Regulation of the G2/M transition in rodent oocytes

Regulation of maturation in meiotically competent mammalian oocytes is a complex process involving the carefully coordinated exchange of signals between the somatic and germ cell compartments of the ovarian follicle via paracrine and cell–cell coupling pathways. This review highlights recent advances in our understanding of how such signaling controls both meiotic arrest and gonadotropin‐triggered meiotic resumption in competent oocytes and relates them to the historical context. Emphasis will be on rodent systems, where many of these new findings have taken place. A regulatory scheme is then proposed that integrates this information into an overall framework for meiotic regulation that demonstrates the complex interplay between different follicular compartments. Mol. Reprod. Dev. 77: 566–585, 2010. © 2010 Wiley‐Liss, Inc.     

Intracellular acidification delays hormonal G2/M transition and inhibits G2/M transition triggered by thiophosphorylated MAPK in Xenopus oocytes

Xenopus oocyte maturation is analogous to G2/M transition and characterized by germinal vesicle breakdown (GVBD), spindle formation, activation of MPF and Mos-Xp42(Mpk1) pathways. It is accompanied prior to GVBD by a transient increase in intracellular pH. We determined that a well known acidifying compound, NH(4)Cl, delayed progesterone-induced GVBD in a dose-dependent manner. GVBD(50) was delayed up to 2.3-fold by 10 mM NH(4)Cl. Cyclin B2 phosphorylation, Cdk1 Tyr15 dephosphorylation as well as p39(Mos) accumulation, Xp42(Mpk1) and p90(Rsk) phosphorylation induced by progesterone were also delayed by incubation of oocyte in NH(4)Cl. The delay induced by NH(4)Cl was prevented by injection of MOPS buffer pH 7.7. In contrast to acidifying medium, alkalyzing treatment such as Tris buffer pH 9 injections, accelerated GVBD, MPF and Xp42(Mpk1) activation, indicating that pHi changes control early steps of G2/M dynamics. When injected in an immature recipient oocyte, egg cytoplasm triggers GVBD through MPF auto-amplification, independently of protein synthesis. In these conditions, GVBD and Xp42(Mpk1) activation were delayed by high concentration of NH(4)Cl, which never prevented or delayed MPF activation. Strickingly, NH(4)Cl strongly inhibited thiophosphorylated active MAPK-induced GVBD and MPF activation. Nevertheless, Tris pH 9 did not have any effects on egg cytoplasm- or active MAPK-induced GVBD. Taken together, our results suggest that dynamic of early events driving Xp42(Mpk1) and MPF activation induced by progesterone may be negatively or positively regulated by pH(i) changes. However Xp42(Mpk1) pathway was inhibited by acidification alone. Finally, MPF auto-amplification loop was not sensitive to pH(i) changes.     

Regulation of the G(2)/M transition in Xenopus oocytes by the cAMP-dependent protein kinase

Vertebrate oocytes are arrested in G(2) phase of the cell cycle at the prophase border of meiosis I. Progesterone treatment of Xenopus oocytes releases the G(2) block and promotes entry into the M phases of meiosis I and II. Substantial evidence indicates that the release of the G(2) arrest requires a decrease in cAMP and reduced activity of the cAMP-dependent protein kinase (PKAc). It has been reported and we confirm here that microinjection of either wild type or kinase-dead K72R PKAc inhibits progesterone-dependent release of the G(2) arrest with equal potency and that inhibition can be reversed by a second injection of the heat-stable inhibitor of PKAc, PKI. However, a mutant enzyme predicted to be completely kinase-dead from the crystal structure of PKAc, K72H PKAc, was much less inhibitory when carrying additional mutations that block interaction with either type I or type II regulatory subunit. Moreover, inhibition by K72H PKAc was reversed by PKI at a 30-fold lower concentration and with more rapid kinetics compared with wild type PKAc. K72R PKAc was found to have low but detectable activity after incubation in an oocyte extract. These results indicate that inhibition of the progesterone-dependent G(2)/M transition in oocytes after microinjection of dead PKAc reflects either low residual activity or binding to regulatory subunits with a resulting net increase in the level of endogenous wild type PKAc. Consistent with this hypothesis, the induction of mitosis in Xenopus egg extracts by the addition of cyclin B was blocked by wild type PKAc but not by K72H PKAc. The identification of substrates for PKAc that maintain cell cycle arrest in G(2) remains an important goal for future work.     

