of target gene expression. Data analysis of major BSPs showed highest relative expression of BSP3 followed by BSP1 and BSP5. The ratio of relative expression of BSP1:BSP3:BSP5 was 10:18:1. The results from the study also showed an altered expression pattern of major BSP genes in buffalo seminal vesicles. Thus, it can be concluded that the altered expression of these genes at mRNA level would also be reflected at protein level in the seminal plasma. This could be one of the probable reasons for poor freezability of buffalo semen as compared to cattle and the finding helps in designing better cryopreservation strategies for buffalo semen.

Protein profile and functionality of spermatozoa from two semen collection methods in Bali bulls

The aim of this study was to evaluate the effect of two semen collection methods (electro-ejaculation [EE] and transrectal massage [RM]) on in vitro sperm functionality and protein composition of seminal plasma in Bali bulls. Ten untrained Bali bulls were selected for semen collection by EE and RM. Parameters analysed were acrosome and plasma membrane integrity, sperm motility (by CASA), normal morphology, functionality (sperm penetration assay), acrosome reaction, total protein content and protein profiles (by 2D-PAGE). Bulls collected by RM had a higher (p<0.05) percentage of spermatozoa with intact acrosome and plasma membrane, functionality and individual motility, and a lower proportion of seminal plasma, total protein content and lower ratio of low molecular weight proteins than those collected by EE. Analysis of 2D-PAGE gel detected about 116 spots in the range of 10–250kDa and isoelectric points (pI) ranging from 3 to 10. Approximately 52% of seminal plasma protein spots were represented by four major protein fractions with molecular weights around 37–45kDa (15.66%), 25–30kDa (12.46%), 14–16kDa (11.73%) and 12–15kDa (11.52%). Ten of the seminal plasma proteins identified by mass spectrometry belonged to major bovine seminal plasma proteins. A very significant finding in this study was related to the two proteins identified, PGK and PLA2, with MW of approximately 37–40 and 50–55kDa and pI of 8.5–8.8 and 5.2–6.0, respectively. These two protein spots can only be detected in the seminal plasma of ejaculates obtained through RM. In conclusion, semen quality as examined by in vitro sperm functionality was found to be better in RM than EE samples after treatment with heparin and calcium ionophore A-23187. In addition some low molecular weight proteins were up-regulated in the seminal plasma obtained from the EE method.

Biophysical characterization of the interaction of O-acylcholines with the major bovine seminal plasma protein, PDC-109.

Thermodynamic parameters characterizing the interaction of O-lauroylcholine (OLC) and O-myristoylcholine (OMC) with PDC-109 have been determined by isothermal titration calorimetric studies. Titration of OLC or OMC with PDC-109 resulted in exothermic peaks, the magnitude of which decreased with subsequent injections, showing saturation of the binding sites at high protein concentration and indicating that the binding process is associated with negative enthalpy change (ΔH° < 0). Association constant (Ka), Stoichiometry (n), and thermodynamic parameters, ΔH°, ΔS° and ΔG° for the binding of OLC to PDC-109 have been obtained at different temperatures, with the values obtained at 25 °C being 5.08 × 105 M−1, 0.47, −4.46 kcal mol−1, 11.13 cal mol−1 K−1 and −7.78 kcal mol−1, respectively. Values of thermodynamic parameters obtained at different temperatures indicate that the binding of PDC-109 to O-lauroylcholine is governed by a negative enthalpy contribution. The interaction of O-lauroylcholine with PDC-109 was also studied by monitoring changes in the protein intrinsic fluorescence emission intensity. The association constant, Ka, for the interaction of PDC-109 with O-lauroylcholine from fluorescence studies at 25 °C was estimated to be 5.46 × 104 M−1, which is somewhat lower than the Ka value determined by ITC. Circular dichroism spectroscopic studies indicate that interaction of O-lauroylcholine with PDC-109 induced changes in the secondary and tertiary structure of PDC-109. Differential scanning calorimetric studies revealed that the thermal unfolding temperature of PDC-109 increases in the presence of OMC, indicating that binding of O-acylcholines stabilizes the protein structure.

