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

Abstract

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.

Artificial activation of mature unfertilized eggs in the malaria vector mosquito, Anopheles stephensi (Diptera, Culicidae)

“…The phosphorylation status of the MEK/MAPK pathway is one of the indicators of egg meiotic arrest and its resumption occurs following egg activation. Our present results indicated that the resumption of meiosis was linked to the MEK/MAPK pathway in A. stephensi, similar to other organisms examined so far (Tachibana et al, 2000;Kondoh et al, 2006;Sensui et al, 2012). In most vertebrates, MEK and MAPK are phosphorylated by the presence of Mos -a key component of cytostatic factor -in mature oocytes, and meiosis is arrested at meiotic metaphase II before fertilization.…”

Release from meiotic arrest in ascidian eggs requires the activity of two phosphatases but not CaMKII

“…2B″). This has also been observed in a different species of ascidian (Sensui et al, 2012). In order to extend this finding, we inhibited Cdk1 activity in fertilised eggs treated with CsA and scored for pronucleus formation, an event that is dependent on the loss of the Mos/MEK/MAPK pathway in ascidians (Dumollard et al, 2011).…”

“…Mos degradation following loss of Cdk1 activity nicely demonstrates how the upstream activating kinase (Mos) is destroyed (Castro et al, 2001;Frank-Vaillant et al, 2001). We and others have also found that maintaining Cdk1 active keeps MAPK active (Sensui et al, 2012). However, our data do not explain how MAPK is dephosphorylated, and this is crucial because phosphorylated, and therefore active, MAPK mediates the effect of the Mos/MEK/MAPK cascade.…”

“…Thus, we conclude that the main role of CN is to activate the APC/C following the spermtriggered Ca 2+ increase, and that loss of Cdk1 activity in turn is necessary for the loss of MAPK activity and efficient meiotic exit. oscillations are maintained during meiosis II owing to a feedback loop from both Cdk1 and MAPK (Levasseur and McDougall, 2000;Dumollard et al, 2011;Sensui et al, 2012). For example, exogenous expression of Δ90 cyclin B or Mos is capable of maintaining spermtriggered Ca 2+ oscillations long after they would normally cease (Levasseur and McDougall, 2000;Sensui et al, 2012).…”

“…oscillations are maintained during meiosis II owing to a feedback loop from both Cdk1 and MAPK (Levasseur and McDougall, 2000;Dumollard et al, 2011;Sensui et al, 2012). For example, exogenous expression of Δ90 cyclin B or Mos is capable of maintaining spermtriggered Ca 2+ oscillations long after they would normally cease (Levasseur and McDougall, 2000;Sensui et al, 2012). To determine whether maintaining endogenous Cdk1 or MAPK activity would also maintain sperm-triggered Ca 2+ oscillations, we measured intracellular Ca 2+ concentrations after fertilisation when CN was inhibited by CsA.…”