To generate haploid gametes, cohesin is removed in a stepwise manner from chromosome arms in meiosis I and the centromere region in meiosis II to segregate chromosomes and sister chromatids, respectively. Meiotic cohesin removal requires cleavage of the meiosis-specific kleisin subunit Rec8 by the protease separase.(1,2) In yeast and C. elegans, Rec8 on chromosome arms has to be phosphorylated to be cleaved in meiosis I,(3-7) whereas Rec8 at the centromere is protected from cleavage by the action of PP2A-B56.(8-10) However, in mammalian meiosis, it is unknown whether Rec8 has to be equally phosphorylated for cleavage, and if so, the identity of the relevant kinase(s). This is due to technical challenges, as Rec8 is poorly conserved, preventing a direct translation of the knowledge gained from model systems such as yeast and C. elegans to mammals. Additionally, there is no turnover of Rec8 after cohesion establishment, preventing phosphomutant analysis of functional Rec8. To address the very basic question of whether Rec8 cleavage requires its phosphorylation in mammals, we adapted a biosensor that detects separase activity to study Rec8 cleavage in single mouse oocytes by live imaging. Crucially, through phosphomutant analysis, we identified phosphorylation sites in Rec8 promoting cleavage. We found that Rec8 cleavage depends on Aurora B/C kinase activities and identified an aminoacid residue that is phosphorylated in vivo. Accordingly, inhibition of Aurora B/C kinases during meiotic maturation impairs endogenous Rec8 phosphorylation and chromosome segregation.

Oocytes are held in meiotic prophase for prolonged periods until hormonal signals trigger meiotic divisions. Key players of M-phase entry are the opposing Cdk1 kinase and PP2A-B55δ phosphatase. In Xenopus, the protein Arpp19, phosphorylated at serine 67 by Greatwall, plays an essential role in inhibiting PP2A-B55δ, promoting Cdk1 activation. Furthermore, Arpp19 has an earlier role in maintaining the prophase arrest through a second serine (S109) phosphorylated by PKA. Prophase release, induced by progesterone, relies on Arpp19 dephosphorylation at S109, owing to an unknown phosphatase. Here, we identified this phosphatase as PP2A-B55δ. In prophase, PKA and PP2A-B55δ are simultaneously active, suggesting the presence of other important targets for both enzymes. The drop in PKA activity induced by progesterone enables PP2A-B55δ to dephosphorylate S109, unlocking the prophase block. Hence, PP2A-B55δ acts critically on Arpp19 on two distinct sites, opposing PKA and Greatwall to orchestrate the prophase release and M-phase entry.

The study of oocytes has made enormous contributions to the understanding of the G(2)/M transition. The complementarity of investigations carried out on various model organisms has led to the identification of the M-phase promoting factor (MPF) and to unravel the basis of cell cycle regulation. Thanks to the power of biochemical approaches offered by frog oocytes, this model has allowed to identify the core signaling components involved in the regulation of M-phase. A central emerging layer of regulation of cell division regards protein translation. Oocytes are a unique model to tackle this question as they accumulate large quantities of dormant mRNAs to be used during meiosis resumption and progression, as well as the cell divisions during early embryogenesis. Since these events occur in the absence of transcription, they require cascades of successive unmasking, translation, and discarding of these mRNAs, implying a fine regulation of the timing of specific translation. In the last years, theXenopusgenome has been sequenced and annotated, enabling the development of omics techniques in this model and starting its transition into the genomic era. This review has critically described how the different phases of meiosis are orchestrated by changes in gene expression. The physiological states of the oocyte have been described together with the molecular mechanisms that control the critical transitions during meiosis progression, highlighting the connection between translation control and meiosis dynamics.

