Role of Mos/MEK/ERK cascade and Cdk1 in Ca2+ oscillations in fertilized ascidian eggs.

Intracellular calcium ion concentration ([Ca(2+)](i)) transients are observed in the fertilized eggs of all species investigated so far, and are critical for initiating several events related to egg activation and cell cycle control. Here, we investigated the role of the Mos/MEK/ERK cascade and Cdk1 on Ca(2+) oscillations in fertilized ascidian eggs. The egg of the ascidian Phallusia nigra shows [Ca(2+)](i) oscillations after fertilization: Ca(2+) waves immediately following fertilization (phase I), and [Ca(2+)](i) oscillations between the first and second polar body extrusions (phase II). Our results show that in P. nigra eggs, ERK activity peaked just before the extrusion of the first polar body, and decreased gradually, eventually disappearing at the extrusion of the second polar body. Cyclin-dependent protein kinase 1(Cdk1) activity decreased to undetectable levels immediately after fertilization, and then periodically increased according to the meiotic and mitotic cell cycle. When the unfertilized eggs were incubated with U0126, an inhibitor of MEK, before insemination, ERK was immediately inactivated, and the phase II [Ca(2+)](i) oscillations disappeared. Alternatively, when the constitutively active Mos protein (GST-Mos) was injected into the unfertilized eggs, ERK activity was preserved for at least 120 min after fertilization, and the phase II [Ca(2+)](i) oscillations lasted for more than 120 min after the second polar body extrusion. These results suggest that ERK activity is necessary for maintaining [Ca(2+)](i) oscillations. GST-ΔN85-cyclin, which maintains Cdk1 activity, caused ERK activity in the eggs to persist for over 120 min after fertilization, and prolonged [Ca(2+)](i) oscillations. Moreover, the effects of GST-ΔN85-cyclin on the egg were abrogated by the application of U0126. Thus, Cdk1-mediated [Ca(2+)](i) oscillations seem to require ERK activity. However, GST-Mos triggered [Ca(2+)](i) oscillations after the second polar body extrusion, whereas GST-ΔN85-cyclin did not, although it prolongs the duration of [Ca(2+)](i) oscillations. Interestingly, GST-ΔN85-cyclin increased the frequency of [Ca(2+)](i) transients in the Mos-induced [Ca(2+)](i) oscillations after the extrusion of the second polar body. Thus, Cdk1 could maintain, but not activate, ERK and [Ca(2+)](i) oscillations. ERK activity and [Ca(2+)](i) oscillations seem to form a negative feedback loop which may be responsible for maintaining the meiotic period.