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First published online October 11, 2005
doi: 10.1242/10.1242/jcs.02604

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Evidence for the involvement of PECAM-1 in a receptor mediated signal-transduction pathway regulating capacitation-associated tyrosine phosphorylation in human spermatozoa

¹ Reproductive Science Group, University of Newcastle, Callaghan, NSW 2308, Australia
² ARC Centre of Excellence in Biotechnology and Development, School of Environmental and Life Sciences, University of Newcastle, Callaghan, NSW 2308, Australia
³ School of Biomedical Sciences, University of Newcastle, Callaghan, NSW 2308, Australia

View larger version (39K): [in a new window]   Fig. 1. Lectin induction of sperm tyrosine phosphorylation. (A) Purified human spermatozoa were incubated with a panel of sperm-binding lectins (Table 1; 0.5 µg/ml) for 30 minutes. Of these lectins, WGA alone demonstrated the ability to consistently up-regulate tyrosine phosphorylation. To examine the specificity of this response, spermatozoa were incubated in a cocktail of the lectins sWGA, MAA and SNA (which collectively possess the same sugar specificity as WGA) either in their native form or as cross-linked complexes (*). (B) The specificity of the WGA response was further examined by pre-incubation of sperm with either neuraminidase and/or N-acteylglucosaminidase (250 mU/ml) prior to the addition of WGA. Control treatments incorporated spermatozoa incubated in BWW prepared without HCO3– (–ve), or in BWW supplemented with dbcAMP and pentoxyfiline (+ve). Following immunodetection of phosphotyrosine residues (anti-py), membranes were stripped and re-probed with anti--tubulin to ensure equivalent protein loading across all treatments. The experiment was replicated three times with pooled semen samples obtained from at least three different donors, and representative blots are depicted.

View larger version (71K): [in a new window]   Fig. 2. Comparison of the WGA-induced phosphotyrosine profile. Purified human spermatozoa were incubated in either BWW alone (BWW), BWW supplemented with 0.5 µg/ml WGA (WGA), or in positive (+ve) and negative (–ve) control media for 30 minutes. Following incubation, spermatozoa were solubilized in either CHAPS (1 hour on ice) or SDS extraction buffers (5 minutes, 100°C) and extracted proteins were prepared for immunodetection of phosphotyrosine residues (anti-py). The SDS-extract was characterized by the presence of two predominant proteins (83 and 105 kDa) however, overexposure of this blot (WGA*) demonstrated a pattern that approximated that seen in CHAPS extracts. This comparison was replicated three times with pooled semen samples obtained from at least three different donors, and representative blots are depicted.

View larger version (49K): [in a new window]   Fig. 3. Characterisation of the dose and time dependency of WGA-stimulated sperm tyrosine phosphorylation. Purified human spermatozoa were incubated in varying concentrations of WGA (0.005-5 µg/ml) or in positive (+ve) and negative (–ve) control media for 30 minutes. Alternatively, spermatozoa were incubated in 0.5 µg/ml WGA for varying times (5-90 minutes). Following incubation, spermatozoa were either (A) solubilized in SDS extraction buffer and prepared for immunoblotting with anti-phosphotyrosine or (B) examined to determine the status of their motility and viability. This experiment was replicated three times with pooled semen samples obtained from at least three different donors and representative blots are depicted. Graphical data is given as the means ± s.e.m. from the three replicate experiments. *P<0.05, **P<0.01.

View larger version (41K): [in a new window]   Fig. 4. Comparison of the WGA-induced tyrosine phosphorylation in normal and defective spermatozoa. (A) Human spermatozoa recovered in the low density (defective) and high density (normal) fractions after Percoll density gradient centrifugation were incubated with either WGA (0.5 µg/ml) or in positive (+ve) and negative (–ve) control media for 30 minutes. An additional control, consisting of sperm incubated in complete BWW supplemented with SJA (a lectin that does not interact with the sperm surface) was also included. Following incubation, spermatozoa were solubilized in SDS extraction buffer and prepared for immunblotting with anti-phosphotyrosine; only the normal cells responded to WGA. (B) To eliminate the possibility of a contaminating leukocyte contribution to the WGA response, viable spermatozoa recovered by Percoll fractionation (pellet) and those isolated by a swim up procedure (swim up) were incubated with WGA (0.5 µg/ml) for 30 minutes at 37°C. After incubation, spermatozoa were solubilized in SDS extraction buffer and prepared for immunblotting with anti-phosphotyrosine. This comparison was replicated three times with pooled semen samples obtained from at least three different donors and representative blots are depicted.

View larger version (36K): [in a new window]   Fig. 5. Evaluation of ROS generation in human spermatozoa following WGA challenge. To investigate ROS generation following WGA challenge, spermatozoa recovered by Percoll fractionation (pellet) and those isolated by a swim up procedure (swim up) were incubated in (A) luminol-peroxidase or (B) lucigenin and chemiluminescence recorded for 20 minutes. WGA (0.5 µg/ml) was then added and chemiluminescence recorded for an additional 30 minutes. Each sample was then tested for leukocyte contamination through the addition of PMA. The resultant ROS generation profile for each treatment was graphed. Control treatments included spermatozoa incubated in the absence of WGA (control) and a cell-free medium control. The latter control generated only basal levels of chemiluminescence and in the interest of clarity is not shown in the figure. (C) The role of ROS generation was further examined by pre-incubating sperm in either SOD and/or catalase (300 and 3000 U respectively) for 1 hour prior to the addition of WGA (0.5 µg/ml). This experiment was replicated three times with pooled semen samples obtained from at least three different donors and representative data are presented.

