Cell cycle progression and cell division are sensitive to hypoxia in Drosophila melanogaster embryos

We and others recently demonstrated that Drosophila melanogaster embryos arrest development and embryonic cells cease dividing when they are deprived of O2. To further characterize the behavior of these embryos in response to O2 deprivation and to define the O2-sensitive checkpoints in the cell cycle, embryos undergoing nuclear cycles 3-13 were subjected to O2 deprivation and examined by confocal microscopy under control, hypoxic, and reoxygenation conditions. In vivo, real-time analysis of embryos carrying green fluorescent protein-kinesin demonstrated that cells arrest at two major points of the cell cycle, either at the interphase (before DNA duplication) or at metaphase, depending on the cell cycle phase at which O2 deprivation was induced. Immunoblot analysis of embryos whose cell divisions are synchronized by inducible String (cdc25 homolog) demonstrated that cyclin B was degraded during low O2 conditions in interphase-arrested embryos but not in those arrested in metaphase. Embryos resumed cell cycle activity within ~20 min of reoxygenation, with very little apparent change in cell cycle kinetics. We conclude that there are specific points during the embryonic cell cycle that are sensitive to the O2 level in D. melanogaster. Given the fact that O2 deprivation also influences the growth and development of other species, we suggest that similar hypoxia-sensitive cell cycle checkpoints may also exist in mammalian cells.     

Cell cycle arrest associated with anoxia-induced quiescence, anoxic preconditioning, and embryonic diapause in embryos of the annual killifish Austrofundulus limnaeus

Embryos of the annual killifish Austrofundulus limnaeus can enter into dormancy associated with diapause and anoxia-induced quiescence. Dormant embryos are composed primarily of cells arrested in the G(1)/G(0) phase of the cell cycle based on flow cytometry analysis of DNA content. In fact, most cells in developing embryos contain only a diploid complement of DNA, with very few cells found in the S, G(2), or M phases of the cell cycle. Diapause II embryos appear to be in a G(0)-like state with low levels of cyclin D1 and p53. However, the active form of pAKT is high during diapause II. Exposure to anoxia causes an increase in cyclin D1 and p53 expression in diapause II embryos, suggesting a possible re-entry into the cell cycle. Post-diapause II embryos exposed to anoxia or anoxic preconditioning have stable levels of cyclin D1 and stable or reduced levels of p53. The amount of pAKT is severely reduced in 12?dpd embryos exposed to anoxia or anoxic preconditioning. This study is the first to evaluate cell cycle control in embryos of A. limnaeus during embryonic diapause and in response to anoxia and builds a foundation for future research on the role of cell cycle arrest in supporting vertebrate dormancy.     

Hypoxia and nitric oxide induce a rapid, reversible cell cycle arrest of the Drosophila syncytial divisions

Cells can respond to reductions in oxygen (hypoxia) by metabolic adaptations, quiescence or cell death. The nuclear division cycles of syncytial stage Drosophila melanogaster embryos reversibly arrest upon hypoxia. We examined this rapid arrest in real time using a fusion of green fluorescent protein and histone 2A. In addition to an interphase arrest, mitosis was specifically blocked in metaphase, much like a checkpoint arrest. Nitric oxide, recently proposed as a hypoxia signal in Drosophila, induced a reversible arrest of the nuclear divisions comparable with that induced by hypoxia. Syncytial stage embryos die during prolonged hypoxia, whereas post-gastrulation embryos (cellularized) survive. We examined ATP levels and morphology of syncytial and cellularized embryos arrested by hypoxia, nitric oxide, or cyanide. Upon oxygen deprivation, the ATP levels declined only slightly in cellularized embryos and more substantially in syncytial embryos. Reversal of hypoxia restored ATP levels and relieved the cell cycle and developmental arrests. However, morphological abnormalities suggested that syncytial embryos suffered irreversible disruption of developmental programs. Our results suggest that nitric oxide plays a role in the response of the syncytial embryo to hypoxia but that it is not the sole mediator of these responses.     

