INOSITOL TRISPHOSPHATE-INDUCED CALCIUM-RELEASE IN THE GENERATION OF CALCIUM OSCILLATIONS IN BOVINE EGGS

Bovine eggs exhibit repetitive rises in intracellular calcium concentration ([Ca2+](i)) in response to fertilization. The signaling pathways and Ca-2 release mechanisms involved in their generation are not well characterized. This study examined the presence of a GTP-binding protein (G-protein) signaling pathway as well as the role of inositol 1,4,5-trisphosphate (InsP3R) receptor (InsP3R)-mediated Ca2+ release and ryanodine receptor (RyR)-mediated Ca2+ release, the two Ca2+ receptors/channels most often thought to participate in the generation of [Ca2+](i) oscillations, by injecting appropriate agonists and antagonists and monitoring their effects on Ca2+ release and pronucleus formation. Injection of guanosine 5'-0-(3-thiotriphosphate) (GTP gamma[S]), which promotes G-protein-mediated phosphoinositide turnover, induced, at high concentrations, repetitive [Ca2+](i) rises. Low concentrations of GTP gamma[S] were ineffective. Injection of inositol trisphosphorothioate (lnsP3S3),a nonmetabolizable analogue of InsP3, evoked an immediate Ca2+ release followed by [Ca2+](i) oscillations. The GTP gamma[S]- and InsP3S3-induced oscillations showed a rapid attenuation in amplitude and were terminated in about 30-60 min. Thimerosal, a thiol oxidizing agent, caused repetitive rises in [Ca2+](i) by sensitizing Ca2+ injection-induced Ca2+ release. Injection of ryanodine, which stimulates Ca2+-induced Ca2+ release via the RyR, did not induce [Ca2+](i) oscillations; and eggs into which it was preinjected exhibited normal [Ca2+](i) oscillations in response to thimerosal. Preinjection of heparin, a competitive InsP3R antagonist, blocked in a dose-dependent manner the Ca2+ response to InsP3 and thimerosal, and its injection into fertilized oscillating eggs inhibited [Ca2+](i) oscillations in all eggs. Heparin also inhibited pronucleus formation after insemination. Preinjection of guanosine 5'-0-(2-thiodiphosphate) (GDP beta[S]), a G-protein antagonist, had similar effects. Together, these results suggest that the InsP3R-mediated Ca2+ release mechanism plays an important role in the generation of fertilization-associated [Ca2+](i) oscillations and egg activation in bovine eggs. The data also suggest the participation of a G-protein pathway during fertilization.