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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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P-selectin antibody binding to sperm cells after acrosomal reaction or permeablization. Porcine sperm cells (2 × 106  cells per ml) without prior washing (A), without prior washing in  the presence of 5 μM A23187 (B), with prior repeated washing  (C), and without prior washing in the presence of 0.01% saponin  (D) were incubated with either FITC-labeled rabbit IgG or  FITC-labeled rabbit P-selectin antibody at 22°C for 1 h (all at 10  μg/ml). After washing, the cells were analyzed by flow cytometry  as above.
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Figure 6: P-selectin antibody binding to sperm cells after acrosomal reaction or permeablization. Porcine sperm cells (2 × 106 cells per ml) without prior washing (A), without prior washing in the presence of 5 μM A23187 (B), with prior repeated washing (C), and without prior washing in the presence of 0.01% saponin (D) were incubated with either FITC-labeled rabbit IgG or FITC-labeled rabbit P-selectin antibody at 22°C for 1 h (all at 10 μg/ml). After washing, the cells were analyzed by flow cytometry as above.

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)

P-selectin antibody binding to sperm cells after acrosomal reaction or permeablization. Porcine sperm cells (2 × 106  cells per ml) without prior washing (A), without prior washing in  the presence of 5 μM A23187 (B), with prior repeated washing  (C), and without prior washing in the presence of 0.01% saponin  (D) were incubated with either FITC-labeled rabbit IgG or  FITC-labeled rabbit P-selectin antibody at 22°C for 1 h (all at 10  μg/ml). After washing, the cells were analyzed by flow cytometry  as above.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2139885&req=5

Figure 6: P-selectin antibody binding to sperm cells after acrosomal reaction or permeablization. Porcine sperm cells (2 × 106 cells per ml) without prior washing (A), without prior washing in the presence of 5 μM A23187 (B), with prior repeated washing (C), and without prior washing in the presence of 0.01% saponin (D) were incubated with either FITC-labeled rabbit IgG or FITC-labeled rabbit P-selectin antibody at 22°C for 1 h (all at 10 μg/ml). After washing, the cells were analyzed by flow cytometry as above.
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