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

Plante G, Lusignan MF, Lafleur M, Manjunath P - Reprod. Biol. Endocrinol. (2015)

Bottom Line: These changes can ultimately be detrimental to sperm storage.They also had affinity for another milk protein fraction (F2) composed mostly of casein micelles.However, stallion BSP showed higher affinity for the fraction (F1).

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Université de Montréal, C.P. 6128, Succ. Centre Ville, Montréal, Québec, Canada, H3C 3J7. genevieve.plante@umontreal.ca.

ABSTRACT

Background: Mammalian semen contains a family of closely related proteins known as Binder of SPerm (BSP proteins) that are added to sperm at ejaculation. BSP proteins extract lipids from the sperm membrane thereby extensively modifying its composition. These changes can ultimately be detrimental to sperm storage. We have demonstrated that bovine BSP proteins interact with major milk proteins and proposed that this interaction could be the basis of sperm protection by milk extenders. In the present study, we investigated if homologous BSP proteins present in boar, stallion and ram seminal plasma display a similar affinity for the milk proteins in order to assess whether the mechanism of sperm protection by milk for these species could be general.

Methods: Skim milk was incubated with seminal plasma proteins (boar, stallion and ram), chromatographed on a Sepharose CL-4B column and protein fractions were analyzed by immunoblotting.

Results: Boar, stallion and ram BSP proteins displayed affinity for a milk protein fraction (F1) mainly composed of α-lactalbumin, β-lactoglobulin, and κ-casein. They also had affinity for another milk protein fraction (F2) composed mostly of casein micelles. However, stallion BSP showed higher affinity for the fraction (F1).

Conclusions: These results further extend our view that the association of BSP proteins with milk proteins could be a general feature of the mechanism of mammalian sperm protection by milk to prevent detrimental effect of prolonged exposure of sperm to seminal plasma.

No MeSH data available.


Related in: MedlinePlus

Isolation and identification of stallion BSP proteins. a Chromatogram of stallion SP proteins (150 mg) on gelatin-agarose column. The gelatin-adsorbed (GA) proteins (tubes 55–75) were pooled, desalted and freeze-dried. b SDS-PAGE pattern of the stallion SP proteins (15 μg) and of the GA fraction (10 μg). c Immunoblot analysis of stallion BSP proteins using the polyclonal antibodies directed against stallion BSP proteins. SP proteins (2 μg) and GA proteins (200 ng). d Specificity of the polyclonal antibodies directed against stallion BSP proteins. SP, stallion seminal plasma proteins (300 ng) and SM, skimmed milk proteins (10 μg). e LC-MS/MS analysis of the GA proteins. Bold letters represent residues identified by LC-MS/MS
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Fig1: Isolation and identification of stallion BSP proteins. a Chromatogram of stallion SP proteins (150 mg) on gelatin-agarose column. The gelatin-adsorbed (GA) proteins (tubes 55–75) were pooled, desalted and freeze-dried. b SDS-PAGE pattern of the stallion SP proteins (15 μg) and of the GA fraction (10 μg). c Immunoblot analysis of stallion BSP proteins using the polyclonal antibodies directed against stallion BSP proteins. SP proteins (2 μg) and GA proteins (200 ng). d Specificity of the polyclonal antibodies directed against stallion BSP proteins. SP, stallion seminal plasma proteins (300 ng) and SM, skimmed milk proteins (10 μg). e LC-MS/MS analysis of the GA proteins. Bold letters represent residues identified by LC-MS/MS

Mentions: In order to study the interaction of BSP proteins with milk components, polyclonal antibodies against BSP proteins from stallion and ram were raised. For polyclonal antibodies against stallion BSP proteins, proteins were purified from SP by affinity chromatography using their ability to bind strongly to gelatin. As seen on Fig. 1a, a large fraction of stallion SP proteins bound specifically to the gelatin-agarose column. We previously showed that stallion seminal plasma contains a group of BSP proteins with molecular mass of 12 kDa, 15–18 kDa, and 22–24 kDa [18]. Analysis of the gelatin-agarose adsorbed proteins by SDS-PAGE (Fig. 1b) revealed the presence of proteins with similar molecular weights. To confirm the identity of the proteins, each band was cut from the acrylamide gel and analyzed by LC-MS/MS spectrometry. The analysis showed that all the proteins recovered belong to BSP protein family. The bands corresponding to 22–24 kDa (generally detected as one broad band in immunoblots), 17 kDa, 15 kDa and 12 kDa were identified as BSP1, whereas that at 16 kDa was identified as BSP2 (Fig. 1e). Immunoblots were performed to test the specificity and sensitivity of the polyclonal antibodies raised against gelatin-adsorbed proteins. Two glycoforms of stallion BSP1 (22–24 kDa, 17 kDa), and stallion BSP2 (16 kDa) were detected (Fig. 1c). However, the antibodies did not detect the 15 kDa and 12 kDa stallion BSP proteins, probably due to their low concentration in SP [18]. As a control, it was validated that none of the milk proteins cross-reacted with antibodies (Fig. 1d).Fig. 1