Characterization of the cDNA and in vitro expression of the ram seminal plasma protein RSVP14

In previous studies we have shown that seminal plasma (SP) proteins can prevent and repair cold-shock membrane damage to ram spermatozoa. Three proteins of approximately 14, 20 and 22kDa, mainly responsible for this protective ability, were identified in ram SP. They are exclusively synthesized in the seminal vesicles and, consequently, named RSVP14, RSVP20 and RSVP22. The aim of this study is to characterize and express the RSVP14 gene to provide new insights into the mechanisms through which SP proteins are able to protect spermatozoa. Additionally, a first approach has been made to the recombinant protein production. The cDNA sequence obtained encodes a 129 amino acid chain and presents a 25-amino acid signal peptide, one potential O-linked glycosylation site and seven phosphorylation sites on tyrosine, serine and threonine residues. The sequence contains two FN-2 domains, the signature characteristic of the bovine seminal plasma (BSP) protein family and related proteins of different species. More interestingly, it was shown that RSVP14 contains four disulphide bonds and a cholesterol recognition/interaction amino acid consensus (CRAC) domain, also found in BSP and similar proteins. Analysis of the relationships between RSVP14 and other mammalian SP proteins revealed a 76–85% identity, particularly with the BSP protein family. The recombinant protein was obtained in insect cell extracts and in Escherichia coli in which RSVP14 was detected in both the pellet and the supernatant. The results obtained corroborate the role of RSVP14 in capacitation and might explain its protective effect against cold-shock injury to the membranes of ram spermatozoa. Furthermore, the biochemical and functional similarities between RSVP14 and BSP proteins suggest that it might play a similar role in sperm functionality.

HSP-1/2, a major protein of equine seminal plasma, exhibits chaperone-like activity

The major bovine seminal plasma protein, PDC-109 exhibits chaperone-like activity (CLA) against a variety of target proteins. The present studies show that the homologous protein from equine seminal plasma, HSP-1/2 also exhibits CLA and inhibits the thermal aggregation of target proteins such as lactate dehydrogenase, and DTT-induced aggregation of insulin in a concentration-dependent manner. Phosphorylcholine binding inhibited the CLA of HSP-1/2, suggesting that aggregation state of the protein is important for this activity. These results demonstrate that HSP-1/2 functions as a molecular chaperone invitro, and suggest that it may protect other proteins of equine seminal plasma from unfolding/misfolding or aggregation. These results suggest that homologous proteins from the seminal plasma of other mammals also exhibit CLA, which will be physiologically relevant.

Top–down mass spectrometry reveals new sequence variants of the major bovine seminal plasma protein PDC‐109

The major protein of bovine seminal plasma, PDC-109, is a 109-residue polypeptide that exists as a polydisperse aggregate under native conditions. The oligomeric state of this aggregate varies with ionic strength and the presence of lipids. Binding of PDC-109 to choline phospholipids on the sperm plasma membrane results in an efflux of cholesterol and choline phospholipids, which is an important step in sperm capacitation. In this study, Fourier transform ion cyclotron resonance mass spectrometry was used to analyze PDC-109 purified from bovine seminal plasma. In addition to the previously known PDC-109 variants, four new sequence variants were identified by top-down mass spectrometry. For example, a protein variant containing point mutations P10L and G14R was identified along with another form having a 14-residue truncation in the N-terminal region. Two other minor variants could also be identified from the affinity-purified PDC-109. These results demonstrate that PDC-109 is naturally produced as a mixture of several protein forms, most of which have not been detected in previous studies. Native mass spectrometry revealed that PDC-109 is exclusively monomeric at low protein concentrations, suggesting that the protein oligomers are weakly bound and can easily be disrupted. Ligand binding to PDC-109 was also investigated, and it was observed that two molecules of O-phosphorylcholine bind to each PDC-109 monomer, consistent with previous reports.

