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Expression of a P-selectin ligand in zona pellucida of porcine oocytes and P-selectin on acrosomal membrane of porcine sperm cells. Potential implications for their involvement in sperm-egg interactions.

Geng JG, Raub TJ, Baker CA, Sawada GA, Ma L, Elhammer AP - J. Cell Biol. (1997)

Bottom Line: In addition, a search for a specific receptor for this ligand leads to the identification of P-selectin on the acrosomal membrane of porcine sperm cells.Moreover, porcine sperm cells were found to be capable of binding to human promyeloid cell line HL-60.Taken together, our findings implicate a potential role for the oocyte P-selectin ligand and the sperm P-selectin in porcine sperm-egg interactions.

View Article: PubMed Central - PubMed

Affiliation: Cell Biology and Inflammation Research, Pharmacia and Upjohn, Inc., Kalamazoo, Michigan 49001, USA. jian-guo.geng@am.pnu.com

ABSTRACT
The selectin family of cell adhesion molecules mediates initial leukocyte adhesion to vascular endothelial cells at sites of inflammation. O-glycan structural similarities between oligosaccharides from human leukocyte P-selectin glycoprotein ligand-1 (PSGL-1) and from zona pellucida glycoproteins of porcine oocytes indicate the possible existence of a P-selectin ligand in the zona pellucida. Here, using biochemical as well as morphological approaches, we demonstrate that a P-selectin ligand is expressed in the porcine zona pellucida. In addition, a search for a specific receptor for this ligand leads to the identification of P-selectin on the acrosomal membrane of porcine sperm cells. In vitro binding of porcine acrosome-reacted sperm cells to oocytes was found to be Ca2+ dependent and inhibitable with either P-selectin, P-selectin receptor-globulin, or leukocyte adhesion blocking antibodies against P-selectin and PSGL-1. Moreover, porcine sperm cells were found to be capable of binding to human promyeloid cell line HL-60. Taken together, our findings implicate a potential role for the oocyte P-selectin ligand and the sperm P-selectin in porcine sperm-egg interactions.

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Immunoblotting of  porcine sperm cells. Porcine  sperm cells (5 × 105 cells per  lane for silver staining and  1 × 106 cells per lane for  immunoblotting), porcine  platelets (1 × 105 cells per  lane), and PUVEC (confluent monolayer of cells from a  35-mm dish per lane) were  mixed with SDS sample  buffer and boiled for 5 min.  After electrophoresis under  reducing (A, R) and nonreducing conditions (A, N; B–D),  proteins were either silver  stained (A) or transferred to  blotting membranes and  probed with 1 μg/ml of biotinylated rabbit P-selectin antibody, biotinylated P7 mAb  against P-selectin (B for  sperm cells and C for platelets), or biotinylated rabbit  E-selectin antibody (D). Immunoreactive proteins were  visualized as outlined in the  legend to Fig. 1.
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Figure 7: Immunoblotting of porcine sperm cells. Porcine sperm cells (5 × 105 cells per lane for silver staining and 1 × 106 cells per lane for immunoblotting), porcine platelets (1 × 105 cells per lane), and PUVEC (confluent monolayer of cells from a 35-mm dish per lane) were mixed with SDS sample buffer and boiled for 5 min. After electrophoresis under reducing (A, R) and nonreducing conditions (A, N; B–D), proteins were either silver stained (A) or transferred to blotting membranes and probed with 1 μg/ml of biotinylated rabbit P-selectin antibody, biotinylated P7 mAb against P-selectin (B for sperm cells and C for platelets), or biotinylated rabbit E-selectin antibody (D). Immunoreactive proteins were visualized as outlined in the legend to Fig. 1.

Mentions: The expression of P-selectin on porcine sperm cells was first established by FACS® analysis, using two different P-selectin antibodies, an FITC-conjugated rabbit P-selectin antibody and an FITC-conjugated P7 mAb. Both antibodies were raised against human platelet P-selectin and both reacted with porcine platelet P-selectin (see Fig. 7 C). As shown in Fig. 5, rabbit P-selectin antibody (A) and P7 mAb (B) bound to repeatedly washed sperm cells whose plasma membranes were no longer intact (see Fig. 8). By contrast, FITC-conjugated rabbit E-selectin antibody did not bind to the sperm cells (Fig. 5 C), although it clearly reacted with the TNF-α–treated PUVEC (D). Interestingly, P-selectin polyclonal antibody did not bind to the unwashed sperm cells (Fig. 6 A) unless they were treated with A23187 (a calcium ionophore known to induce the acrosomal reaction; Fig. 6 B), repeated washing (causing disruption of the plasma membranes, as demonstrated in Fig. 8; Fig. 6 C), or saponin (a detergent that selectively permeabilizes the plasma membrane; Fig. 6 D). Together, these results suggest that porcine sperm cells express P-selectin, but not E-selectin, and that P-selectin is expressed on the acrosomal membrane of the sperm cells, but not on the plasma membrane.