O-linked N-acetylglucosaminyltransferase inhibition prevents G2/M transition in Xenopus laevis oocytes

Full-grown Xenopus oocytes are arrested at the prophase of the first meiotic division in a G(2)-like state. Progesterone triggers meiotic resumption also called the G(2)/M transition. This event is characterized by germinal vesicle breakdown (GVBD) and by a burst in phosphorylation level that reflects activation of M-phase-promoting factor (MPF) and MAPK pathways. Besides phosphorylation and ubiquitin pathways, increasing evidence has suggested that the cytosolic and nucleus-specific O-GlcNAc glycosylation also contributes to cell cycle regulation. To investigate the relationship between O-GlcNAc and cell cycle, Xenopus oocyte, in which most of the M-phase regulators have been discovered, was used. Alloxan, an O-GlcNAc transferase inhibitor, blocked G(2)/M transition in a concentration-dependent manner. Alloxan prevented GVBD and both MPF and MAPK activations, either triggered by progesterone or by egg cytoplasm injection. The addition of detoxifying enzymes (SOD and catalase) did not rescue GVBD, indicating that the alloxan effect did not occur through reactive oxygen species production. These results were strengthened by the use of a benzoxazolinone derivative (XI), a new O-GlcNAc transferase inhibitor. Conversely, injection of O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino-N-phenylcarbamate, an O-GlcNAcase inhibitor, accelerated the maturation process. Glutamine:fructose-6-phosphate amidotransferase inhibitors, azaserine and 6-diazo-5-oxonorleucine, failed to prevent GVBD. Such a strategy appeared to be inefficient; indeed, UDP-GlcNAc assays in mature and immature oocytes revealed a constant pool of the nucleotide sugar. Finally, we observed that cyclin B2, the MPF regulatory subunit, was associated with an unknown O-GlcNAc partner. The present work underlines a crucial role for O-GlcNAc in G(2)/M transition and strongly suggests that its function is required for cell cycle regulation.     

Regulation of human c-Abl tyrosine kinase activity in Xenopus oocytes and acceleration of progesterone-induced G2/M transition by oncogenic forms

Deregulated activity of the Abl protein tyrosine kinase is oncogenic in humans and in animals. The normal cellular form of the enzyme is maintained at a low state of activity by mechanisms that have not yet been entirely elucidated. In particular, little is known about the trans-acting cellular factors involved. We have tested the activity of human c-Abl microinjected into oocytes of Xenopus laevis. In contrast to versions of Abl capable of transforming mammalian cells, which were highly active when introduced into oocytes, the activity of wild type c-Abl was inhibited. Oncogenic forms of Abl efficiently enhanced the ability of Xenopus oocytes to enter M phase following stimulation by progesterone. Abl-enhanced maturation was normal as judged by accumulation of Mos as well as activation of MAP kinase and Cdc2/CyclinB (MPF). Concomitant with maturation and activation of these kinases, Abl became extensively phosphorylated. Altogether, this suggests that an SH3 domain-dependent Abl regulation mechanism similar to the one observed in mammalian cells operates in Xenopus oocytes. Maturation enhancement by microinjection into Xenopus oocytes represents a useful novel assay for analyzing Abl activity. Moreover, the Xenopus oocyte may be a convenient source of trans-acting Abl regulators for biochemical studies.     

CDC6 regulates both G2/M transition and metaphase-to-anaphase transition during the first meiosis of mouse oocytes

Cell division cycle protein, CDC6, is essential for the initiation of DNA replication. CDC6 was recently shown to inhibit the microtubule-organizing activity of the centrosome. Here, we show that CDC6 is localized to the spindle from pro-metaphase I (MI) to MII stages of oocytes, and it plays important roles at two critical steps of oocyte meiotic maturation. CDC6 depletion facilitated the G2/M transition (germinal vesicle breakdown [GVBD]) through regulation of Cdh1 and cyclin B1 expression and CDK1 (CDC2) phosphorylation in a GVBD-inhibiting culture system containing milrinone. Furthermore, GVBD was significantly decreased after knockdown of cyclin B1 in CDC6-depleted oocytes, indicating that the effect of CDC6 loss on GVBD stimulation was mediated, at least in part, by raising cyclin B1. Knockdown of CDC6 also caused abnormal localization of γ-tubulin, resulting in defective spindles, misaligned chromosomes, cyclin B1 accumulation, and spindle assembly checkpoint (SAC) activation, leading to significant pro-MI/MI arrest and PB1 extrusion failure. These phenotypes were also confirmed by time-lapse live cell imaging analysis. The results indicate that CDC6 is indispensable for maintaining G2 arrest of meiosis and functions in G2/M checkpoint regulation in mouse oocytes. Moreover, CDC6 is also a key player regulating meiotic spindle assembly and metaphase-to-anaphase transition in meiotic oocytes.     