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

Plante G, Lusignan MF, Lafleur M, Manjunath P - Reprod. Biol. Endocrinol. (2015)

Isolation and identification of stallion BSP proteins. a Chromatogram of stallion SP proteins (150 mg) on gelatin-agarose column. The gelatin-adsorbed (GA) proteins (tubes 55–75) were pooled, desalted and freeze-dried. b SDS-PAGE pattern of the stallion SP proteins (15 μg) and of the GA fraction (10 μg). c Immunoblot analysis of stallion BSP proteins using the polyclonal antibodies directed against stallion BSP proteins. SP proteins (2 μg) and GA proteins (200 ng). d Specificity of the polyclonal antibodies directed against stallion BSP proteins. SP, stallion seminal plasma proteins (300 ng) and SM, skimmed milk proteins (10 μg). e LC-MS/MS analysis of the GA proteins. Bold letters represent residues identified by LC-MS/MS
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4536704&req=5

Fig1: Isolation and identification of stallion BSP proteins. a Chromatogram of stallion SP proteins (150 mg) on gelatin-agarose column. The gelatin-adsorbed (GA) proteins (tubes 55–75) were pooled, desalted and freeze-dried. b SDS-PAGE pattern of the stallion SP proteins (15 μg) and of the GA fraction (10 μg). c Immunoblot analysis of stallion BSP proteins using the polyclonal antibodies directed against stallion BSP proteins. SP proteins (2 μg) and GA proteins (200 ng). d Specificity of the polyclonal antibodies directed against stallion BSP proteins. SP, stallion seminal plasma proteins (300 ng) and SM, skimmed milk proteins (10 μg). e LC-MS/MS analysis of the GA proteins. Bold letters represent residues identified by LC-MS/MS
Mentions: In order to study the interaction of BSP proteins with milk components, polyclonal antibodies against BSP proteins from stallion and ram were raised. For polyclonal antibodies against stallion BSP proteins, proteins were purified from SP by affinity chromatography using their ability to bind strongly to gelatin. As seen on Fig. 1a, a large fraction of stallion SP proteins bound specifically to the gelatin-agarose column. We previously showed that stallion seminal plasma contains a group of BSP proteins with molecular mass of 12 kDa, 15–18 kDa, and 22–24 kDa [18]. Analysis of the gelatin-agarose adsorbed proteins by SDS-PAGE (Fig. 1b) revealed the presence of proteins with similar molecular weights. To confirm the identity of the proteins, each band was cut from the acrylamide gel and analyzed by LC-MS/MS spectrometry. The analysis showed that all the proteins recovered belong to BSP protein family. The bands corresponding to 22–24 kDa (generally detected as one broad band in immunoblots), 17 kDa, 15 kDa and 12 kDa were identified as BSP1, whereas that at 16 kDa was identified as BSP2 (Fig. 1e). Immunoblots were performed to test the specificity and sensitivity of the polyclonal antibodies raised against gelatin-adsorbed proteins. Two glycoforms of stallion BSP1 (22–24 kDa, 17 kDa), and stallion BSP2 (16 kDa) were detected (Fig. 1c). However, the antibodies did not detect the 15 kDa and 12 kDa stallion BSP proteins, probably due to their low concentration in SP [18]. As a control, it was validated that none of the milk proteins cross-reacted with antibodies (Fig. 1d).Fig. 1

Bottom Line: These changes can ultimately be detrimental to sperm storage.They also had affinity for another milk protein fraction (F2) composed mostly of casein micelles.However, stallion BSP showed higher affinity for the fraction (F1).

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Université de Montréal, C.P. 6128, Succ. Centre Ville, Montréal, Québec, Canada, H3C 3J7. genevieve.plante@umontreal.ca.

ABSTRACT

Background: Mammalian semen contains a family of closely related proteins known as Binder of SPerm (BSP proteins) that are added to sperm at ejaculation. BSP proteins extract lipids from the sperm membrane thereby extensively modifying its composition. These changes can ultimately be detrimental to sperm storage. We have demonstrated that bovine BSP proteins interact with major milk proteins and proposed that this interaction could be the basis of sperm protection by milk extenders. In the present study, we investigated if homologous BSP proteins present in boar, stallion and ram seminal plasma display a similar affinity for the milk proteins in order to assess whether the mechanism of sperm protection by milk for these species could be general.

Methods: Skim milk was incubated with seminal plasma proteins (boar, stallion and ram), chromatographed on a Sepharose CL-4B column and protein fractions were analyzed by immunoblotting.

Results: Boar, stallion and ram BSP proteins displayed affinity for a milk protein fraction (F1) mainly composed of α-lactalbumin, β-lactoglobulin, and κ-casein. They also had affinity for another milk protein fraction (F2) composed mostly of casein micelles. However, stallion BSP showed higher affinity for the fraction (F1).

Conclusions: These results further extend our view that the association of BSP proteins with milk proteins could be a general feature of the mechanism of mammalian sperm protection by milk to prevent detrimental effect of prolonged exposure of sperm to seminal plasma.

No MeSH data available.


Related in: MedlinePlus