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.     

MAPK inactivation is required for the G2 to M-phase transition of the first mitotic cell cycle.

Down-regulation of MAP kinase (MAPK) is a universal consequence of fertilization in the animal kingdom, although its role is not known. Here we show that MAPK inactivation is essential for embryos, both vertebrate and invertebrate, to enter first mitosis. Suppressing down-regulation of MAPK at fertilization, for example by constitutively activating the upstream MAPK cascade, specifically suppresses cyclin B-cdc2 kinase activation and its consequence, entry into first mitosis. It thus appears that MAPK functions in meiotic maturation by preventing unfertilized eggs from proceeding into parthenogenetic development. The most general effect of artificially maintaining MAPK activity after fertilization is prevention of the G2 to M-phase transition in the first mitotic cell cycle, even though inappropriate reactivation of MAPK after fertilization may lead to metaphase arrest in vertebrates. Advancing the time of MAPK inactivation in fertilized eggs does not, however, speed up their entry into first mitosis. Thus, sustained activity of MAPK during part of the first mitotic cell cycle is not responsible for late entry of fertilized eggs into first mitosis.     

Regulation of the G2/M transition in oocytes of xenopus tropicalis

The molecular events regulating hormone-induced oocyte activation and meiotic maturation are probably best understood in Xenopus laevis. In X. laevis, progesterone activates the G2-arrested oocyte, induces entry into M phase of meiosis I (MI) and resumption of the meiotic cell cycles, and leads to the formation of a mature, fertilizable egg. Oocytes of Xenopus tropicalis offer several practical advantages over those of X. laevis, including faster and more synchronous meiotic cell cycle progression, less seasonal variability, and the availability of transgenic approaches. Previous work found several similarities in the pathways regulating oocyte maturation in the two species. Here, we report several additional ones that are conserved in X. tropicalis. (1). Injection of Mos mRNA into G2-arrested oocytes activates the MAP kinase cascade and induces the G2/MI transition. (2). Injection of the beta subunit of the kinase CK2 (a negative regulator of Mos and oocyte activation) delays the G2/MI transition. (3). Elevating PKA activity blocks progesterone-induced maturation; repressing PKA activity induces entry into MI in the absence of progesterone. (4). LF (anthrax lethal factor), which cleaves certain MAP kinase kinases, strongly reduces both the rate and extent of entry into MI. In contrast to the one previously reported major difference between oocytes of the two species, we find that injection of egg cytoplasm ("MPF activity") into G2-arrested X. tropicalis oocytes induces entry into meiosis I even when protein synthesis is blocked, just as it does in oocytes of X. laevis. These results indicate that much of what we have learned from studies of X. laevis oocytes holds for those of X. tropicalis, and suggest that X. tropicalis oocytes offer a good experimental system for investigating certain questions that require a rapid, synchronous progression through the G2/meiosis I transition.     

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.     

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.     

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 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.     

Okadaic acid-sensitive phosphatase is related to MII/G1 transition in mouse oocytes

It is reported that okadaic acid (OA)-sensitive phosphatase is related to mitogen-activated protein kinase (MAPK)/p90rsk activation in mammalian oocytes. OA is also involved in the positive feedback loop between M phase-promoting factor (MPF) and cdc25c in Xenopus oocytes during meiotic maturation. However, the effect of phosphatase inhibition by OA on MPF and MAPK activities at the MII/G1 in oocytes remains unknown. The aim of this study is to clarify the relationship between OA-sensitive phosphatase and mitosis MII/G1 transition in mouse oocytes. MII-arrested oocytes were, isolated from mice, inseminated and cultured in TYH medium (control group) or TYH medium supplemented with 2.5 μM of OA (OA group). Histone H1 kinase and myelin basic protein (MBP) kinase activities were measured as indicators of MPF and p42 MAPK activities after insemination. Phosphorylation of cdc25c after insemination was analized in OA and control group by western blotting. Seven hours after insemination a pronucleus (PN) was formed in 84.1% (69/85) of oocytes in the control group. However, no PN was formed in oocytes of the OA group (p < 0.001). Although MPF and MAPK activities in the control group significantly decreased at 3, 4, 5, and 7 h after insemination, these decreases were significantly inhibited by OA addition (p < 0.05). Furthermore, OA addition prevented cdc25c dephosphorylation 7 h after insemination. In conclusion, OA-sensitive phosphatase correlates with inactivation of MPF and MAPK, and with the dephosphorylation of cdc25c at the MII/G1 transition in mouse oocytes.     