Expression of a P-selectin ligand in zona pellucida of porcine oocytes and P-selectin on acrosomal membrane of porcine sperm cells. Potential implications for their involvement in sperm-egg interactions.

Geng JG, Raub TJ, Baker CA, Sawada GA, Ma L, Elhammer AP - J. Cell Biol. (1997)

Immunoblotting of  porcine sperm cells. Porcine  sperm cells (5 × 105 cells per  lane for silver staining and  1 × 106 cells per lane for  immunoblotting), porcine  platelets (1 × 105 cells per  lane), and PUVEC (confluent monolayer of cells from a  35-mm dish per lane) were  mixed with SDS sample  buffer and boiled for 5 min.  After electrophoresis under  reducing (A, R) and nonreducing conditions (A, N; B–D),  proteins were either silver  stained (A) or transferred to  blotting membranes and  probed with 1 μg/ml of biotinylated rabbit P-selectin antibody, biotinylated P7 mAb  against P-selectin (B for  sperm cells and C for platelets), or biotinylated rabbit  E-selectin antibody (D). Immunoreactive proteins were  visualized as outlined in the  legend to Fig. 1.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2139885&req=5

Figure 7: Immunoblotting of porcine sperm cells. Porcine sperm cells (5 × 105 cells per lane for silver staining and 1 × 106 cells per lane for immunoblotting), porcine platelets (1 × 105 cells per lane), and PUVEC (confluent monolayer of cells from a 35-mm dish per lane) were mixed with SDS sample buffer and boiled for 5 min. After electrophoresis under reducing (A, R) and nonreducing conditions (A, N; B–D), proteins were either silver stained (A) or transferred to blotting membranes and probed with 1 μg/ml of biotinylated rabbit P-selectin antibody, biotinylated P7 mAb against P-selectin (B for sperm cells and C for platelets), or biotinylated rabbit E-selectin antibody (D). Immunoreactive proteins were visualized as outlined in the legend to Fig. 1.
Mentions: The expression of P-selectin on porcine sperm cells was first established by FACS® analysis, using two different P-selectin antibodies, an FITC-conjugated rabbit P-selectin antibody and an FITC-conjugated P7 mAb. Both antibodies were raised against human platelet P-selectin and both reacted with porcine platelet P-selectin (see Fig. 7 C). As shown in Fig. 5, rabbit P-selectin antibody (A) and P7 mAb (B) bound to repeatedly washed sperm cells whose plasma membranes were no longer intact (see Fig. 8). By contrast, FITC-conjugated rabbit E-selectin antibody did not bind to the sperm cells (Fig. 5 C), although it clearly reacted with the TNF-α–treated PUVEC (D). Interestingly, P-selectin polyclonal antibody did not bind to the unwashed sperm cells (Fig. 6 A) unless they were treated with A23187 (a calcium ionophore known to induce the acrosomal reaction; Fig. 6 B), repeated washing (causing disruption of the plasma membranes, as demonstrated in Fig. 8; Fig. 6 C), or saponin (a detergent that selectively permeabilizes the plasma membrane; Fig. 6 D). Together, these results suggest that porcine sperm cells express P-selectin, but not E-selectin, and that P-selectin is expressed on the acrosomal membrane of the sperm cells, but not on the plasma membrane.

Bottom Line: In addition, a search for a specific receptor for this ligand leads to the identification of P-selectin on the acrosomal membrane of porcine sperm cells.Moreover, porcine sperm cells were found to be capable of binding to human promyeloid cell line HL-60.Taken together, our findings implicate a potential role for the oocyte P-selectin ligand and the sperm P-selectin in porcine sperm-egg interactions.

View Article: PubMed Central - PubMed

Affiliation: Cell Biology and Inflammation Research, Pharmacia and Upjohn, Inc., Kalamazoo, Michigan 49001, USA. jian-guo.geng@am.pnu.com

ABSTRACT
The selectin family of cell adhesion molecules mediates initial leukocyte adhesion to vascular endothelial cells at sites of inflammation. O-glycan structural similarities between oligosaccharides from human leukocyte P-selectin glycoprotein ligand-1 (PSGL-1) and from zona pellucida glycoproteins of porcine oocytes indicate the possible existence of a P-selectin ligand in the zona pellucida. Here, using biochemical as well as morphological approaches, we demonstrate that a P-selectin ligand is expressed in the porcine zona pellucida. In addition, a search for a specific receptor for this ligand leads to the identification of P-selectin on the acrosomal membrane of porcine sperm cells. In vitro binding of porcine acrosome-reacted sperm cells to oocytes was found to be Ca2+ dependent and inhibitable with either P-selectin, P-selectin receptor-globulin, or leukocyte adhesion blocking antibodies against P-selectin and PSGL-1. Moreover, porcine sperm cells were found to be capable of binding to human promyeloid cell line HL-60. Taken together, our findings implicate a potential role for the oocyte P-selectin ligand and the sperm P-selectin in porcine sperm-egg interactions.

Show MeSH
Related in: MedlinePlus