Cell division is orchestrated by the phosphorylation and dephosphorylation of thousands of proteins. These post-translational modifications underlie the molecular cascades converging to the activation of the universal mitotic kinase, Cdk1, and entry into cell division. They also govern the structural events that sustain the mechanics of cell division. While the role of protein kinases in mitosis has been well documented by decades of investigations, little was known regarding the control of protein phosphatases until the recent years. However, the regulation of phosphatase activities is as essential as kinases in controlling the activation of Cdk1 to enter M-phase. The regulation and the function of phosphatases result from post-translational modifications but also from the combinatorial association between conserved catalytic subunits and regulatory subunits that drive their substrate specificity, their cellular localization and their activity. It now appears that sequential dephosphorylations orchestrated by a network of phosphatase activities trigger Cdk1 activation and then order the structural events necessary for the timely execution of cell division. This review discusses a series of recent works describing the important roles played by protein phosphatases for the proper regulation of meiotic division. Many breakthroughs in the field of cell cycle research came from studies on oocyte meiotic divisions. Indeed, the meiotic division shares most of the molecular regulators with mitosis. The natural arrests of oocytes in G2 and in M-phase, the giant size of these cells, the variety of model species allowing either biochemical or imaging as well as genetics approaches explain why the process of meiosis has served as an historical model to decipher signalling pathways involved in the G2-to-M transition. The review especially highlights how the phosphatase PP2A-B55δ critically orchestrates the timing of meiosis resumption in amphibian oocytes. By opposing the kinase PKA, PP2A-B55δ controls the release of the G2 arrest through the dephosphorylation of their substrate, Arpp19. Few hours later, the inhibition of PP2A-B55δ by Arpp19 releases its opposing kinase, Cdk1, and triggers M-phase. In coordination with a variety of phosphatases and kinases, the PP2A-B55δ/Arpp19 duo therefore emerges as the key effector of the G2-to-M transition.

Journal of Cell Science was made aware by a reader of issues regarding background cloning in Fig. 4D in J. Cell Sci. (2013) 126, 3916-3926 (doi:10.1242/jcs.126599).The journal contacted the authors, who said that keratin spots in two control lanes of Fig. 4D had been blurred. After discussion with the corresponding author, Olivier Haccard, the journal referred this matter to Université Pierre et Marie Curie (UPMC, now Sorbonne Université). The UPMC committee decided that the conclusions of the paper were not affected by the error and recommended correction of the paper (full reports available at: http://www2.cnrs.fr/sites/communique/fichier/rapport_conclusions.pdf and http://www2.cnrs.fr/sites/communique/fichier/rapport_analyse_detaillee.pdf).The editorial policies of Journal of Cell Science state that: "Should an error appear in a published article that affects scientific meaning or author credibility but does not affect the overall results and conclusions of the paper, our policy is to publish a Correction…" and that a Retraction should be published when "…a published paper contain[s] one or more significant errors or inaccuracies that change the overall results and conclusions of the paper…". Journal of Cell Science follows the guidelines of the Committee on Publication Ethics (COPE), which state: "Retraction should usually be reserved for publications that are so seriously flawed (for whatever reason) that their findings or conclusions should not be relied upon". The standards of figure assembly and data presentation in this paper fall short of good scientific practice. However, given that the investigating committee at UPMC decided that the conclusions of the paper were not affected by the errors, the appropriate course of action – according to COPE guidelines – is to publish a Correction, which the journal has made as detailed as possible.The original blot for Fig. 4D was available and the corrected figure panel is shown below. During analysis of the original data for the UPMC investigating committee, the authors also found a problem with Fig. 2C. The autoradiographic films used did not arise from the samples illustrated in Fig. 2B. Although this error did not affect the overall scientific conclusions, the authors would like to correct this panel as shown below. Readers should note that, in addition to a splice in Fig. S4, which was highlighted by a reader, there are several other instances of unmarked splicing of lanes in western blots in this paper. The authors state that splicing was performed solely for presentation purposes, and provided the journal with all the original data from which the figures were assembled. Although such unindicated splicing is not acceptable by today's standards, it was still widespread in 2013 when this paper was published. Journal of Cell Science guidelines for figure preparation regarding splicing were introduced early in 2013, after this paper was accepted.The authors apologise to the journal and readers for the errors.Journal of Cell Science refers readers to other notices related to the UPMC investigation:doi:10.1242/jcs.166553doi:10.1242/jcs.222240doi:10.1242/jcs.222190doi:10.1242/dev.169573

Entry into mitosis and meiosis is orchestrated by the phosphorylation of thousands of mitotic substrates under the control of active Cdk1-cyclin B complexes. To avoid futile cycles of phosphorylation/dephosphorylation, the specific Cdk1-antagonizing phosphatase, PP2A-B55δ, must be simultaneously inactivated. This process is achieved by the activation of the kinase Greatwall (Gwl), which phosphorylates ARPP19. Gwlphosphorylated ARPP19 then inactivates PP2A-B55δ to allow Cdk1 activation as well as to secure the phosphorylation state of mitotic substrates. This chapter discusses a series of recent works showing that ARPP19 is also phosphorylated by another kinase, PKA. Phosphorylated by PKA, ARPP19 arrests Xenopus oocytes in G2 before the first meiotic division. Therefore, depending on its phosphorylation state by either PKA or Gwl, ARPP19 either restrains or activates Cdk1 in Xenopus oocytes. Beyond the understanding of the mechanisms of meiotic and mitotic cell division, the control of ARPP19 by its dual phosphorylation enlightens the cAMP-regulated signalization pathways that control vital functions in numerous eukaryotic cell types.