Germanium oxide inhibits the transition from G2 to M phase of CHO cells

We report here for the first time that germanium oxide (GeO(2)) blocks cell progression. GeO(2) is not genotoxic to Chinese hamster ovary (CHO) cells and has limited cytotoxicity. However, GeO(2) arrests cells at G2/M phase. The proportion of cells stopped at G2/M phase increased dose-dependently up to 5 mM GeO(2) when treated for 12 h, but decreased at GeO(2) concentration was greater than 5 mM. Analysis of 5-bromodeoxyuridine-labeled cells indicated that GeO(2) delayed S phase progression in a dose-dependent manner, and blocked cells at G2/M phase. Microscopic examination confirmed that GeO(2) treatment arrested cells at G2 phase. Similar to several other events that cause G2 block, the GeO(2)-induced G2 block can also be ameliorated by caffeine in a dose- and time-dependent manner. To explore the mechanism of G2 arrest by GeO(2), cyclin content and cyclin-dependent kinase activity were examined. Cyclin B1 level was not affected after GeO(2) treatment in CHO cells. However, GeO(2) decreased p34(cdc2) kinase (Cdk1) activity. The kinase activity recovered within 9 h after GeO(2) removal and correlated with the transition of G2/M-G1 phase of the cells. This result suggests that GeO(2) treatment reduces Cdk1 activity and causing the G2 arrest in CHO cells.     

H2AX foci in late S/G2- and M-phase cells after hydroxyurea- and aphidicolin-induced DNA replication stress in Vicia

Immunocytochemistry using α-phospho-H2AX antibodies shows that hydroxyurea (HU), an inhibitor of ribonucleotide reductase, and aphidicolin (APH), an inhibitor of DNA-polymerases α and δ, may promote formation of phospho-H2AX foci in late S/G2-phase cells in root meristems of Vicia faba. Although fluorescent foci spread throughout the whole area of nucleoplasm, large phospho-H2AX aggregates in HU-treated cells allocate mainly in perinucleolar regions. A strong tendency of ATR/ATM-dependent phospho-Chk1^S317 kinase to focus in analogous compartments, as opposed to phospho-Chk2^T68 and to both effector kinases in APH-treated cells, may suggest that selected elements of the intra-S-phase cell cycle checkpoints share overlapping locations with DNA repair factors known to concentrate in phospho-H2AX aggregates. APH-induced phosphorylation of H2AX exhibits little or no overlap with the areas positioned close to nucleoli. Following G2-M transition of the HU- and APH-pretreated cells, altered chromatin structures are still discernible as large phospho-H2AX foci in the vicinity of chromosomes. Both in HU- and APH-treated roots, immunofluorescence analysis revealed a dominant fraction of small foci and a less frequent population of large phospho-H2AX agregates, similar to those observed in animal cells exposed to ionizing radiation. The extent of H2AX phosphorylation has been found considerably reduced in root meristem cells treated with HU and caffeine. The frequencies of phospho-H2AX foci observed during mitosis and caffeine-mediated premature chromosome condensation (PCC) suggest that there may be functional links between the checkpoint mechanisms that control genome integrity and those activities which operate throughout the unperturbed mitosis in plants.     