Dephosphorylation of cell cycle-regulated proteins correlates with anoxia-induced suspended animation in Caenorhabditis elegans

Some metazoans have evolved the capacity to survive severe oxygen deprivation. The nematode, Caenorhabditis elegans, exposed to anoxia (0 kPa, 0% O(2)) enters into a recoverable state of suspended animation during all stages of the life cycle. That is, all microscopically observable movement ceases including cell division, developmental progression, feeding, and motility. To understand suspended animation, we compared oxygen-deprived embryos to nontreated embryos in both wild-type and hif-1 mutants. We found that hif-1 mutants survive anoxia, suggesting that the mechanisms for anoxia survival are different from those required for hypoxia. Examination of wild-type embryos exposed to anoxia show that blastomeres arrest in interphase, prophase, metaphase, and telophase but not anaphase. Analysis of the energetic state of anoxic embryos indicated a reversible depression in the ATP to ADP ratio. Given that a decrease in ATP concentrations likely affects a variety of cellular processes, including signal transduction, we compared the phosphorylation state of several proteins in anoxic embryos and normoxic embryos. We found that the phosphorylation state of histone H3 and cell cycle-regulated proteins recognized by the MPM-2 antibody were not detectable in anoxic embryos. Thus, dephosphorylation of specific proteins correlate with the establishment and/or maintenance of a state of anoxia-induced suspended animation.     

Characterization of sub-nuclear changes in <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis> embryos exposed to brief,intermediate and long-term anoxia to analyze anoxia-induced cell cycle arrest

Background  

The soil nematode C. elegans survives oxygen-deprived conditions (anoxia; <.001 kPa O₂) by entering into a state of suspended animation in which cell cycle progression reversibly arrests. The majority of blastomeres of embryos exposed to anoxia arrest at interphase, prophase and metaphase. The spindle checkpoint proteins SAN-1 and MDF-2 are required for embryos to survive 24 hours of anoxia. To further investigate the mechanism of cell-cycle arrest we examined and compared sub-nuclear changes such as chromatin localization pattern, post-translational modification of histone H3, spindle microtubules, and localization of the spindle checkpoint protein SAN-1 with respect to various anoxia exposure time points. To ensure analysis of embryos exposed to anoxia and not post-anoxic recovery we fixed all embryos in an anoxia glove box chamber.     

Hypoxia-induced irreversible S-phase arrest involves down-regulation of cyclin A

We have studied hypoxia-induced cell cycle arrest in human cells where the retinoblastoma tumour suppressor protein (pRB) is either functional (T-47D cells) or abrogated by expression of the HPV18 E7 oncoprotein (NHIK 3025 cells). All cells in S phase are immediately arrested upon exposure to extreme hypoxia. During an 18-h extreme hypoxia regime, the cyclin A protein level is down-regulated in cells of both types when in S-phase, and, as we have previously shown, pRB re-binds in the nuclei of all T-47D cells (Amellem et al. 1996). Hence, pRB is not necessary for the down-regulation of cyclin A during hypoxia. However, our findings indicate that re-oxygenation cannot release pRB from its nuclear binding following this prolonged exposure. The result is permanent S-phase arrest even after re-oxygenation, and this is correlated with a complete and permanent down-regulation of cyclin A in the pRB functional T-47D cells. In contrast, both cell cycle arrest and cyclin A down-regulation in S phase are reversed upon re-oxygenation in non-pRB-functional NHIK 3025 cells after prolonged exposure to extreme hypoxia. Our results indicate that pRB is involved in permanent S-phase arrest and down-regulation of cyclin A after extreme hypoxia.     

Exercise training prevents the inflammatory response to hypoxia in cremaster venules.

Systemic hypoxia produces microvascular inflammation in several tissues, including skeletal muscle. Exercise training (ET) has been shown to reduce the inflammatory component of several diseases. Alternatively, ET could influence hypoxia-induced inflammation by improving tissue oxygenation or increasing mechanical antiadhesive forces at the leukocyte-endothelial interface. The effect of 5 wk of treadmill ET on hypoxia-induced microvascular inflammation was studied in the cremaster microcirculation of rats using intravital microscopy. In untrained rats, hypoxia (arterial Po(2) = 32.3 +/- 2.1 Torr) increased leukocyte-endothelial adherence from 2.3 +/- 0.4 to 10.2 +/- 0.3 leukocytes per 100 microm of venule (P < 0.05) and was accompanied by extravasation of FITC-labeled albumin after 4 h of hypoxia (extra-/intravascular fluorescence intensity ratio = 0.50 +/- 0.07). These responses were attenuated in ET (leukocyte adherence was 1.5 +/- 0.4 during normoxia and 1.8 +/- 0.7 leukocytes per 100 mum venule after 10 min of hypoxia; extra-/intravascular fluorescence intensity ratio = 0.11 +/- 0.02; P < 0.05 vs. untrained) despite similar reductions of arterial (32.4 +/- 1.8 Torr) and microvascular Po(2) (measured with an oxyphor-quenching method) in both groups. Shear rate decreased during hypoxia to similar extents in ET and untrained rats. In addition, circulating blood leukocyte count was similar in ET and untrained rats. The effects of ET on hypoxia-induced leukocyte-endothelial adherence remained up to 4 wk after discontinuing training. Thus ET attenuated hypoxia-induced inflammation despite similar effects of hypoxia on tissue Po(2), venular shear rate, and circulating leukocyte count.     

