Limits...
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.

Show MeSH

Related in: MedlinePlus

Binding of porcine sperm cells to human myeloid cell  line HL-60. Freshly collected sperm cells were loaded with a fluorescent dye, BCECF-AM. The labeled sperm cells (1 × 106 cells  in 0.5-ml aliquots) were mixed with either Ramos cells or HL-60  cells (2 × 105 cells in 0.1-ml aliquots) at 22°C for 1 h in the presence of 5 μM A23187, 1.26 mM CaCl2, and 0.81 mM MgCl2, unless otherwise indicated. After removal of unbound sperm cells,  the cell pellets were fixed with 2% paraformaldehyde. Binding of  the fluorescence-labeled sperm cells to Ramos or to HL-60 cells  was measured by flow cytometric analysis (FACScan®). (A) Binding of labeled sperm cells to Ramos cells in the presence of  EDTA (A1) or A23187 (A2). Binding of labeled sperm cells to  HL-60 cells in the absence (A3) or presence (A4) of A23187. (B)  Binding of labeled sperm cells to HL-60 cells in the presence of  mouse IgG (B1), P23 mAb (a leukocyte adhesion nonblocking  mAb against P-selectin; B2), or P7 mAb (a leukocyte adhesion  blocking mAb against P-selectin; B3). (C) Binding of labeled  sperm cells to HL-60 cells in the presence of mouse IgM (C1),  CSLEX (an mAb against SLex; C2), or PL5 (an mAb against  PSGL-1; C3). (D) Binding of labeled sperm cells to HL-60 cells in  the presence of human IgG (D1), E-selectin Rg (D2), or P-selectin Rg (D3).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2139885&req=5

Figure 9: Binding of porcine sperm cells to human myeloid cell line HL-60. Freshly collected sperm cells were loaded with a fluorescent dye, BCECF-AM. The labeled sperm cells (1 × 106 cells in 0.5-ml aliquots) were mixed with either Ramos cells or HL-60 cells (2 × 105 cells in 0.1-ml aliquots) at 22°C for 1 h in the presence of 5 μM A23187, 1.26 mM CaCl2, and 0.81 mM MgCl2, unless otherwise indicated. After removal of unbound sperm cells, the cell pellets were fixed with 2% paraformaldehyde. Binding of the fluorescence-labeled sperm cells to Ramos or to HL-60 cells was measured by flow cytometric analysis (FACScan®). (A) Binding of labeled sperm cells to Ramos cells in the presence of EDTA (A1) or A23187 (A2). Binding of labeled sperm cells to HL-60 cells in the absence (A3) or presence (A4) of A23187. (B) Binding of labeled sperm cells to HL-60 cells in the presence of mouse IgG (B1), P23 mAb (a leukocyte adhesion nonblocking mAb against P-selectin; B2), or P7 mAb (a leukocyte adhesion blocking mAb against P-selectin; B3). (C) Binding of labeled sperm cells to HL-60 cells in the presence of mouse IgM (C1), CSLEX (an mAb against SLex; C2), or PL5 (an mAb against PSGL-1; C3). (D) Binding of labeled sperm cells to HL-60 cells in the presence of human IgG (D1), E-selectin Rg (D2), or P-selectin Rg (D3).

Mentions: These results strongly suggest that both the P-selectin ligand in the zona pellucida of porcine oocytes and P-selectin on the acrosome-reacted sperm cells are biologically functional. To corroborate this finding, we investigated whether porcine sperm cells could bind to HL-60 cells, a human promyeloid cell line that expresses the functional PSGL-1 (Moore et al., 1992; Sako et al., 1993; Lenter et al., 1994; Ma et al., 1994; Asa et al., 1995; Spertini et al., 1996; Tu et al., 1996). Fig. 9 shows that, in the presence of Ca2+, Mg2+, and A23187, fluorescently labeled sperm cells avidly bound to HL-60 cells (A4), but not to Ramos cells (A1 and A2), a human lymphoblast cell line that does not express the functional PSGL-1 (Vachino et al., 1995). This binding was reduced when the experiment was carried out in the absence of A23187 (A3), a calcium ionophore that induces the sperm acrosomal reaction (Aarons et al., 1991; Tao et al., 1993). The partial binding observed in the absence of A23187 (A3) was likely attributed to the broken cytoplasmic membrane on some sperm cells, caused by the repeated washing procedures used in their preparation (Fig. 8). The requirement for A23187 for full binding activity is consistent with the expression of P-selectin on the acrosomal membrane of sperm cells (Figs. 6 and 8).


