Role of two series of Ca²⁺ oscillations in activation of ascidian eggs

Changes in [Ca²⁺](i) are an essential factor regulating egg activation. Matured ascidian eggs are arrested at metaphase I, and two series of [Ca²⁺](i) transients have been observed after fertilization: Ca²⁺ waves just after fertilization (Series I) and [Ca²⁺](i) oscillation between the first and second polar body extrusion (Series II). We investigated mechanisms involved in the elevation of [Ca²⁺](i) and the role of the [Ca²⁺](i) transients during egg activation in Ciona savignyi. The monoclonal antibody 18A10 against IP₃ receptor type 1, which inhibits IP₃- induced Ca²⁺ release in hamster and mouse eggs, did not show substantial inhibitory effects on series I or egg deformation, whereas Series II and the first cell division were inhibited by the antibody. Ruthenium red, an inhibitor of ryanodine receptor-mediated Ca²⁺ release, had no apparent effect of [Ca²⁺](i) transients and other events related to the egg activation. Microinjection of IP₃ into unfertilized eggs induced [Ca²⁺](i) transients similar to those seen in Series I, whereas injection of cyclic ADP ribose, an agonist of ryanodine receptors, rarely induced [Ca²⁺](i) transient. Adenophostin B, a potent nonmetabolizable agonist of IP₃ receptors, induced [Ca²⁺](i) oscillations which continued after first polar body extrusion, without separation to two series, and led to extrusion of first and second polar bodies. These results suggest that Series II is driven by the mouse type 1-like IP₃ receptor while Series I seems to be mediated by another type of IP₃ receptor. Injection of IP₃ only induced the first polar body extrusion and the egg was arrested at metaphase II even when a higher amount of IP₃ was injected. On the other hand, reinjection of IP₃ after the first polar body extrusion led to emission of the second polar body. Thus, Series I and II of [Ca²⁺](i) transients are likely to be required for metaphase-anaphase transition in meiosis.