The metaphase II arrest in mouse oocytes is controlled through microtubule-dependent destruction of cyclin B in the presence of CSF.

In unfertilized eggs from vertebrates, the cell cycle is arrested in metaphase of the second meiotic division (metaphase II) until fertilization or activation. Maintenance of the long-term meiotic metaphase arrest requires mechanisms preventing the destruction of the maturation promoting factor (MPF) and the migration of the chromosomes. In frog oocytes, arrest in metaphase II (M II) is achieved by cytostatic factor (CSF) that stabilizes MPF, a heterodimer formed of cdc2 kinase and cyclin. At the metaphase/anaphase transition, a rapid proteolysis of cyclin is associated with MPF inactivation. In Drosophila, oocytes are arrested in metaphase I (M I); however, only mechanical forces generated by the chiasmata seem to prevent chromosome separation. Thus, entirely different mechanisms may be involved in the meiotic arrests in various species. We report here that in mouse oocytes a CSF-like activity is involved in the M II arrest (as observed in hybrids composed of fragments of metaphase II-arrested oocytes and activated mitotic mouse oocytes) and that the high activity of MPF is maintained through a continuous equilibrium between cyclin B synthesis and degradation. In addition, the presence of an intact metaphase spindle is required for cyclin B degradation. Finally, MPF activity is preferentially associated with the spindle after bisection of the oocyte. Taken together, these observations suggest that the mechanism maintaining the metaphase arrest in mouse oocytes involves an equilibrium between cyclin synthesis and degradation, probably controlled by CSF, and which is also dependent upon the three-dimensional organization of the spindle.     

NPP-16/Nup50 Function and CDK-1 Inactivation Are Associated with Anoxia-induced Prophase Arrest in Caenorhabditis elegans

Oxygen, an essential nutrient, is sensed by a multiple of cellular pathways that facilitate the responses to and survival of oxygen deprivation. The Caenorhabditis elegans embryo exposed to severe oxygen deprivation (anoxia) enters a state of suspended animation in which cell cycle progression reversibly arrests at specific stages. The mechanisms regulating interphase, prophase, or metaphase arrest in response to anoxia are not completely understood. Characteristics of arrested prophase blastomeres and oocytes are the alignment of condensed chromosomes at the nuclear periphery and an arrest of nuclear envelope breakdown. Notably, anoxia-induced prophase arrest is suppressed in mutant embryos lacking nucleoporin NPP-16/NUP50 function, indicating that this nucleoporin plays an important role in prophase arrest in wild-type embryos. Although the inactive form of cyclin-dependent kinase (CDK-1) is detected in wild-type–arrested prophase blastomeres, the inactive state is not detected in the anoxia exposed npp-16 mutant. Furthermore, we found that CDK-1 localizes near chromosomes in anoxia-exposed embryos. These data support the notion that NPP-16 and CDK-1 function to arrest prophase blastomeres in C. elegans embryos. The anoxia-induced shift of cells from an actively dividing state to an arrested state reveals a previously uncharacterized prophase checkpoint in the C. elegans embryo.     