There was an error published in J. Cell Sci. (2015) 128 , [2482-2496][1] ([doi: 10.1242/jcs.166553][2]). In [Fig. 4B][3], the western blot used to illustrate the elution profile of Cdk1 in the control was mistakenly used a second time to illustrate the samples incubated with anti-Cdc6 antibody.

The small protein ARPP19 plays a dual role during oocyte meiosis resumption. In Xenopus, ARPP19 phosphorylation at S109 by PKA is necessary for maintaining oocytes arrested in prophase of the first meiotic division. Progesterone downregulates PKA, leading to the dephosphorylation of ARPP19 at S109. This initiates a transduction pathway ending with the activation of the universal inducer of M-phase, the kinase Cdk1. This last step depends on ARPP19 phosphorylation at S67 by the kinase Greatwall. Hence, phosphorylated by PKA at S109, ARPP19 restrains Cdk1 activation while when phosphorylated by Greatwall at S67, ARPP19 becomes an inducer of Cdk1 activation. Here, we investigate the functional interplay between S109 and S67-phosphorylations of ARPP19. We show that both PKA and Gwl phosphorylate ARPP19 independently of each other and that Cdk1 is not directly involved in regulating the biological activity of ARPP19. We also show that the phosphorylation of ARPP19 at S67 that activates Cdk1, is dominant over the inhibitory S109 phosphorylation. Therefore our results highlight the importance of timely synchronizing ARPP19 phosphorylations at S109 and S67 to fully activate Cdk1.

Entry into mitosis and meiosis is orchestrated by the phosphorylation of thousands of mitotic substrates under the control of active Cdk1-cyclin B complexes. To avoid futile cycles of phosphorylation/dephosphorylation, the specific Cdk1-antagonizing phosphatase, PP2A-B55δ, must be simultaneously inactivated. This process is achieved by the activation of the kinase Greatwall (Gwl), which phosphorylates ARPP19. Gwl-phosphorylated ARPP19 then inactivates PP2A-B55δ to allow Cdk1 activation as well as to secure the phosphorylation state of mitotic substrates. This chapter discusses a series of recent works showing that ARPP19 is also phosphorylated by another kinase, PKA. Phosphorylated by PKA, ARPP19 arrests Xenopus oocytes in G2 before the first meiotic division. Therefore, depending on its phosphorylation state by either PKA or Gwl, ARPP19 either restrains or activates Cdk1 in Xenopus oocytes. Beyond the understanding of the mechanisms of meiotic and mitotic cell division, the control of ARPP19 by its dual phosphorylation enlightens the cAMP-regulated signalization pathways that control vital functions in numerous eukaryotic cell types.

Vertebrate oocytes proceed through the first and the second meiotic division without an intervening S-phase to become haploid. Although DNA replication does not take place, unfertilized oocytes acquire the competence to replicate DNA one hour after the first meiotic division by accumulating an essential factor of the replicative machinery, Cdc6. Here, we show that the turnover of Cdc6 is precisely regulated in oocytes to avoid inhibition of Cdk1. At meiosis resumption, Cdc6 is expressed but cannot accumulate owing to a degradation mechanism that is activated through Cdk1. During transition from the first to the second meiotic division, Cdc6 is under the antagonistic regulation of B-type cyclins (which interact with and stabilize Cdc6) and the Mos-MAPK pathway (which negatively controls Cdc6 accumulation). Because overexpressing Cdc6 inhibits Cdk1 reactivation and drives oocytes into a replicative interphasic state, the fine-tuning of Cdc6 accumulation is essential to ensure two meiotic waves of Cdk1 activation and to avoid unscheduled DNA replication during meiotic maturation.

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.