Early replicating DNA involved in the G2 to M transition in Allium cepa L meristematic cells

Summary— The involvement of genome portions replicating at different times of the S phase in the control of cell cycle events was analysed in Allium cepa L meristematic cells. 5-Azacytidine (5-azaC) was incorporated into discrete replicating DNA portions in synchronous cells. Cells treated with 5-azaC during the late S phase went through normal cell cycles while cells treated with 5-azaC during the early S phase were able to go through the immediately succeeding mitosis, as well as to begin the subsequent S phase. However, they were unable to enter a second mitosis. Thus, sequences replicating in the early S phase appear to code for a mitotic inhibitor which represses the emergence of a positive mitotic signal. This inhibition took place in the first half of the interphase (ie during G1) and resulted in a cell cycle blockage in G2.     

Regulation of M-phase progression in Chaetopterus oocytes by protein kinase C.

We have examined the presence of protein kinase C in oocytes of Chaetopterus pergamentaceus and its role in the initiation of germinal vesicle breakdown (GVBD). First, we demonstrated that the oocytes contain a phospholipid- and calcium-dependent protein kinase, protein kinase C (PKC). Since PKC is the primary intracellular receptor for phorbol esters, we tested the ability of phorbol 12,13-dibutyrate (PDBu) to induce GVBD and compared several critical events and processes involved in GVBD induced by PDBu to those induced normally (by seawater). Seawater and 100-200 nM PDBu induced chromosome condensation, spindle formation, and spindle migration over a similar time course. Both treatments induced similar alterations in the SDS-PAGE pattern of newly synthesized proteins. The synthesis of polypeptides of approximately 46 and 54 kDa increased specifically. Both treatments increased oocyte protein phosphorylation, especially of proteins of 22, 32, 46, 55, 64, and 84 kDa. Both treatments resulted in the activation of an M-phase-specific histone H1 kinase activity, which demonstrates the appearance of maturation-promoting factor. Staurosporine, a potent protein kinase C inhibitor, blocked GVBD and the activation of M-phase-specific H1 kinase, whereas HA1004, which preferentially antagonizes protein kinase A, had no effect. The results of this study demonstrate that protein kinase C can activate a wide spectrum of essential biochemical and morphological processes involved in GVBD. Further, these studies suggest that protein kinase C elicits GVBD by activating maturation-promoting factor and support the hypothesis that protein kinase C plays an essential role in oocyte maturation in this species.     

Abrogation of G(2)/M-phase block enhances the cytotoxicity of daunorubicin, melphalan and cisplatin in TP53 mutant human tumor cells

Irradiation of human melanoma (MeWo, Be11) and squamous cell carcinoma (4451, 4197) cells induces cell cycle blocks from which the cells recover to re-enter mitosis after 40-60 h. In the TP53 mutant cell lines, MeWo and 4451, irradiation induces a G(2)-phase block, where the fraction of cells in G(2) phase reaches a maximum after 18-20 h. In the TP53 wild-type cell lines, 4197 and Be11, a G(1)- and G(2)-phase block is reached 12 and 16 h postirradiation, respectively. Addition of pentoxifylline after irradiation at the time when the number of cells in G(2) phase has reached a maximum shortens the normal recovery from G(2)-phase block to approximately 7 h. Addition of daunorubicin, melphalan and cisplatin under these conditions markedly enhanced drug toxicity. In the TP53-mutated cell lines MeWo and 4451, the survival ratio at 7 Gy measured by colony formation was 2.3-2.8, 8.6-85 and 52-74 for daunorubicin, melphalan and cisplatin, respectively. In the TP53 wild-type cell lines, the corresponding survival ratios were found to be 1.3-1.4, 2.3-3.0 and 1.2-2.6, respectively. The survival ratios are for clonogenic survival after 7 Gy and 2 mM pentoxifylline and measure the influence of drug doses that ensure 95% survival in nonirradiated controls. The results indicate that the G(2)-phase block is a crucial event in the damage response that can be manipulated to achieve a significant enhancement of drug toxicity. These effects are particularly pronounced in TP53 mutant cells and are observed at drug doses well below the clinical range.     