Seminal Plasma Proteome: Promising Biomarkers for Bull Fertility

The seminal plasma is a mixture of secretions from the testis, epididymis and male accessory sex glands. A number of seminal plasma proteins are associated with male fertility but most of these have not been studied in detail till now. Recently, proteomics has been used to show the differences in proteins profile in seminal plasma from high and low fertility bulls. For example, osteopontin, phospholipase A2, P25b, acidic seminal fluid proteins, α-l-fucosidase and cathepsin D are positively correlated with fertility of bulls and may act as useful fertility marker (s), while lipocalin-type prostaglandin D synthase, spermadhesin Z13, clusterin and ubiquitin are negatively correlated with fertility in bulls. Bovine seminal plasma proteins in seminal plasma act like a double-edged sword and showed a quadratic association with bull fertility. The physiological roles of the metalloproteinase-2 (TIMP-2), ecto-ADP-ribosyltransferase 5, nuclobindin, Niemann-Pick C2 and epididymal sperm-binding protein 1 and their relationship to bull fertility need further studies. This review summarizes the physiological functions of proteins of seminal plasma and their relation to bull fertility.

Isothermal Titration Calorimetric Studies on the Interaction of the Major Bovine Seminal Plasma Protein, PDC-109 with Phospholipid Membranes

The interaction of the major bovine seminal plasma protein, PDC-109 with lipid membranes was investigated by isothermal titration calorimetry. Binding of the protein to model membranes made up of diacyl phospholipids was found to be endothermic, with positive values of binding enthalpy and entropy, and could be analyzed in terms of a single type of binding sites on the protein. Enthalpies and entropies for binding to diacylphosphatidylcholine membranes increased with increase in temperature, although a clear-cut linear dependence was not observed. The entropically driven binding process indicates that hydrophobic interactions play a major role in the overall binding process. Binding of PDC-109 with dimyristoylphosphatidylcholine membranes containing 25 mol% cholesterol showed an initial increase in the association constant as well as enthalpy and entropy of binding with increase in temperature, whereas the values decreased with further increase in temperature. The affinity of PDC-109 for phosphatidylcholine increased at higher pH, which is physiologically relevant in view of the basic nature of the seminal plasma. Binding of PDC-109 to Lyso-PC could be best analysed in terms of two types of binding interactions, a high affinity interaction with Lyso-PC micelles and a low-affinity interaction with the monomeric lipid. Enthalpy-entropy compensation was observed for the interaction of PDC-109 with phospholipid membranes, suggesting that water structure plays an important role in the binding process.

Biophysical Investigations on the Interaction of the Major Bovine Seminal Plasma Protein, PDC-109, with Heparin

PDC-109, the major bovine seminal plasma protein, binds to sperm plasma membrane and modulates capacitation in the presence of heparin. In view of this, the PDC-109/heparin interaction has been investigated employing various biophysical approaches. Isothermal titration calorimetric studies yielded the association constant and changes in enthalpy and entropy for the interaction at 25 °C (pH 7.4) as 1.92 (±0.2) × 10(5) M(-1), 18.6 (±1.6) kcal M(-1), and 86.5 (±5.1) cal M(-1) K(-1), respectively, whereas differential scanning calorimetric studies indicated that heparin binding results in a significant increase in the thermal stability of PDC-109. The affinity decreases with increase in pH and ionic strength, consistent with the involvement of electrostatic forces in this interaction. Circular dichroism spectroscopic studies indicated that PDC-109 retains its conformational features even up to 70-75 °C in the presence of heparin, whereas the native protein unfolds at about 55 °C. Atomic force microscopic studies demonstrated that large oligomeric structures are formed upon binding of PDC-109 to heparin, indicating an increase in the local density of the protein, which may be relevant to the ability of heparin to potentiate PDC-109 induced sperm capacitation.

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.