View larger version (39K): [in a new window]   Fig. 6. Evaluation of cAMP levels and tyrosine phosphorylation status following WGA treatment. To investigate the production of intracellular cAMP following WGA challenge, human spermatozoa were incubated with either WGA (0.5 µg/ml) or SJA (0.5 µg/ml), or in positive (+ve) or negative (–ve) control media for 30 minutes. Following incubation, the sperm sample was split and one half was prepared for (A) the measurement of [cAMP]i and the remaining cells were prepared for (B) immunodetection of phosphotyrosine residues (anti-py). This experiment was replicated three times with pooled semen samples obtained from at least three different donors and representative blots are presented. Graphical data represent the means ± s.e.m. from the three replicate experiments. **P<0.01.

View larger version (58K): [in a new window]   Fig. 7. Impact of inhibitors on WGA-induced tyrosine phosphorylation. Spermatozoa were pre-incubated for 1 hour in genistein, herbamycin, H-89, PP1, PP2, SU6656 or the vehicle control (DMSO). WGA (0.5 µg/ml) was then added and the incubations continued for an additional 30 minutes. The sperm sample was then prepared for analysis of phosphotyrosine levels by immunoblotting. This experiment was replicated three times with pooled semen samples obtained from at least three different donors and representative blots are depicted.

View larger version (71K): [in a new window]   Fig. 8. Identification of sperm surface WGA binding proteins. Sperm surface proteins were biotinylated, purified using streptavidin-conjugated magnetic beads and resolved using duplicate 2D SDS-PAGE. (A) The preparative gel was stained with Coomassie Brilliant Blue (G-250) and (B) the analytical gel was transferred to a nitrocellulose membrane and blotted with streptavidin HRP to assess the efficacy of removal of the biotin tag, before (C) being stripped and reprobed with biotinylated WGA. Proteins that cross-reacted with WGA were excised from the preparative gel and subjected to MALDI-ToF mass spectrometry. Three of the four predominant WGA-binding proteins were identified as platelet endothelial cell adhesion molecule-1 (125 kDa, pI 6.5; acc. no. P16284), Zinc-2-glycoprotein (32 kDa, pI 5; acc. no. P25311) and prostate-specific antigen (28 kDa, pI 6; acc. no. AAA60193). A minimum of three matching peptides (bold and underlined) were used to assign an identity to each protein.

View larger version (36K): [in a new window]   Fig. 9. Detection of PECAM-1 in human spermatozoa. The presence of a PECAM-1 homologue in human spermatozoa was confirmed by immunoblotting and immunolocalization using anti-PECAM-1 antibodies. (A) Human sperm and platelets were solubilized in SDS extraction buffer and the proteins resolved by 1D SDS-PAGE before being transferred to nitrocellulose. Blots were successively probed with normal rabbit serum, polyclonal anti-PECAM-1 antibodies and with biotinylated WGA lectin. Cross-reactive bands of approximately 125 kDa were observed in both preparations using the anti-PECAM and WGA probes. (B) A protein band of identical molecular mass was also detected using a panel of three additional anti-PECAM-1 monoclonal antibodies. (C) To confirm the affinity of the sperm PECAM-1 molecule for WGA, this lectin was used to isolate binding partners from a pool of solubilized platelet and sperm proteins. These proteins were resolved by SDS-PAGE, blotted onto nitrocellulose and probed successively with normal rabbit serum and anti-PECAM-1 antibodies. An additional control in which streptavidin beads were incubated directly with solubilized sperm and platelet proteins in the absence of biotinylated WGA was also included to preclude the possibility of non-specific affinity purification. (D) Histochemical staining of live human spermatozoa with anti-PECAM-1 and WGA revealed the respective antigens were present on the majority of the surface of greater than 90% of spermatozoa.

View larger version (54K): [in a new window]   Fig. 10. Investigation of the role of sperm PECAM-1 in WGA induced signalling. To investigate the role of sperm PECAM-1 as a functional receptor for WGA signalling in human spermatozoa we examined whether anti-PECAM-1 antibodies could suppress the response to WGA. For this purpose, human spermatozoa were incubated with either anti-PECAM-1 antibodies alone or with anti-PECAM-1 antibodies followed by WGA. Control treatments in which sperm were incubated with normal rabbit serum were also included. Each incubation was conducted for a period of 30 minutes. After incubation solubilized sperm proteins were prepared for immunodetection of phosphotyrosine residues (anti-py). Blots were then stripped and reprobed with anti--tubulin to ensure equivalent protein loading across all treatments. The experiment was replicated three times with pooled semen samples obtained from at least three different donors and representative blots are depicted.

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