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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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Distribution of a P-selectin ligand in porcine zona pellucida. Porcine oocytes were washed and incubated sequentially with  platelet P-selectin, rabbit P-selectin antibody, and an HRP-conjugated secondary antibody. P-selectin binding was visualized by the  electron-dense reaction product. P-selectin is bound to the membrane vesicles and fragments embedded within the zona pellucida at or  near the (A) inner surface (arrows) of the zona pellucida, which is adjacent to the oocyte removed during the staining procedures, and  (B) outer surface. (C) Zona pellucida stained in the absence of P-selectin. (D) Zona pellucida stained with P-selectin in the presence of  EDTA. Bar, 500 nm.
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Figure 3: Distribution of a P-selectin ligand in porcine zona pellucida. Porcine oocytes were washed and incubated sequentially with platelet P-selectin, rabbit P-selectin antibody, and an HRP-conjugated secondary antibody. P-selectin binding was visualized by the electron-dense reaction product. P-selectin is bound to the membrane vesicles and fragments embedded within the zona pellucida at or near the (A) inner surface (arrows) of the zona pellucida, which is adjacent to the oocyte removed during the staining procedures, and (B) outer surface. (C) Zona pellucida stained in the absence of P-selectin. (D) Zona pellucida stained with P-selectin in the presence of EDTA. Bar, 500 nm.

Mentions: Three additional weaker bands (at ∼210, ∼180, and ∼140 kD under reducing conditions) were also recognized by P-selectin (Fig. 1 C, lane 1). The presence of the ∼210kD band may be due to incomplete reduction of the ∼210kD dimer, a phenomenon frequently observed for human leukocyte PSGL-1 (Moore et al., 1992; Sako et al., 1993; Lenter et al., 1994). The nature of ∼180- and ∼140-kD bands is unclear; they were not recognized by P-selectin (Fig. 1 B, lane 1) and PSGL-1 peptide antibody (see Fig. 3 A) when proteins were separated under nonreducing conditions. Plus, they were not recognized by PSGL-1 peptide antibody (Fig. 3, B and C), under reducing conditions. In addition, there was some background staining spots on the blots, which were not similar to the protein bands (Fig. 1 B, lane 2).


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)

Distribution of a P-selectin ligand in porcine zona pellucida. Porcine oocytes were washed and incubated sequentially with  platelet P-selectin, rabbit P-selectin antibody, and an HRP-conjugated secondary antibody. P-selectin binding was visualized by the  electron-dense reaction product. P-selectin is bound to the membrane vesicles and fragments embedded within the zona pellucida at or  near the (A) inner surface (arrows) of the zona pellucida, which is adjacent to the oocyte removed during the staining procedures, and  (B) outer surface. (C) Zona pellucida stained in the absence of P-selectin. (D) Zona pellucida stained with P-selectin in the presence of  EDTA. Bar, 500 nm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2139885&req=5

Figure 3: Distribution of a P-selectin ligand in porcine zona pellucida. Porcine oocytes were washed and incubated sequentially with platelet P-selectin, rabbit P-selectin antibody, and an HRP-conjugated secondary antibody. P-selectin binding was visualized by the electron-dense reaction product. P-selectin is bound to the membrane vesicles and fragments embedded within the zona pellucida at or near the (A) inner surface (arrows) of the zona pellucida, which is adjacent to the oocyte removed during the staining procedures, and (B) outer surface. (C) Zona pellucida stained in the absence of P-selectin. (D) Zona pellucida stained with P-selectin in the presence of EDTA. Bar, 500 nm.
Mentions: Three additional weaker bands (at ∼210, ∼180, and ∼140 kD under reducing conditions) were also recognized by P-selectin (Fig. 1 C, lane 1). The presence of the ∼210kD band may be due to incomplete reduction of the ∼210kD dimer, a phenomenon frequently observed for human leukocyte PSGL-1 (Moore et al., 1992; Sako et al., 1993; Lenter et al., 1994). The nature of ∼180- and ∼140-kD bands is unclear; they were not recognized by P-selectin (Fig. 1 B, lane 1) and PSGL-1 peptide antibody (see Fig. 3 A) when proteins were separated under nonreducing conditions. Plus, they were not recognized by PSGL-1 peptide antibody (Fig. 3, B and C), under reducing conditions. In addition, there was some background staining spots on the blots, which were not similar to the protein bands (Fig. 1 B, lane 2).

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