Regulation of Cdc25C by ERK-MAP kinases during the G2/M transition

Induction of G(2)/M phase transition in mitotic and meiotic cell cycles requires activation by phosphorylation of the protein phosphatase Cdc25. Although Cdc2/cyclin B and polo-like kinase (PLK) can phosphorylate and activate Cdc25 in vitro, phosphorylation by these two kinases is insufficient to account for Cdc25 activation during M phase induction. Here we demonstrate that p42 MAP kinase (MAPK), the Xenopus ortholog of ERK2, is a major Cdc25 phosphorylating kinase in extracts of M phase-arrested Xenopus eggs. In Xenopus oocytes, p42 MAPK interacts with hypophosphorylated Cdc25 before meiotic induction. During meiotic induction, p42 MAPK phosphorylates Cdc25 at T48, T138, and S205, increasing Cdc25's phosphatase activity. In a mammalian cell line, ERK1/2 interacts with Cdc25C in interphase and phosphorylates Cdc25C at T48 in mitosis. Inhibition of ERK activation partially inhibits T48 phosphorylation, Cdc25C activation, and mitotic induction. These findings demonstrate that ERK-MAP kinases are directly involved in activating Cdc25 during the G(2)/M transition.     

The xenopus Suc1/Cks protein promotes the phosphorylation of G(2)/M regulators

The entry into mitosis is controlled by Cdc2/cyclin B, also known as maturation or M-phase promoting factor (MPF). In Xenopus egg extracts, the inhibitory phosphorylations of Cdc2 on Tyr-15 and Thr-14 are controlled by the phosphatase Cdc25 and the kinases Myt1 and Wee1. At mitosis, Cdc25 is activated and Myt1 and Wee1 are inactivated through phosphorylation by multiple kinases, including Cdc2 itself. The Cdc2-associated Suc1/Cks1 protein (p9) is also essential for entry of egg extracts into mitosis, but the molecular basis of this requirement has been unknown. We find that p9 strongly stimulates the regulatory phosphorylations of Cdc25, Myt1, and Wee1 that are carried out by the Cdc2/cyclin B complex. Overexpression of the prolyl isomerase Pin1, which binds to the hyperphosphorylated forms of Cdc25, Myt1, and Wee1 found at M-phase, is known to block the initiation of mitosis in egg extracts. We have observed that Pin1 specifically antagonizes the stimulatory effect of p9 on phosphorylation of Cdc25 by Cdc2/cyclin B. This observation could explain why overexpression of Pin1 inhibits mitotic initiation. These findings suggest that p9 promotes the entry into mitosis by facilitating phosphorylation of the key upstream regulators of Cdc2.     

Regulation of the G2-mitosis transition.

The cell cycle is regulated by pathways composed of a dependent series of steps, by timers, and by checkpoint controls which ensure the completion of one event before the initiation of another. This review focuses on the regulation of the initiation of mitosis, with particular emphasis on the regulation of p34cdc2 activity at this point in the cell cycle. The review draws on data from various organisms, but strongly emphasizes the genetic framework as seen in the fission yeast Schizosaccharomyces pombe and the biology and biochemistry of maturation promoting factor in frog oocytes. An attempt is made to include all known genes and proteins where a link can be made to the initiation event. The nutritional size control and its major known controlling elements, the wee1/mik1 protein kinases, and cdc25 protein tyrosine phosphatase are considered in detail along with their regulation. In addition, the checkpoint control pathways which mediate G2 delay in response to failure of DNA replication or DNA damage are examined.     

Spatiotemporal regulations of Wee1 at the G2/M transition

Wee1 is a protein kinase that negatively regulates mitotic entry in G2 phase by suppressing cyclin B-Cdc2 activity, but its spatiotemporal regulations remain to be elucidated. We observe the dynamic behavior of Wee1 in Schizosaccharomyces pombe cells and manipulate its localization and kinase activity to study its function. At late G2, nuclear Wee1 efficiently suppresses cyclin B-Cdc2 around the spindle pole body (SPB). During the G2/M transition when cyclin B-Cdc2 is highly enriched at the SPB, Wee1 temporally accumulates at the nuclear face of the SPB in a cyclin B-Cdc2-dependent manner and locally suppresses both cyclin B-Cdc2 activity and spindle assembly to counteract a Polo kinase-dependent positive feedback loop. Then Wee1 disappears from the SPB during spindle assembly. We propose that regulation of Wee1 localization around the SPB during the G2/M transition is important for proper mitotic entry and progression.     

Multiple roles for separase auto-cleavage during the G2/M transition

The cysteine protease separase triggers anaphase onset by cleaving chromosome-bound cohesin. In humans, separase also cleaves itself at multiple sites, but the biological significance of this reaction has been elusive. Here we show that preventing separase auto-cleavage, via targeted mutagenesis of the endogenous hSeparase locus in somatic cells, interferes with entry into and progression through mitosis. The initial delay in mitotic entry was not dependent on the G2 DNA damage checkpoint, but rather involved improper stabilization of the mitosis-inhibiting kinase Wee1. During M phase, cells deficient in separase auto-cleavage exhibited striking defects in spindle assembly and metaphase chromosome alignment, revealing an additional early mitotic function for separase. Both the G2 and M phase phenotypes could be recapitulated by separase RNA interference and corrected by re-expressing wild-type separase in trans. We conclude that separase auto-cleavage coordinates multiple aspects of the G2/M programme in human cells, thus contributing to the timing and efficiency of chromosome segregation.     