Maturation/M-phase promoting factor: a regulator of aging in porcine oocytes

Deterioration in the quality of mammalian oocytes during the metaphase-II arrest period is well known as "oocyte aging." Oocytes in which aging has occurred are called aged oocytes, and these oocytes show enhanced activation and higher fragmentation rates after parthenogenetic activation. Previously we showed that porcine aged oocytes had low maturation/M-phase promoting factor (MPF) activity, and we suggested that this low MPF activity contributed at least in part to the aging phenomena. In the present study, we examined the relationship between MPF activity and these aging phenomena by artificially regulating MPF activity in porcine metaphase-II-arrested oocytes. Since we have shown recently that aged porcine oocytes contain abundant phosphorylated inactive MPF, so-called pre-MPF, we used vanadate and caffeine, which affect the phosphorylation status of MPF, to regulate MPF activity. Incubation of 48-h-matured oocytes with vanadate for 1 h increased the phosphorylation of MPF and decreased MPF activity. The parthenogenetic activation and fragmentation rates were significantly increased compared with those of control oocytes. Conversely, treatment of 72-h-cultured aged oocytes with caffeine (last 10 h of culture) decreased the level of pre-MPF and elevated MPF activity. These oocytes revealed significantly lower parthenogenetic activation rates and a lower percentage of fragmentation than did untreated aged oocytes. These results indicate that not only the increased ability for parthenogenetic activation but also the increased fragmentation rate observed in porcine aged oocytes may be attributable in part to the gradual decrease in MPF activity during prolonged culture. Control of MPF phosphorylation with these agents may allow for some degree of manipulation of oocyte aging.     

Maturation/M-phase promoting factor regulates aging of porcine oocytes matured in vitro

Control of oocyte aging during manipulation of matured oocytes should have advantages for recently developed reproductive technologies, such as cloning after nuclear transfer. We have shown that the enhanced activation ability and fragmentation of porcine in vitro matured and aged oocytes bore a close relationship to the gradual decrease in maturation/M-phase promoting factor (MPF) activity and that porcine aged oocytes contained plenty of MPF, but it was in an inactive form, pre-MPF, as a result of phosphorylation of its catalytic subunit p34(cdc2) and, therefore, had low MPF activity. We incubated porcine oocytes with vanadate and caffeine, which affected the phosphorylation status and MPF activity, and evaluated their activation abilities and fragmentation frequencies. Incubation of nonaged oocytes with vanadate increased p34(cdc2) phosphorylation and reduced MPF activity to levels similar to those of aged oocytes and increased their parthenogenetic activation and fragmentation rates compared with those of the control oocytes. Conversely, treating aged oocytes with caffeine reduced p34(cdc2) phosphorylation and increased MPF activity. These oocytes showed significantly lower parthenogenetic activation and fragmentation rates than aged mature oocytes. These results suggest that MPF activity is a key mechanism of oocyte aging and controlling MPF activity by altering p34(cdc2) phosphorylation with these chemicals may enable oocyte aging to be manipulated in vitro. We expect those ideas will be applied practically to pig cloning.     

G2/M-phase arrest and death by apoptosis of HL60 cells irradiated with exponentially decreasing low-dose-rate gamma radiation.

