Under arrest!

During the development of a vertebrate oocyte, there's a pause in metaphase of meiosis II to prevent parthenogenetic activation — only when the egg has been fertilized can development resume. This arrest is maintained by the so-called 'cytostatic factor' (CSF) and, reporting in Nature, Julie Reimann and Peter Jackson now describe the long-sought mediator of CSF activity.

The existence of the CSF was proposed by Yoshio Masui and Clement Markert in 1971. Then, in 1989, George Vande Woude and colleagues showed that the Mos signalling pathway is a critical component of CSF activity. Subsequent studies found that Mos is indeed needed to establish CSF arrest, but that it is not required to maintain it.

Enter Emi1. Last year, Jackson's group showed that this protein inhibits the anaphase-promoting complex (APC)/Cdc20 in Xenopus laevis. This is significant because the APC-mediated degradation of cyclin B/Cdc2 is the trigger that releases eggs from metaphase arrest. Cdc20 fits into this picture because it is required for the activation of APC after fertilization (see diagram).

Fertilization leads to an increase in the levels of cytoplasmic Ca²⁺ and, ultimately, release from CSF-mediated arrest. So, to test whether Emi1 is indeed a component of CSF, the authors checked whether its overexpression is enough to prevent release from metaphase arrest in the presence of Ca²⁺. Sure enough, the addition of purified Emi1 to arrested eggs ('CSF extracts') prevented the Ca²⁺-induced destruction of cyclin B. The authors then showed that this effect did not require the Mos pathway.

Reimann and Jackson next asked whether Emi1 is needed to maintain the metaphase arrest. They depleted Emi1 from CSF extracts using magnetic beads coupled to anti-Emi1 antibodies, and observed the destruction of cyclin B and release from arrest in these extracts. When purified Emi1 was added back to these extracts, the remaining cyclin B was stabilized and metaphase arrest resumed. Moreover, pre-incubation with a carboxy-terminal fragment of Emi1, which can inhibit the activation of APC, rescued cyclin B stability and metaphase arrest, indicating that these effects indeed occur through APC.

If Emi1 inhibits the Cdc20-mediated activation of APC, then addition of excess Cdc20 might be expected to override the inhibitor effect of Emi1. And this is what the authors saw — Cdc20 induced the spontaneous degradation of cyclin B in a dose-dependent fashion. But what acts upstream of Cdc20? And how is the Emi1-dependent inhibition removed after fertilization?

On fertilization, the Ca²⁺ signal is transduced by calmodulin-dependent protein kinase II (CaMKII). So the authors wondered whether the activation of CaMKII might lead to a change in the interaction between Emi1 and Cdc20. They found that, just 2.5 minutes after the addition of Ca²⁺, the binding of endogenous Emi1 and Cdc20 to one another was blocked. This was accompanied by a rapid increase in the electrophoretic mobility of Cdc20, which is consistent with dephosphorylation.

Is the mystery finally solved then? Not quite. We still do not know how Emi1 is activated during oocyte maturation, or whether it is involved at an earlier stage too (possibly under the control of the Mos pathway). But this study takes us a big step closer to understanding this very elusive factor.