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)

Binding of porcine sperm cells to human myeloid cell  line HL-60. Freshly collected sperm cells were loaded with a fluorescent dye, BCECF-AM. The labeled sperm cells (1 × 106 cells  in 0.5-ml aliquots) were mixed with either Ramos cells or HL-60  cells (2 × 105 cells in 0.1-ml aliquots) at 22°C for 1 h in the presence of 5 μM A23187, 1.26 mM CaCl2, and 0.81 mM MgCl2, unless otherwise indicated. After removal of unbound sperm cells,  the cell pellets were fixed with 2% paraformaldehyde. Binding of  the fluorescence-labeled sperm cells to Ramos or to HL-60 cells  was measured by flow cytometric analysis (FACScan®). (A) Binding of labeled sperm cells to Ramos cells in the presence of  EDTA (A1) or A23187 (A2). Binding of labeled sperm cells to  HL-60 cells in the absence (A3) or presence (A4) of A23187. (B)  Binding of labeled sperm cells to HL-60 cells in the presence of  mouse IgG (B1), P23 mAb (a leukocyte adhesion nonblocking  mAb against P-selectin; B2), or P7 mAb (a leukocyte adhesion  blocking mAb against P-selectin; B3). (C) Binding of labeled  sperm cells to HL-60 cells in the presence of mouse IgM (C1),  CSLEX (an mAb against SLex; C2), or PL5 (an mAb against  PSGL-1; C3). (D) Binding of labeled sperm cells to HL-60 cells in  the presence of human IgG (D1), E-selectin Rg (D2), or P-selectin Rg (D3).
© Copyright Policy
Related In: Results  -  Collection

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

Figure 9: Binding of porcine sperm cells to human myeloid cell line HL-60. Freshly collected sperm cells were loaded with a fluorescent dye, BCECF-AM. The labeled sperm cells (1 × 106 cells in 0.5-ml aliquots) were mixed with either Ramos cells or HL-60 cells (2 × 105 cells in 0.1-ml aliquots) at 22°C for 1 h in the presence of 5 μM A23187, 1.26 mM CaCl2, and 0.81 mM MgCl2, unless otherwise indicated. After removal of unbound sperm cells, the cell pellets were fixed with 2% paraformaldehyde. Binding of the fluorescence-labeled sperm cells to Ramos or to HL-60 cells was measured by flow cytometric analysis (FACScan®). (A) Binding of labeled sperm cells to Ramos cells in the presence of EDTA (A1) or A23187 (A2). Binding of labeled sperm cells to HL-60 cells in the absence (A3) or presence (A4) of A23187. (B) Binding of labeled sperm cells to HL-60 cells in the presence of mouse IgG (B1), P23 mAb (a leukocyte adhesion nonblocking mAb against P-selectin; B2), or P7 mAb (a leukocyte adhesion blocking mAb against P-selectin; B3). (C) Binding of labeled sperm cells to HL-60 cells in the presence of mouse IgM (C1), CSLEX (an mAb against SLex; C2), or PL5 (an mAb against PSGL-1; C3). (D) Binding of labeled sperm cells to HL-60 cells in the presence of human IgG (D1), E-selectin Rg (D2), or P-selectin Rg (D3).
Mentions: These results strongly suggest that both the P-selectin ligand in the zona pellucida of porcine oocytes and P-selectin on the acrosome-reacted sperm cells are biologically functional. To corroborate this finding, we investigated whether porcine sperm cells could bind to HL-60 cells, a human promyeloid cell line that expresses the functional PSGL-1 (Moore et al., 1992; Sako et al., 1993; Lenter et al., 1994; Ma et al., 1994; Asa et al., 1995; Spertini et al., 1996; Tu et al., 1996). Fig. 9 shows that, in the presence of Ca2+, Mg2+, and A23187, fluorescently labeled sperm cells avidly bound to HL-60 cells (A4), but not to Ramos cells (A1 and A2), a human lymphoblast cell line that does not express the functional PSGL-1 (Vachino et al., 1995). This binding was reduced when the experiment was carried out in the absence of A23187 (A3), a calcium ionophore that induces the sperm acrosomal reaction (Aarons et al., 1991; Tao et al., 1993). The partial binding observed in the absence of A23187 (A3) was likely attributed to the broken cytoplasmic membrane on some sperm cells, caused by the repeated washing procedures used in their preparation (Fig. 8). The requirement for A23187 for full binding activity is consistent with the expression of P-selectin on the acrosomal membrane of sperm cells (Figs. 6 and 8).

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