Aurora B/C-dependent phosphorylation promotes Rec8 cleavage in mammalian oocytes.

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.

The M-phase regulatory phosphatase PP2A-B55δ opposes protein kinase A on Arpp19 to initiate meiotic division.

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 G2-to-M transition from a phosphatase perspective: a new vision of the meiotic division.

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.

Translational Control of Xenopus Oocyte Meiosis: Toward the Genomic Era.

The study of oocytes has made enormous contributions to the understanding of the G2/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, the Xenopus genome 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.

The greatwall kinase is dominant over PKA in controlling the antagonistic function of ARPP19 in Xenopus oocytes.

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.

Control of Cdc6 accumulation by Cdk1 and MAPK is essential for completion of oocyte meiotic divisions in Xenopus.

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.

Phosphorylation of ARPP19 by protein kinase A prevents meiosis resumption in Xenopus oocytes.

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.

The phosphorylation of ARPP19 by Greatwall renders the auto-amplification of MPF independently of PKA in Xenopus oocytes.

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 known as MPF for M-phase promoting factor. Vertebrate oocytes are blocked in prophase of the first meiotic division, an arrest that is tightly controlled by high PKA activity. Re-entry into meiosis depends on activation of Cdk1, which obeys a two-step mechanism: a catalytic amount of Cdk1 is generated in a PKA and protein-synthesis-dependent manner; then a regulatory network known as the 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 act independently of PKA. Therefore, the protein rendering this process independent of PKA in oocytes remains unknown. Using a physiologically 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-B55δ phosphatase, thus inhibiting its activity. This process is controlled by Cdk1 and has 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 activation of MPF independently of protein synthesis, provided that 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 with respect to the PKA-negative control.

Naturally occurring steroids in Xenopus oocyte during meiotic maturation. Unexpected presence and role of steroid sulfates.

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.

Unfertilized Xenopus eggs die by Bad-dependent apoptosis under the control of Cdk1 and JNK.

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.

Mos in the oocyte: how to use MAPK independently of growth factors and transcription to control meiotic divisions.

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.

A forward chemical genetic screen reveals an inhibitor of the Mre11-Rad50-Nbs1 complex.

The MRN (Mre11-Rad50-Nbs1)-ATM (ataxia-telangiectasia mutated) pathway is essential for sensing and signaling from DNA double-strand breaks. The MRN complex acts as a DNA damage sensor, maintains genome stability during DNA replication, promotes homology-dependent DNA repair and activates ATM. MRN is essential for cell viability, which has limited functional studies of the complex. Small-molecule inhibitors of MRN could circumvent this experimental limitation and could also be used as cellular radio- and chemosensitization compounds. Using cell-free systems that recapitulate faithfully the MRN-ATM signaling pathway, we designed a forward chemical genetic screen to identify inhibitors of the pathway, and we isolated 6-(4-hydroxyphenyl)-2-thioxo-2,3-dihydro-4(1H)-pyrimidinone (mirin, 1) as an inhibitor of MRN. Mirin prevents MRN-dependent activation of ATM without affecting ATM protein kinase activity, and it inhibits Mre11-associated exonuclease activity. Consistent with its ability to target the MRN complex, mirin abolishes the G2/M checkpoint and homology-dependent repair in mammalian cells.

Rad50 adenylate kinase activity regulates DNA tethering by Mre11/Rad50 complexes.

Mre11 and Rad50 are the catalytic components of a highly conserved DNA repair complex that functions in many aspects of DNA metabolism involving double-strand breaks. The ATPase domains in Rad50 are related to the ABC transporter family of ATPases, previously shown to share structural similarities with adenylate kinases. Here we demonstrate that Mre11/Rad50 complexes from three organisms catalyze the reversible adenylate kinase reaction in vitro. Mutation of the conserved signature motif reduces the adenylate kinase activity of Rad50 but does not reduce ATP hydrolysis. This mutant resembles a rad50 null strain with respect to meiosis and telomere maintenance in S. cerevisiae, correlating adenylate kinase activity with in vivo functions. An adenylate kinase inhibitor blocks Mre11/Rad50-dependent DNA tethering in vitro and in cell-free extracts, indicating that adenylate kinase activity by Mre11/Rad50 promotes DNA-DNA associations. We propose a model for Rad50 that incorporates both ATPase and adenylate kinase reactions as critical activities that regulate Rad50 functions.

The DNA damage response during an unperturbed S-phase.

DNA replication is a highly conserved and controlled process. To maintain genome integrity, the DNA must be faithfully duplicated once before chromosomes are segregated to daughter cells. Experimental insults to cells during DNA replication trigger an array of responses to help cells cope with DNA damage and replication stress. This has been coined the DNA damage response. During an unperturbed S-phase, DNA lesions and aberrant DNA structures arise as a consequence of normal DNA replication. Recent data suggest that the same pathways regulating the response to acute DNA damage also operate during normal S-phase to maintain genome integrity in the face of low levels of damage. This review will focus on the role of key proteins and signaling pathways, originally identified by their requirement to maintain genome stability during DNA replication following experimental insults, in the regulation of progression through normal S-phase.

Two-step activation of ATM by DNA and the Mre11-Rad50-Nbs1 complex.

DNA double-strand breaks (DSBs) trigger activation of the ATM protein kinase, which coordinates cell-cycle arrest, DNA repair and apoptosis. We propose that ATM activation by DSBs occurs in two steps. First, dimeric ATM is recruited to damaged DNA and dissociates into monomers. The Mre11-Rad50-Nbs1 complex (MRN) facilitates this process by tethering DNA, thereby increasing the local concentration of damaged DNA. Notably, increasing the concentration of damaged DNA bypasses the requirement for MRN, and ATM monomers generated in the absence of MRN are not phosphorylated on Ser1981. Second, the ATM-binding domain of Nbs1 is required and sufficient to convert unphosphorylated ATM monomers into enzymatically active monomers in the absence of DNA. This model clarifies the mechanism of ATM activation in normal cells and explains the phenotype of cells from patients with ataxia telangiectasia-like disorder and Nijmegen breakage syndrome.

Xenopus H-RasV12 promotes entry into meiotic M phase and cdc2 activation independently of Mos and p42(MAPK).

In the Xenopus oocyte, progesterone triggers M phase Promoting Factor (MPF) activation in a protein synthesis dependent manner. Although the synthesis of the p42(MAPK) activator Mos appears to be required for MPF activation, p42(MAPK) activity has been shown to be dispensable. To clarify this paradox, we attempted to activate the p42(MAPK) pathway independently of Mos synthesis by cloning and using Xenopus H-Ras in the oocyte. We demonstrate that the injection of the constitutively active Xe H-RasV12 mutant induces p42(MAPK) and MPF activation through two independent pathways. Xe H-RasV12 induces only a partial activation of p42(MAPK) when protein synthesis and MPF activation are prevented. A full level of p42(MAPK) activation is reached when MPF is activated and Mos is present. In contrast, MPF activation induced by Xe H-RasV12 is achieved independently of Mos synthesis and p42(MAPK) activation but still depends on protein synthesis. Therefore, the amphibian oocyte represents a new model system to analyse an original H-Ras pathway ending to MPF activation and distinct from the p42(MAPK) pathway. The identification of the proteins synthesized in response to Xe H-RasV12 and required for MPF activation, represents an important clue in understanding the mechanism of progesterone action.

Mos is not required for the initiation of meiotic maturation in Xenopus oocytes.

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.