The natural arrest of vertebrate unfertilized eggs in second meiotic metaphase results from the activity of cytostatic factor (CSF). The product of the c-mos(xe) proto-oncogene is thought to be a component of CSF and can induce metaphase arrest when injected into blastomeres of two-cell embryos. The c-Mos(xe) protein can directly activate the mitogen-activated protein kinase kinase (MAP kinase kinase) in vitro, leading to activation of MAP kinase. MAP kinase and c-Mos(xe) are active in unfertilized eggs and are rapidly inactivated after fertilization. Microinjection of thiophosphorylated MAP kinase into one blastomere of a two-cell embryo induced metaphase arrest similar to that induced by c-Mos(xe). However, only arrest with c-Mos(xe) was associated with activation of endogenous MAP kinase. These results indicate that active MAP kinase is a component of CSF in Xenopus and suggest that the CSF activity of c-Mos(xe) is mediated by MAP kinase.

In Xenopus oocytes, the c‐ mos proto‐oncogene product has been proposed to act downstream of progesterone to control the entry into meiosis I, the transition from meiosis I to meiosis II, which is characterized by the absence of S phase, and the metaphase II arrest seen prior to fertilization. Here, we report that inhibition of Mos synthesis by morpholino antisense oligonucleotides does not prevent the progesterone‐induced initiation of Xenopus oocyte meiotic maturation, as previously thought. Mos‐depleted oocytes complete meiosis I but fail to arrest at metaphase II, entering a series of embryonic‐like cell cycles accompanied by oscillations of Cdc2 activity and DNA replication. We propose that the unique and conserved role of Mos is to prevent mitotic cell cycles of the female gamete until the fertilization in Xenopus , starfish and mouse oocytes.

We previously reported that immunodepletion of Greatwall kinase prevents Xenopus egg extracts from entering or maintaining M phase due to the accumulation of inhibitory phosphorylations on Thr14 and Tyr15 of Cdc2. M phase–promoting factor (MPF) in turn activates Greatwall, implying that Greatwall participates in an MPF autoregulatory loop. We show here that activated Greatwall both accelerates the mitotic G2/M transition in cycling egg extracts and induces meiotic maturation in G2-arrested Xenopus oocytes in the absence of progesterone. Activated Greatwall can induce phosphorylations of Cdc25 in the absence of the activity of Cdc2, Plx1 (Xenopus Polo-like kinase) or mitogen-activated protein kinase, or in the presence of an activator of protein kinase A that normally blocks mitotic entry. The effects of active Greatwall mimic in many respects those associated with addition of the phosphatase inhibitor okadaic acid (OA); moreover, OA allows cycling extracts to enter M phase in the absence of Greatwall. Taken together, these findings support a model in which Greatwall negatively regulates a crucial phosphatase that inhibits Cdc25 activation and M phase induction.

The tyrosine phosphorylation/dephosphorylation of p34cdc2 was estimated by immunoblotting with antiphosphotyrosine antibody during meiotic maturation of Xenopus oocytes. At the time of germinal vesicle breakdown (GVBD), p34cdc2 is tyrosine dephosphorylated whereas a p42 protein, which might correspond to a MAP2 kinase, becomes tyrosine phosphorylated. No modification in the level of tyrosine phosphorylation of either proteins was noticed during the whole maturation process from GVBD until metaphase H. When added to prophase oocytes, 6-DMAP (6-dimethyl-aminopurine) blocks GVBD, M-phase-promoting factor (MPF) activation and Hl-histone, kinase activation induced by either progesterone, MPF transfer or okadaic acid microinjection. In each case, the tyrosine déphosphorylation reaction of p34cdc2 is inhibited. In meiosis I oocytes (just after the initiation of GVBD), 6-DMAP provokes the rephosphorylation of p34cdc2 on tyrosine residue(s), inactivation of MPF and Hl-histone kinase and re-entry of the cell into an interphase-like state. These processes are reversible by simply removing the agent. In contrast to the observations in prophase oocytes, okadaic acid is able to reverse the inhibitory effect of 6-DMAP in meiosis I oocytes on MPF and Hl-histone kinase activities and to initiate dephosphorylation of p34cdc2 on tyrosyl residue(s) even in the presence of 6-DMAP. Altogether, our results show that 6-DMAP and okadaic acid antagonistically control in vivo the level of tyrosine phosphorylation of p34cdc2.

During oogenesis, oocytes are arrested in prophase and resume meiosis by activating the kinase Cdk1 upon hormonal stimulation. In all vertebrates, release from prophase arrest relies on protein kinase A (PKA) downregulation and on the dephosphorylation of a long sought but still unidentified substrate. Here we show that ARPP19 is the PKA substrate whose phosphorylation at serine 109 is necessary and sufficient for maintaining Xenopus oocytes arrested in prophase. By downregulating PKA, progesterone, the meiotic inducer in Xenopus, promotes partial dephosphorylation of ARPP19 that is required for the formation of a threshold level of active Cdk1. Active Cdk1 then initiates the MPF autoamplification loop that occurs independently of both PKA and ARPP19 phosphorylation at serine 109 but requires the Greatwall (Gwl)-dependent phosphorylation of ARPP19 at serine 67. Therefore, ARPP19 stands at a crossroads in the meiotic M-phase control network by integrating differential effects of PKA and Gwl, two kinases essential for meiosis resumption.