Inositol 1,4,5-trisphosphate receptors selectively localized to the acrosomes of mammalian sperm

Calcium flux is required for the mammalian sperm acrosome reaction, an exocytotic event triggered by egg binding, which results in a dramatic rise in sperm intracellular calcium. Calcium-dependent membrane fusion results in the release of enzymes that facilitate sperm penetration through the zona pellucida during fertilization. We have characterized inositol 1,4,5- trisphosphate (IP₃)-gated calcium channels and upstream components of the phosphoinositide signaling system in mammalian sperm. Peptide antibodies colocalized G{aq/11) and the β1 isoform of phospholipase C (PLCβ1) to the anterior acrosomal region of mouse sperm. Western blotting using a polyclonal antibody directed against purified brain IP₃ receptor (IP₃R) identified a specific 260 kD band in 1% Triton X-100 extracts of rat, hamster, mouse and dog sperm. In each species, IP₃R immunostaining localized to the acrosome cap. Scatchard analysis of [³H]IP₃ binding to rat sperm sonicates revealed a curvilinear plot with high affinity (K(d) = 26 nM, B(max) = 30 pmol/mg) and low affinity (K(d) = 1.6 μM,B(max) = 550 pmol/mg) binding sites, reflecting among the highest receptor densities in mammalian tissue. Immunoelectron microscopy confirmed the acrosomal localization in rat sperm. The IP₃R fractionated with acrosomes by discontinuous sucrose gradient centrifugation and was enriched in the medium of acrosome-reacted sperm. ATP-dependent ⁴⁵Ca²⁺ loading of digitonin permeabilized rat sperm was decreased by 45 % in the presence of 10 μM IP₃. The IP₃-mediated release of calcium was blocked by heparin. Thapsigargin, a sequiterpene lactone inhibitor of the microsomal Ca²⁺-ATPase, stimulated the acrosome reaction of mouse sperm to the same extent as the Ca²⁺ ionophore, A23187. The failure of caffeine and ryanodine to affect calcium accumulation suggested that thapsigargin acted through an IP₃-sensitive store. The presence of G(αq/11), PLCβ1 and a functional IP₃R in the anterior acrosomal region of mammalian sperm, as well as thapsigargin's induction of the acrosome reaction, implicate IP₃-gated calcium release in the mammalian acrosome reaction.