Monoclonal antibodies increase intracellular Ca2+ in sea urchin spermatozoa

Changes in intracellular free Ca2+ ([Ca2+]i) of sea urchin (Strongylocentrotus purpuratus) spermatozoa were measured using the fluorescent Ca2+ indicators fura-2 and indo-1. The intracellular pH (pHi) of sperm was also determined. The fucose sulfate-rich glycoconjugate component of egg jelly induced increases in [Ca2+]i and pHi in sperm and induced the acrosome reaction. Monoclonal antibodies (mAbs) to external domains of a 210-kDa glycoprotein of the sperm plasma membrane induced a 23-fold increase in [Ca2+]i (vs. 9-fold for fucose sulfate-rich glycoconjugate), but the mAbs did not cause the pHi to increase and did not induce the acrosome reaction. When the mAb treatment which induced an increase in [Ca2+]i was combined with an NH4Cl treatment, which increased the pHi, the acrosome reaction was induced. mAb-induced increases in [Ca2+]i were dependent on millimolar concentrations of extracellular Ca2+ and were reversed by placing sperm in Ca2+-free seawater or by chelating Ca2+ with EGTA. The mAb-induced [Ca2+]i increase was sensitive to the pH of the seawater, although mAb binding was not. The data show that increased [Ca2+]i and pHi are necessary for induction of the acrosome reaction and suggest that the 210-kDa protein may play a role in regulating Ca2+ entry into the spermatozoan. These mAbs make it possible to separate the increase in [Ca2+]i from the increase in pHi and may be useful in the elucidation of the regulatory role of Ca2+ in sperm physiology.