Oxygen-induced changes in hemoglobin expression in Drosophila

The hemoglobin gene 1 (dmeglob1) of the fruit fly Drosophila melanogaster is expressed in the tracheal system and fat body, and has been implicated in hypoxia resistance. Here we investigate the expression levels of dmeglob1 and lactate dehydrogenase (a positive control) in embryos, third instar larvae and adult flies under various regimes of hypoxia and hyperoxia. As expected, mRNA levels of lactate dehydrogenase increased under hypoxia. We show that expression levels of dmeglob1 are decreased under both short- and long-term hypoxia, compared with the normoxic (21% O2) control. By contrast, a hypoxia/reoxygenation regime applied to third instar larvae elevated the level of dmeglob1 mRNA. An excess of O2 (hyperoxia) also triggered an increase in dmeglob1 mRNA. The data suggest that Drosophila hemoglobin may be unlikely to function merely as a myoglobin-like O2 storage protein. Rather, dmeglob1 may protect the fly from an excess of O2, either by buffering the flux of O2 from the tracheoles to the cells or by degrading noxious reactive oxygen species.     

The cyclin-dependent kinase inhibitor Roughex is involved in mitotic exit in Drosophila

Background: Exit from mitosis is a tightly regulated event. This process has been studied in greatest detail in budding yeast, where several activities have been identified that cooperate to downregulate activity of the cyclin-dependent kinase (CDK) Cdc28 and force an exit from mitosis. Cdc28 is inactivated through proteolysis of B-type cyclins by the multisubunit ubiquitin ligase termed the anaphase promoting complex/cyclosome (APC/C) and inhibition by the cyclin-dependent kinase inhibitor (CKI) Sic1. In contrast, the only mechanism known to be essential for CDK inactivation during mitosis in higher eukaryotes is cyclin destruction.Results: We now present evidence that the Drosophila CKI Roughex (Rux) contributes to exit from mitosis. Observations of fixed and living embryos show that metaphase is significantly longer in rux mutants than in wild-type embryos. In addition, Rux overexpression is sufficient to drive cells experimentally arrested in metaphase into interphase. Furthermore, rux mutant embryos are impaired in their ability to overcome a transient metaphase arrest induced by expression of a stable cyclin A. Rux has numerous functional similarities with Sic1. While these proteins share no sequence similarity, we show that Sic1 inhibits mitotic Cdk1-cyclin complexes from Drosophila in vitro and in vivo.Conclusions: Rux inhibits Cdk1-cyclin A kinase activity during metaphase, thereby contributing to exit from mitosis. To our knowledge, this is the first mitotic function ascribed to a CKI in a multicellular organism and indicates the existence of a novel regulatory mechanism for the metaphase to anaphase transition during development.     

Cellular effects of olomoucine,an inhibitor of cyclin-dependent kinases

Olomoucine (2-(2-hydroxyethylamino)-6-benzylamino-9-methylpurine) has been recently described as a competitive inhibitor (ATP-binding site) of the cell cycle regulating p34^cdc2/cyclin B, p33^cdk2/cyclin A and p33^cdk2/cyclin E kinases, the brain $\text{p33}{}^{\mathrm{cdk5}}\text{p35}$ kinase and the $\text{ERK1}\text{AP}\text{-kinase}$. The unusual specificity of this compound towards cell cycle regulating enzymes suggests that it could inhibit certain steps of the cell cycle. The cellular effects of olomoucine were investigated in a large variety of plant and animal models. This compound inhibits the $\text{G1}\text{S}$ transition of unicellular algae (dinoflagellate and diatom). It blocks Fucus zygote cleavage and development of Laminaria gametophytes. Stimulated Petunia mesophyl protoplasts are arrested in G1 by olomoucine. By arresting cleavage it blocks the development of Calanus copepod larvae. It reversibly inhibits the early cleavages of Caenorhabditis elegans embryos and those of ascidian embryos. Olomoucine inhibits the serotonin-induced prophase/metaphase transition of clam oocytes; furthermore, it triggers the release of these oocytes from their meiotic metaphase I arrest, and induces nuclei reformation. Olomoucine slows down the prophase/metaphase transition in cleaving sea urchin embryos, but does not affect the duration of the metaphase/anaphase and anaphase/telophase transitions. It also inhibits the prophase/metaphase transition of starfish oocytes triggered by various agonists. Xenopus oocyte maturation, the in vivo and in vitro phosphorylation of elongation factor EF-1 are inhibited by olomoucine. Mouse oocyte maturation is delayed by this compound, whereas parthenogenetic release from metaphase II arrest is facilitated. Growth of a variety of human cell lines (rhabdomyosarcoma cell lines Rh1, Rh18, Rh28 and Rh30; MCF-7, KB-3-1 and their adriamycin-resistant counterparts; National Cancer Institute 60 human tumor cell lines comprising nine tumor types) is inhibited by olomoucine. Cell cycle parameter analysis of the non-small cell lung cancer cell line MR65 shows that olomoucine affects G1 and S phase transits. Olomoucine inhibits DNA synthesis in interleukin-2-stimulated T lymphocytes (CTLL-2 cells) and triggers a G1 arrest similar to interleukin-2 deprivation. Both cdc2 and cdk2 kinases (immunoprecipitated from nocodazole- and hydroxyurea-treated CTLL-2 cells, respectively) are inhibited by olomoucine. Both yeast and Drosophila embryos were insensitive to olomoucine. Taken together the results of this Noah's Ark approach show that olomoucine arrests cells both at the $\text{G1}\text{S}$ and the $\text{G2}\text{M}$ boundaries, consistent with the hypothesis of a prevalent effect on the cdk2 and cdc2 kinases, respectively.     

