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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

Gel filtration analysis of milk proteins and interaction between boar BSP1 and milk proteins. a Gel filtration chromatogram of 3 ml of heated skimmed milk alone. b Gel filtration chromatogram of 35 mg of boar SP proteins alone (open circles) or after incubation with skimmed milk (solid circles). Following the elution, tubes were pooled to provide five fractions for boar SP proteins alone (1, tubes 42–51; 2, tubes 52–78; 3, tubes 79–103; 4, tubes 104–128 and 5, tubes 129–139) or seven fractions for SP incubated with milk (I, tubes 42–54; II, tubes 55–71; III, tubes 72–88; IV, tubes 89–94; V, tubes 95–106; VI, tubes 107–124 and VII, tubes 125–138). c Immunoblot analysis of pooled fractions from the elution of boar SP proteins alone. Aliquots equivalent to 0.3 % of each fraction were precipitated and used for immunoblots. d Immunoblot analysis of pooled fractions from the elution of boar SP proteins incubated with skimmed milk. Proteins were precipitated and used for immunoblots (top panel: 0.015 % of fraction VI and 0.03 % of other fractions; bottom panel: 0.3 % of fractions). E) Immunoblot analysis of milk proteins with boar BSP1 polyclonal antibodies. Boar SP proteins (10 μg) and milk proteins (20 μg) were separated by SDS-PAGE, transferred to PVDF and probed with polyclonal antibodies against boar BSP1. The experiments were carried out in triplicates and the results of one typical experiment are shown
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Fig3: Gel filtration analysis of milk proteins and interaction between boar BSP1 and milk proteins. a Gel filtration chromatogram of 3 ml of heated skimmed milk alone. b Gel filtration chromatogram of 35 mg of boar SP proteins alone (open circles) or after incubation with skimmed milk (solid circles). Following the elution, tubes were pooled to provide five fractions for boar SP proteins alone (1, tubes 42–51; 2, tubes 52–78; 3, tubes 79–103; 4, tubes 104–128 and 5, tubes 129–139) or seven fractions for SP incubated with milk (I, tubes 42–54; II, tubes 55–71; III, tubes 72–88; IV, tubes 89–94; V, tubes 95–106; VI, tubes 107–124 and VII, tubes 125–138). c Immunoblot analysis of pooled fractions from the elution of boar SP proteins alone. Aliquots equivalent to 0.3 % of each fraction were precipitated and used for immunoblots. d Immunoblot analysis of pooled fractions from the elution of boar SP proteins incubated with skimmed milk. Proteins were precipitated and used for immunoblots (top panel: 0.015 % of fraction VI and 0.03 % of other fractions; bottom panel: 0.3 % of fractions). E) Immunoblot analysis of milk proteins with boar BSP1 polyclonal antibodies. Boar SP proteins (10 μg) and milk proteins (20 μg) were separated by SDS-PAGE, transferred to PVDF and probed with polyclonal antibodies against boar BSP1. The experiments were carried out in triplicates and the results of one typical experiment are shown

Mentions: When chromatographed on a Sepharose CL-4B column, heated skimmed milk separates into three peaks (Fig. 3a). As previously described, the first peak (milk F1; tubes 40–60) contains mainly α-lactalbumin, β-lactoglobulin, κ-casein and some high-molecular-weight corresponding to albumin, lactoferrin, and immunoglobulins; the second peak (milk F2; tubes 80–110) contains mainly caseins (α- and β-caseins); and the third peak (milk F3; tubes 130–150) contains small-molecular-weight components such as amino acids, salts, sugars and vitamins [36].Fig. 3


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)

Gel filtration analysis of milk proteins and interaction between boar BSP1 and milk proteins. a Gel filtration chromatogram of 3 ml of heated skimmed milk alone. b Gel filtration chromatogram of 35 mg of boar SP proteins alone (open circles) or after incubation with skimmed milk (solid circles). Following the elution, tubes were pooled to provide five fractions for boar SP proteins alone (1, tubes 42–51; 2, tubes 52–78; 3, tubes 79–103; 4, tubes 104–128 and 5, tubes 129–139) or seven fractions for SP incubated with milk (I, tubes 42–54; II, tubes 55–71; III, tubes 72–88; IV, tubes 89–94; V, tubes 95–106; VI, tubes 107–124 and VII, tubes 125–138). c Immunoblot analysis of pooled fractions from the elution of boar SP proteins alone. Aliquots equivalent to 0.3 % of each fraction were precipitated and used for immunoblots. d Immunoblot analysis of pooled fractions from the elution of boar SP proteins incubated with skimmed milk. Proteins were precipitated and used for immunoblots (top panel: 0.015 % of fraction VI and 0.03 % of other fractions; bottom panel: 0.3 % of fractions). E) Immunoblot analysis of milk proteins with boar BSP1 polyclonal antibodies. Boar SP proteins (10 μg) and milk proteins (20 μg) were separated by SDS-PAGE, transferred to PVDF and probed with polyclonal antibodies against boar BSP1. The experiments were carried out in triplicates and the results of one typical experiment are shown
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig3: Gel filtration analysis of milk proteins and interaction between boar BSP1 and milk proteins. a Gel filtration chromatogram of 3 ml of heated skimmed milk alone. b Gel filtration chromatogram of 35 mg of boar SP proteins alone (open circles) or after incubation with skimmed milk (solid circles). Following the elution, tubes were pooled to provide five fractions for boar SP proteins alone (1, tubes 42–51; 2, tubes 52–78; 3, tubes 79–103; 4, tubes 104–128 and 5, tubes 129–139) or seven fractions for SP incubated with milk (I, tubes 42–54; II, tubes 55–71; III, tubes 72–88; IV, tubes 89–94; V, tubes 95–106; VI, tubes 107–124 and VII, tubes 125–138). c Immunoblot analysis of pooled fractions from the elution of boar SP proteins alone. Aliquots equivalent to 0.3 % of each fraction were precipitated and used for immunoblots. d Immunoblot analysis of pooled fractions from the elution of boar SP proteins incubated with skimmed milk. Proteins were precipitated and used for immunoblots (top panel: 0.015 % of fraction VI and 0.03 % of other fractions; bottom panel: 0.3 % of fractions). E) Immunoblot analysis of milk proteins with boar BSP1 polyclonal antibodies. Boar SP proteins (10 μg) and milk proteins (20 μg) were separated by SDS-PAGE, transferred to PVDF and probed with polyclonal antibodies against boar BSP1. The experiments were carried out in triplicates and the results of one typical experiment are shown
Mentions: When chromatographed on a Sepharose CL-4B column, heated skimmed milk separates into three peaks (Fig. 3a). As previously described, the first peak (milk F1; tubes 40–60) contains mainly α-lactalbumin, β-lactoglobulin, κ-casein and some high-molecular-weight corresponding to albumin, lactoferrin, and immunoglobulins; the second peak (milk F2; tubes 80–110) contains mainly caseins (α- and β-caseins); and the third peak (milk F3; tubes 130–150) contains small-molecular-weight components such as amino acids, salts, sugars and vitamins [36].Fig. 3

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