Xp38gamma/SAPK3 promotes meiotic G(2)/M transition in Xenopus oocytes and activates Cdc25C

We have studied the role of p38 mitogen-activated protein kinases (MAPKs) in the meiotic maturation of Xenopus oocytes. Overexpression of a constitutively active mutant of the p38 activator MKK6 accelerates progesterone-induced maturation. Immunoprecipit ation experiments indicate that p38gamma/SAPK3 is the major p38 activated by MKK6 in the oocytes. We have cloned Xenopus p38gamma (Xp38gamma) and show that co-expression of active MKK6 with Xp38gamma induces oocyte maturation in the absence of progesterone. The maturation induced by Xp38gamma requires neither protein synthesis nor activation of the p42 MAPK-p90Rsk pathway, but it is blocked by cAMP-dependent protein kinase. A role for the endogenous Xp38gamma in progesterone-induced maturation is supported by the inhibitory effect of kinase-dead mutants of MKK6 and Xp38gamma. Furthermore, MKK6 can rescue the inhibition of oocyte maturation by anthrax lethal factor, a protease that inactivates MAPK kinases. We also show that Xp38gamma can activate the phosphatase XCdc25C, and we identified Ser205 of XCdc25C as a major phosphorylation site for Xp38gamma. Our results indicate that phosphorylation of XCdc25C by Xp38gamma/SAPK3 is important for the meiotic G(2)/M progression of Xenopus oocytes.     

A constitutively active form of the protein kinase p90Rsk1 is sufficient to trigger the G2/M transition in Xenopus oocytes

The protein kinase p90(Rsk) has previously been implicated as a key target of the MAPK pathway during M phase of meiosis II in Xenopus oocytes. To determine whether Rsk is a mediator of MAPK for stimulation of the G(2)/M transition early in meiosis I, we sought to generate a form of Rsk that would be constitutively active in resting, G(2) phase oocytes. Initial studies revealed that an N-terminal truncation of 43 amino acids conferred enhanced specific activity on the enzyme in G(2) phase, and stability was highest if the C terminus was not truncated. The full-length enzyme is known to be activated by phosphorylation at five sites. Two of these sites and flanking residues were replaced with either aspartic or glutamic acid, and Tyr(699) was mutated to alanine. The resulting construct, termed fully activated (FA) Rsk, had constitutive activity in G(2) phase, with a specific activity equivalent to that of wild type Rsk in M phase. In eight independent experiments approximately 45% of oocytes expressing FA-Rsk underwent germinal vesicle breakdown (GVBD, the G(2)/M transition) in the absence of progesterone, and this effect could be observed even in the presence of the MAPK kinase inhibitor U0126. Moreover, the specific activity of FA-Rsk in vivo was unaffected by U0126. In oocytes that did not undergo GVBD with FA-Rsk expression, subsequent treatment with progesterone resulted in a very rapid rate of GVBD even in the presence of U0126 to inhibit the endogenous MAPK/Rsk pathway. These results indicate that Rsk is the mediator of MAPK effects for the G(2)/M transition in meiosis I and in a subpopulation of oocytes Rsk is sufficient to trigger the G(2)/M transition.     

Cyclin A2 is phosphorylated during the G2/M transition in mouse two-cell embryos

In the present study, we investigated the expression of cyclin A2 in mouse two-cell embryos to elucidate the role of cyclin A2 at the G2/M transition. Two forms of cyclin A2 on SDS-PAGE (an upper and a lower band) were detected in two-cell embryos synchronized at the M phase by nocodazole. To investigate the nature of this shift, embryos synchronized at the M phase were treated with alkaline phosphatase (AP). The upper band of cyclin A2 was fainter in AP-treated embryos than in nontreated embryos. This result indicates that cyclin A2 in mouse two-cell embryos is phosphorylated and the band on SDS-PAGE shifts up during the G2/M transition. In addition, we examined the sequential expression of cyclin A2 in two-cell blocked embryos after OA treatment. The upper band of cyclin A2 was first detected at 2 hr after the treatment, corresponding to the timing of Cdc2 kinase activation. In two-cell embryos after removal from nocodazole treatment, the phosphorylated form of cyclin A2 protein decreased abruptly just before cytokinesis. These results suggest that the mechanism of cyclin A2 degradation in mouse two-cell embryos may be different from that in somatic cells.