Time-dependent modulation of carotid body afferent activity during and after intermittent hypoxia

The ventilatory response to several minutes of hypoxia consists of various time-dependent phenomena, some of which occur during hypoxia (e.g., short-term depression), whereas others appear on return to normoxia (e.g., posthypoxic frequency decline). Additional phenomena can be elicited by acute, intermittent hypoxia (e.g., progressive augmentation, long-term facilitation). Current data suggest that these phenomena originate centrally. We tested the hypothesis that carotid body afferent activity undergoes time-dependent modulation, consistent with a direct role in these ventilatory phenomena. Using an in vitro rat carotid body preparation, we found that 1) afferent activity declined during the first 5 min of severe (40 Torr Po(2)), moderate (60 Torr Po(2)), or mild (80 Torr Po(2)) hypoxia; 2) after return to normoxia (100 Torr Po(2)) and after several minutes of moderate or severe hypoxia, afferent activity was transiently reduced compared with prehypoxic levels; and 3) with successive 5-min bouts of mild, moderate, or severe hypoxia, afferent activity during bouts increased progressively. We call these phenomena sensory hypoxic decline, sensory posthypoxic decline, and sensory progressive augmentation, respectively. These phenomena were stimulus specific: similar phenomena were not seen with 5-min bouts of normoxic hypercapnia (100 Torr Po(2) and 50-60 Torr Pco(2)) or hypoxic hypocapnia (60 Torr Po(2) and 30 Torr Pco(2)). However, bouts of either normoxic hypercapnia or hypocapnic hypoxia resulted in sensory long-term facilitation. We suggest time-dependent carotid body activity acts in parallel with central mechanisms to shape the dynamics of ventilatory responses to respiratory chemostimuli.     

Oxygen-dependent PAF receptor binding and intracellular signaling in ovine fetal pulmonary vascular smooth muscle

Circulating levels of platelet-activating factor (PAF) are high in the fetus, and PAF is active in maintaining high PVR in fetal hypoxia (Ibe BO, Hibler S, Raj J. J Appl Physiol 85: 1079-1085, 1998). PAF synthesis by fetal pulmonary vascular smooth muscle cells (PVSMC) is high in hypoxia, but how oxygen tension affects PAF receptor (PAF-r) binding in PVSMC is not known. We studied the effect of oxygen tension on PAF-r binding and signaling in fetal PVSMC. PAF binding was saturable. PAF-r density (B(max): fmol/10(6) cells; means +/- SE, n = 6), 25.2 +/- 0.77 during hypoxia (Po(2) <40 Torr), was higher than 13.9 +/- 0.44 during normoxia (Po(2) approximately 100 Torr). K(d) was twofold lower in hypoxia than normoxia. PAF-r protein expression, 35-40% greater in hypoxia, was inhibited by cycloheximide, a protein synthesis inhibitor, suggesting translational regulation. IP(3) release, an index of PAF-r-mediated cell signaling, was greater in hypoxia (EC(50): hypoxia, 2.94 +/- 0.61; normoxia, 5.85 +/- 0.51 nM). Exogenous PAF induced 50-90% greater intracellular calcium flux in cells during hypoxia, indicating hypoxia augments PAF-r-mediated cell signaling. PAF-r phosphorylation, with or without 5 nM PAF, was 40% greater in hypoxia. These data show 1) hypoxia upregulates PAF-r binding, PAF-r phosphorylation, and PAF-r-mediated intracellular signaling, evidenced by augmented IP(3) production and intracellular Ca(2+) flux; and 2) hypoxia-induced PAF-r phosphorylation results in activation of PAF-r-mediated signal transduction. The data suggest the fetal hypoxic environment facilitates PAF-r binding and signaling, thereby promoting PAF-mediated pulmonary vasoconstriction and maintenance of high PVR in utero.     

