Intracellular Ca2+ during fertilization and artificial activation in mouse oocytes

AIM: To study the mechanism of oocyte activation in mammals. METHODS: Mouse oocytes arrested at metaphase of the second meiotic division were loaded with Fura 2-AM and then activated with ethanol, calcimycin, electric pulse or fertilization. Intracellular free Ca2+ during activation were measured by Spex AR-CM-MIC cation system. Cortical granule exocytosis after' activation was detected under electron microscopy. RESULTS: Sperm penetration initiated a long lasting Ca2+ oscillation in Ca2+ containing IVF medium in mouse ova. The Ca2+ oscillation lasted for over 3 - 4 h until the ova developed to pronuclear stage. The Ca2+ oscillated faster as extracellular Ca2+ concentration was increased from normal 1.7 mmol . L(-1) to 5.0 mmol . L(-1) and ceased to oscillate when extracellular Ca2+ was removed. The ova resumed Ca2+ oscillation after transferred back to IVF (Ca2+ 1.7 mmol . L(-1)). The ova which exhibited Ca2+ oscillation all extruded second polar body and formed pronuclei. Suppression the Ca2+ oscillation by intracellular injection of egtazic acid (2 - 10 pL, 200 mu mol . L(-1)) blocked the activation of oocytes. Heparin (100 mu mol ., L(-1)) injection failed to prevent the Ca2+ oscillation. In artificial activation, ethanol, calcimycin, and a single electric pulse usually induced a monotonic Ca2+ rise and resulted in the activation only in older oocytes (>18 h after CG injection). Activation of freshly ovulated oocytes required multiple intracellular Ca2+ increases induced by repetitive electric pulses. Artificial activation elicited the similar cortical granule exocytosis in oocytes as occuring at fertilization. Suppression of the intracellular Ca2+ elevation by introduction of egtazic acid into the oocytes blocked the activation process. CONCLUSIONS: The increase of intracellular free Ca2+ is the primary signal responsible for the initiation of oocyte activation but there are distinct differences between fertilization and artificial activation both in Ca2+ change patterns and Ca2+. sources. Young oocytes require oscillatory Ca2+ signals for activation.