Entry into mitosis or meiosis relies on the coordinated action of kinases and phosphatases that ultimately leads to the activation of Cyclin B-Cdk1, also called MPF for M-phase promoting factor. Vertebrate oocytes are blocked in prophase of the first meiotic division, an arrest tightly controlled by a high PKA activity. Reentry into meiosis depends on Cdk1 activation that obeys a two steps mechanism: a catalytic amount of Cdk1 is generated in a PKA and protein synthesis-dependent manner; then a regulatory network called MPF auto-amplification loop is initiated. This second step is independent of PKA and protein synthesis. However, none of the molecular components of the auto-amplification loop identified so far acts independently of PKA. Therefore, the protein rendering this process independent of PKA in oocytes remains unknown. Using a physiological intact cell system, the Xenopus oocyte, we show that the phosphorylation of ARPP19 at S67 by the Greatwall kinase promotes its binding to the PP2A-B55 δ phosphatase, thus inhibiting its activity. This process is controlled by Cdk1 and plays an essential role within the Cdk1 auto-amplification loop for entry into the first meiotic division. Moreover, once phosphorylated by Greatwall, ARPP19 escapes the negative regulation exerted by PKA. It also promotes MPF activation independently of protein synthesis, provided a small amount of Mos is present. Taken together, these findings reveal that PP2A-B55δ, Greatwall and ARPP19 are not only required for entry into meiotic divisions, but are also pivotal effectors within the Cdk1 auto-regulatory loop responsible for its independence toward PKA negative control.

In the ovary, oocytes are surrounded by follicle cells and arrested in prophase of meiosis I. Although steroidogenic activity of follicle cells is involved in oogenesis regulation, clear qualitative and quantitative data about the steroid content of follicles are missing. We measured steroid levels of Xenopus oocytes and follicles by gas chromatography-mass spectrometry. We show that dehydroepiandrosterone sulfate is the main steroid present in oocytes. Lower levels of free steroids are also detected, e.g., androgens, whereas progesterone is almost undetectable. We propose that sulfatation is a protective mechanism against local variations of active steroids that could be deleterious for follicle-enclosed oocytes. Steroid levels were measured after LH stimulation, responsible for the release by follicle cells of a steroid signal triggering oocyte meiosis resumption. Oocyte levels of androgens rise slowly during meiosis re-entry whereas progesterone increases abruptly to micromolar concentration, therefore representing the main physiological mediator of meiosis resumption in Xenopus oocyte.

Ovulated eggs possess maternal apoptotic execution machinery that is inhibited for a limited time. The fertilized eggs switch off this time bomb whereas aged unfertilized eggs and parthenogenetically activated eggs fail to stop the timer and die. To investigate the nature of the molecular clock that triggers the egg decision of committing suicide, we introduce here Xenopus eggs as an in vivo system for studying the death of unfertilized eggs. We report that after ovulation, a number of eggs remains in the female body where they die by apoptosis. Similarly, ovulated unfertilized eggs recovered in the external medium die within 72 h. We showed that the death process depends on both cytochrome c release and caspase activation. The apoptotic machinery is turned on during meiotic maturation, before fertilization. The death pathway is independent of ERK but relies on activating Bad phosphorylation through the control of both kinases Cdk1 and JNK. In conclusion, the default fate of an unfertilized Xenopus egg is to die by a mitochondrial dependent apoptosis activated during meiotic maturation.

In many cell types, the mitogen-activated protein kinase (MAPK) also named extracellular signal-regulated kinase (ERK) is activated in response to a variety of extracellular growth factor-receptor interactions and leads to the transcriptional activation of immediate early genes, hereby influencing a number of tissue-specific biological activities, as cell proliferation, survival and differentiation. In one specific cell type however, the female germ cell, MAPK does not follow this canonical scheme. In oocytes, MAPK is activated independently of growth factors and tyrosine kinase receptors, acts independently of transcriptional regulation, plays a crucial role in controlling meiotic divisions, and is under the control of a peculiar upstream regulator, the kinase Mos. Mos was originally identified as the transforming gene of Moloney murine sarcoma virus and its cellular homologue was the first proto-oncogene to be molecularly cloned. What could be the specific roles of Mos that render it necessary for meiosis? Which unique functions could explain the evolutionary cost to have selected one gene to only serve for few hours in one very specific cell type? This review discusses the original features of MAPK activation by Mos and the roles of this module in oocytes.

Nat. Chem. Biol. 4, 119–125 (2008); published online 6 January 2008; corrected after print 13 February 2009 In the version of this article initially published, the reported chemical structure and systematic name for mirin (compound 1), identified from a high-throughput screen, were incorrectly assigned in the commercial library.