| There is increasing evidence that complement components might play a role in fertilization. C1q, the first component of the classical complement cascade, has the ability to promote sperm agglutination in a capacitation-dependent manner as well as having an effect on sperm-oolemma binding and fusion. We have previously detected gC1qR, the receptor for the globular heads of C1q, on the surface of capacitated sperm. Here we describe an apparent increase in receptor expression over the rostral portion of the sperm head occurring after capacitation using Western blot techniques, flow cytometric analysis and confocal imaging of immunofluorescent stained human sperm. In addition, the ability of live spermatozoa to bind to both C1q and monoclonal anti-gC1qaR antibody coated microtiter wells was also increased after capacitation. Since these results suggest that gC1qR may play a role in human fertilization, we sought to determine the role of gC1qR in the Ca2+-mobilization and signal transduction events occurring after capacitation. Spectrofluorimetric analysis of fluo-3 loaded capacitated human spermatozoa, collected from known fertile donors, reveals a sharp and sustained increase in intracellular calcium when sperm surface gC1qR is cross-linked with 100 mug/ml polyclonal antibody or aggregated with 22.5 mug/ml of its multivalent ligand, C1q. Since a cytoplasmic calcium increase in capacitated sperm is accompanied by protein tyrosine phosphorylation during the acrosome reaction, we performed Western blotting analysis and showed that both treatments lead to the increase in protein tyrosine phosphorylation of 4 proteins of apparent molecular weights 65, 89, 97 and 120 kDa, respectively. Furthermore, ELISA studies illustrate an increase in serine and threonine phosphorylation. Cell signaling via gC1qR may be transduced to the cytoplasm through the alphavbeta3 integrin since antigen capture ELISA using anti-gC1qR monoclonal antibody was able to pull down both this integrin as well as its ligand, vitronectin, from solubilized, capacitated sperm. Finally, the increase in intracellular calcium resulting from gC1qR ligation is sufficient to initiate the acrosome reaction. The addition of anti-gC1qR polyclonal antibody or C1q leads to a loss of acrosome as assessed by both fluorescein isothiocyanate-conjugated Pisum sativum agglutinin staining and flow cytometric analysis of the acrosomal marker CD46. Neither non-immune control nor heat inactivated C1q caused any significant change in acrosomal status. Taken together, our data implicate gC1qR as a novel candidate molecule regulating signal transduction events that precede the acrosome reaction. |