Activation of mast cells by systemic hypoxia, but not by local hypoxia, mediates increased leukocyte-endothelial adherence in cremaster venules.

Systemic hypoxia, produced by lowering inspired Po2, induces a rapid inflammation in several microcirculations, including cremaster muscle. Mast cell activation is a necessary element of this response. Selective reduction of cremaster microvascular Po2 (PmO2) with normal systemic arterial Po2 (PaO2; cremaster hypoxia/systemic normoxia), however, does not elicit increased leukocyte-endothelial adherence (LEA) in cremaster venules. This could be due to a short time of leukocyte exposure to the hypoxic cremaster environment. Conversely, LEA increases when PaO2 is lowered, while cremaster PmO2 remains high (cremaster normoxia/systemic hypoxia). An alternative explanation of these results is that a mediator released from a central site during systemic hypoxia initiates the inflammatory cascade. We hypothesized that if this is the case, cremaster mast cells would be activated during cremaster normoxia/systemic hypoxia, but not during cremaster hypoxia/systemic normoxia. The microcirculation of rat cremaster muscles was visualized by using intravital microscopy. Cremaster PmO2 was measured with a phosphorescence quenching method. Cremaster hypoxia/systemic normoxia (PmO2 7 +/- 1 Torr, PaO2 87 +/- 2 Torr) did not increase LEA; however, topical application of the mast cell activator compound 48/80 under these conditions did increase LEA. The effect of compound 48/80 on LEA was blocked by topical cromolyn, a mast cell stabilizer. LEA increased during cremaster normoxia/systemic hypoxia, (PmO2 64 +/- 5 Torr, PaO2 33 +/- 2 Torr); this increase was blocked by topical cromolyn. The results suggest that mast cell stimulation occurs only when PaO2 is reduced, independent of cremaster PmO2, and support the idea of a mediator that is released during systemic hypoxia and initiates the inflammatory cascade.     

Signaling and apoptosis differences between severe hypoxia and desferoxamine treatment of human epithelial cells

The mechanisms by which cells undergo proliferation arrest or cell death in response to hypoxia are still not completely understood. Originally, we showed that HeLa and Hep3B carcinoma cells undergo different proliferation responses in hypoxia. We now show that these 2 cell lines also have different cell death responses to severe hypoxia, with HeLa showing both cell cycle arrest and apoptosis (as early as 12 h after hypoxia treatment), and Hep3B showing resistance to both. Hypoxia-induced apoptosis in Hela was associated with decreases of both phospho-S473- and -T308-AKT and loss of AKT function, whereas Hep3B cells were resistant to hypoxia-induced apoptosis and did not lose phospho-AKT or AKT function. We then decided to test if our observations were confirmed using a hypoxia mimic, desferoxamine. Desferoxamine treatment yielded cell cycle arrest in HeLa and moderate arrest in Hep3B but, surprisingly, did not induce notable apoptosis of either cell line with up to 24 h of treatment. Hypoxia-treated normal human mammary epithelial cells also showed hypoxia-induced apoptosis. Interestingly, in these cell lines, there was a complete correlation between loss of phospho-AKT and (or) total AKT, and susceptibility to hypoxia-induced apoptosis. Our data suggests a model in which regulated loss of active AKT at a precise time point in hypoxia may be associated with apoptosis in susceptible cells.     

