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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  zona pellucida proteins with  PSGL-1 peptide antibody.  Porcine zona pellucida proteins, fractionated by 7%  SDS-PAGE under nonreducing (A) and reducing (B  and C) conditions and transferred to blotting membranes,  were probed with rabbit preimmune IgG or PSGL-1 peptide antibody followed by biotinylated antibody to rabbit  IgG. Immunoreactive proteins were visualized as outlined in the legend to Fig. 1.
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Figure 2: Immunoblotting of zona pellucida proteins with PSGL-1 peptide antibody. Porcine zona pellucida proteins, fractionated by 7% SDS-PAGE under nonreducing (A) and reducing (B and C) conditions and transferred to blotting membranes, were probed with rabbit preimmune IgG or PSGL-1 peptide antibody followed by biotinylated antibody to rabbit IgG. Immunoreactive proteins were visualized as outlined in the legend to Fig. 1.

Mentions: To corroborate the above findings, the porcine zona pellucida proteins were also probed with an antibody against a synthetic peptide encoding residues 41–55 of the amino acid sequence of PSGL-1. Fig. 2 shows that this antibody, but not preimmune IgG, bound to the ∼210-kD protein under nonreducing conditions (A, arrow) and the ∼80-kD protein under reducing conditions (B, arrow). Preincubation of the antibody with the synthetic peptide abrogated this binding (C, arrow). The protein bands at ∼50–60 kD were most likely due to nonspecific binding, since (a) they existed in the blots probed with both preimmune IgG and PSGL-1 peptide antibody (A and B), and (b) they were not inhibited with the respective peptide antigen (C).


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  zona pellucida proteins with  PSGL-1 peptide antibody.  Porcine zona pellucida proteins, fractionated by 7%  SDS-PAGE under nonreducing (A) and reducing (B  and C) conditions and transferred to blotting membranes,  were probed with rabbit preimmune IgG or PSGL-1 peptide antibody followed by biotinylated antibody to rabbit  IgG. Immunoreactive proteins were visualized as outlined in the legend to Fig. 1.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2139885&req=5

Figure 2: Immunoblotting of zona pellucida proteins with PSGL-1 peptide antibody. Porcine zona pellucida proteins, fractionated by 7% SDS-PAGE under nonreducing (A) and reducing (B and C) conditions and transferred to blotting membranes, were probed with rabbit preimmune IgG or PSGL-1 peptide antibody followed by biotinylated antibody to rabbit IgG. Immunoreactive proteins were visualized as outlined in the legend to Fig. 1.
Mentions: To corroborate the above findings, the porcine zona pellucida proteins were also probed with an antibody against a synthetic peptide encoding residues 41–55 of the amino acid sequence of PSGL-1. Fig. 2 shows that this antibody, but not preimmune IgG, bound to the ∼210-kD protein under nonreducing conditions (A, arrow) and the ∼80-kD protein under reducing conditions (B, arrow). Preincubation of the antibody with the synthetic peptide abrogated this binding (C, arrow). The protein bands at ∼50–60 kD were most likely due to nonspecific binding, since (a) they existed in the blots probed with both preimmune IgG and PSGL-1 peptide antibody (A and B), and (b) they were not inhibited with the respective peptide antigen (C).

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