Cells of the TP53-deficient human leukemia cell line HL60 continue to progress throughout the cell cycle and arrest in the G2/M phase during protracted exposure to exponentially decreasing low-dose-rate radiation. We have hypothesized that G2/M-phase arrest contributes to the extent of radiation-induced cell death by apoptosis as well as to overall cell killing. To test this hypothesis, we used caffeine and nocodazole to alter the duration of G2/M-phase arrest of HL60 cells exposed to exponentially decreasing low-dose-rate irradiation and measured the activity of G2/M-phase checkpoint proteins, redistribution of cells in the phases of the cell cycle, cell death by apoptosis, and overall survival after irradiation. The results from these experiments demonstrate that concomitant exposure of HL60 cells to caffeine (2 mM) during irradiation inhibited radiation-induced tyrosine 15 phosphorylation of the G2/M-phase transition checkpoint protein CDC2/p34 kinase and reduced G2/M-phase arrest by 40-46% compared to cells irradiated without caffeine. Radiation-induced apoptosis also decreased by 36-50% in cells treated with caffeine and radiation compared to cells treated with radiation alone. Radiation survival was significantly increased by exposure to caffeine. In contrast, prolongation of G2/M-phase arrest by pre-incubation with nocodazole enhanced radiation-induced apoptosis and overall radiation-induced cell killing. To further study the role of cell death by apoptosis in the response to exponentially decreasing low-dose-rate irradiation, HL60 cells were transfected with the BCL2 proto-oncogene. The extent of G2/M-phase arrest was similar for parental, neomycin-transfected control and BCL2-transfected cells during and after exponentially decreasing low-dose-rate irradiation. However, there were significant differences (P < 0.01) in the extent of radiation-induced apoptosis of parental and neomycin- and BCL2-transfected cells after irradiation, with significantly less radiation-induced apoptosis and higher overall survival in BCL2-transfected cells than similarly irradiated control cells. These data demonstrate that radiation-induced G2/M-phase arrest and subsequent induction of apoptosis play an important role in the response of HL60 cells to low-dose-rate irradiation and suggest that it may be possible to increase radiation-induced apoptosis by altering the extent of G2/M-phase arrest. These findings are clinically relevant and suggest a novel therapeutic strategy for increasing the efficacy of brachytherapy and radioimmunotherapy.     

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.     

Meiotic competence acquisition is associated with the appearance of M-phase characteristics in growing mouse oocytes

To determine whether the acquisition of meiotic competence during the growth phase of oogenesis is associated with the appearance of M-phase characteristics, oocytes obtained from 13- to 30-day-old mice were evaluated by fluorescence microscopy with respect to chromatin and microtubule organization , in vitro maturation ability, and the distribution of M-phase phosphoproteins. Meiotically incompetent oocytes were distinguished from their competent counterparts in displaying elaborate interphase-like arrays of cytoplasmic microtubules and dispersed germinal vesicle chromatin. Meiotically competent oocytes were larger in size, exhibited condensation of chromatin around the nucleolus, and displayed a progressive diminution of cytoplasmic microtubules in conjunction with the appearance of multiple microtubule organizing centers. After 24 hr in culture, medium- to large-sized oocytes exhibiting perinucleolar chromatin condensation resume meiosis whereas smaller meiotically incompetent oocytes retain GVs with diffuse chromatin. Moreover, indirect immunofluorescence studies using the M-phase phosphoprotein specific monoclonal antibody MPM-2 indicate that the appearance of reactive cytoplasmic foci is directly correlated with nuclear changes characteristic of meiotically competent oocytes. Thus, the earliest transition to a meiotically competent state during oocyte growth in the immature mouse ovary is characterized by stage-specific and coordinated modifications of nuclear and cytoplasmic components.     

Loss of p53 induces M-phase retardation following G2 DNA damage checkpoint abrogation

Most cell lines that lack functional p53 protein are arrested in the G2 phase of the cell cycle due to DNA damage. When the G2 checkpoint is abrogated, these cells are forced into mitotic catastrophe. A549 lung adenocarcinoma cells, in which p53 was eliminated with the HPV16 E6 gene, exhibited efficient arrest in the G2 phase when treated with adriamycin. Administration of caffeine to G2-arrested cells induced a drastic change in cell phenotype, the nature of which depended on the status of p53. Flow cytometric and microscopic observations revealed that cells that either contained or lacked p53 resumed their cell cycles and entered mitosis upon caffeine treatment. However, transit to the M phase was slower in p53-negative cells than in p53-positive cells. Consistent with these observations, CDK1 activity was maintained at high levels, along with stable cyclin B1, in p53-negative cells. The addition of butyrolactone I, which is an inhibitor of CDK1 and CDK2, to the p53-negative cells reduced the floating round cell population and induced the disappearance of cyclin B1. These results suggest a relationship between the p53 pathway and the ubiquitin-mediated degradation of mitotic cyclins and possible cross-talk between the G2-DNA damage checkpoint and the mitotic checkpoint.