Spindle checkpoint proteins Mad1 and Mad2 are required for cytostatic factor-mediated metaphase arrest

In cells containing disrupted spindles, the spindle assembly checkpoint arrests the cell cycle in metaphase. The budding uninhibited by benzimidazole (Bub) 1, mitotic arrest-deficient (Mad) 1, and Mad2 proteins promote this checkpoint through sustained inhibition of the anaphase-promoting complex/cyclosome. Vertebrate oocytes undergoing meiotic maturation arrest in metaphase of meiosis II due to a cytoplasmic activity termed cytostatic factor (CSF), which appears not to be regulated by spindle dynamics. Here, we show that microinjection of Mad1 or Mad2 protein into early Xenopus laevis embryos causes metaphase arrest like that caused by Mos. Microinjection of antibodies to either Mad1 or Mad2 into maturing oocytes blocks the establishment of CSF arrest in meiosis II, and immunodepletion of either protein blocked the establishment of CSF arrest by Mos in egg extracts. A Mad2 mutant unable to oligomerize (Mad2 R133A) did not cause cell cycle arrest in blastomeres or in egg extracts. Once CSF arrest has been established, maintenance of metaphase arrest requires Mad1, but not Mad2 or Bub1. These results suggest a model in which CSF arrest by Mos is mediated by the Mad1 and Mad2 proteins in a manner distinct from the spindle checkpoint.     

Ca(2+)-promoted cyclin B1 degradation in mouse oocytes requires the establishment of a metaphase arrest

CDK1-cyclin B1 is a universal cell cycle kinase required for mitotic/meiotic cell cycle entry and its activity needs to decline for mitotic/meiotic exit. During their maturation, mouse oocytes proceed through meiosis I and arrest at second meiotic metaphase with high CDK1-cyclin B1 activity. Meiotic arrest is achieved by the action of a cytostatic factor (CSF), which reduces cyclin B1 degradation. Meiotic arrest is broken by a Ca2+ signal from the sperm that accelerates it. Here we visualised degradation of cyclin B1::GFP in oocytes and found that its degradation rate was the same for both meiotic divisions. Ca2+ was the necessary and sufficient trigger for cyclin B1 destruction during meiosis II; but it played no role during meiosis I and furthermore could not accelerate cyclin B1 destruction during this time. The ability of Ca2+ to trigger cyclin B1 destruction developed in oocytes following a restabilisation of cyclin B1 levels at about 12 h of culture. This was independent of actual first polar body extrusion. Thus, in metaphase I arrested oocytes, Ca2+ would induce cyclin B1 destruction and the first polar body would be extruded. In contrast to some reports in lower species, we found no evidence that oocyte activation was associated with an increase in 26S proteasome activity. We therefore conclude that Ca2+ mediates cyclin B1 degradation by increasing the activity of an E3 ubiquitin ligase. However, this stimulation occurs only in the presence of the ubiquitin ligase inhibitor CSF. We propose a model in which Ca2+ directly stimulates destruction of CSF during mammalian fertilisation.     

Effect of acute hypoxia on glomus cell Em and psi m as measured by fluorescence imaging.

We have reinvestigated the hypothesis of the relative importance of glomus cell plasma and mitochondrial membrane potentials (E(m) and psi(m), respectively) in acute hypoxia by a noninvasive fluorescence microimaging technique using the voltage-sensitive dyes bis-oxonol and JC-1, respectively. Short-term (24 h)-cultured rat glomus cells and cultured PC-12 cells were used for the study. Glomus cell E(m) depolarization was indirectly confirmed by an increase in bis-oxonol (an anionic probe) fluorescence due to a graded increase in extracellular K(+). Fluorescence responses of glomus cell E(m) to acute hypoxia (approximately 10 Torr Po(2)) indicated depolarization in 20%, no response in 45%, and hyperpolarization in 35% of the cells tested, whereas all PC-12 cells consistently depolarized in response to hypoxia. Furthermore, glomus cell E(m) hyperpolarization was confirmed with high CO (approximately 500 Torr). Glomus cell psi(m) depolarization was indirectly assessed by a decrease in JC-1 (a cationic probe) fluorescence. Accordingly, 1 microM carbonyl cyanide p-trifluoromethoxyphenylhydrazone (an uncoupler of oxidative phosphorylation), high CO (a metabolic inhibitor), and acute hypoxia (approximately 10 Torr Po(2)) consistently depolarized the mitochondria in all glomus cells tested. Likewise, all PC-12 cell mitochondria depolarized in response to FCCP and hypoxia. Thus, although bis-oxonol could not show glomus cell depolarization consistently, JC-1 monitored glomus cell mitochondrial depolarization as an inevitable phenomenon in hypoxia. Overall, these responses supported our "metabomembrane hypothesis" of chemoreception.