Activated M-phase-promoting factor (MPF) is exported from the nucleus of starfish oocytes to increase the sensitivity of the Ins(1,4,5)P3 receptors

2003 ◽  
Vol 31 (1) ◽  
pp. 79-82 ◽  
Author(s):  
L. Santella ◽  
E. Ercolano ◽  
D. Lim ◽  
G.A. Nusco ◽  
F. Moccia
Keyword(s):  
Germinal Vesicle ◽  
Actin Binding ◽  
Cyclin Dependent Kinase ◽  
Ip3 Receptors ◽  
Large Nucleus ◽  
Nuclear Compartment ◽  
M Phase ◽  
Increased Sensitivity ◽  
Meiotic Cycle

Starfish oocytes that are extracted from the ovaries are arrested at the prophase of the first meiotic division. At this stage of maturation, they are characterized by a large nucleus called the germinal vesicle. Meiosis resumption (maturation) can be induced in vitro by adding the hormone 1-methyladenine (1-MA) to the seawater in which the oocytes are suspended. Earlier work in our laboratory had detected Ca2+ increases in both the cytoplasm and the nucleus of the oocytes approx. 2 min after the 1-MA challenge. The nuclear Ca2+ increase was found to be essential for the continuation of the meiotic cycle, since the injection of bis-(o-aminophenoxy)ethane-N,N,N´,N´-tetra-acetic acid (BAPTA) into the nuclear compartment completely blocked the re-initiation of the cell cycle. We have recently confirmed, using confocal microscopy, that the cytoplasmic and nuclear Ca2+ pools are regulated independently and that the nuclear envelope in starfish oocytes is not freely permeated by the Ca2+ wave that sweeps across the nuclear region. Studies by others have shown that the sensitivity of the Ins(1,4,5)P3 (IP3) receptors (IP3Rs) to IP3 increases during oocyte maturation, so that they release progressively more calcium in response to the injection of IP3, as maturation proceeds. We have now shown that the increased sensitivity of the IP3Rs may depend on the activation of the cyclin-dependent kinase, MPF (M-phase-promoting factor) that occurs in the nucleus. MPF does not directly phosphorylate IP3Rs but phosphorylates instead the actin-binding protein actin depolymerization factor (ADF)/cofilin.

scholarly journals Cell cycle dynamics of an M-phase-specific cytoplasmic factor in Xenopus laevis oocytes and eggs.

1984 ◽  
Vol 98 (4) ◽  
pp. 1247-1255 ◽  
Author(s):  
J Gerhart ◽  
M Wu ◽  
M Kirschner
Keyword(s):  
Cell Cycle ◽  
Xenopus Laevis ◽  
Germinal Vesicle ◽  
Small Scale ◽  
Xenopus Laevis Oocytes ◽  
Germinal Vesicle Breakdown ◽  
Cytoplasmic Factor ◽  
M Phase ◽  
Cytostatic Factor ◽  
Meiotic Cycle

We have examined the regulation of maturation-promoting factor (MPF) activity in the mitotic and meiotic cell cycles of Xenopus laevis eggs and oocytes. To this end, we developed a method for the small scale extraction of eggs and oocytes and measured MPF activity in extracts by a dilution end point assay. We find that in oocytes, MPF activity appears before germinal vesicle breakdown and then disappears rapidly at the end of the first meiotic cycle. In the second meiotic cycle, MPF reappears before second metaphase, when maturation arrests. Thus, MPF cycling coincides with the abbreviated cycles of meiosis. When oocytes are induced to mature by low levels of injected MPF, cycloheximide does not prevent the appearance of MPF at high levels in the first cycle. This amplification indicates that an MPF precursor is present in the oocyte and activated by posttranslational means, triggered by the low level of injected MPF. Furthermore, MPF disappears approximately on time in such oocytes, indicating that the agent for MPF inactivation is also activated by posttranslational means. However, in the absence of protein synthesis, MPF never reappears in the second meiotic cycle. Upon fertilization or artificial activation of normal eggs, MPF disappears from the cytoplasm within 8 min. For a period thereafter, the inactivating agent remains able to destroy large amounts of MPF injected into the egg. It loses activity just as endogenous MPF appears at prophase of the first mitotic cycle. The repeated reciprocal cycling of MPF and the inactivating agent during cleavage stages is unaffected by colchicine and nocodazole and therefore does not require the effective completion of spindle formation, mitosis, or cytokinesis. However, MPF appearance is blocked by cycloheximide applied before mitosis; and MPF disappearance is blocked by cytostatic factor. In all these respects, MPF and the inactivating agent seem to be tightly linked to, and perhaps participate in, the cell cycle oscillator previously described for cleaving eggs of Xenopus laevis (Hara, K., P. Tydeman, and M. Kirschner, 1980, Proc. Natl. Acad. Sci. USA, 77:462-466).

Effect of dehydroleucodine on meiosis reinitiation in Bufo arenarum denuded oocytes

Zygote ◽  
2007 ◽  
Vol 15 (2) ◽  
pp. 183-187 ◽  
Author(s):  
G. Sánchez Toranzo ◽  
O.S. Giordano ◽  
L.A. López ◽  
M.I. Bühler
Keyword(s):  
Germinal Vesicle ◽  
Inhibitory Effect ◽  
Follicle Cells ◽  
Dependent Manner ◽  
Bufo Arenarum ◽  
M Phase ◽  
Amplification Loop ◽  
Activation Cascade ◽  
P34 Cdc2

SummaryIn amphibian oocytes meiosis, the transition from G2 to M phase is regulated by the maturation promoting factor (MPF), a complex of the cyclin-dependent kinase p34/cdc2 and cyclin B. In immature oocytes there is an inactive complex (pre-MPF), in which cdc2 is phosphorylated on both Thr-161 and Thr-14/Tyr-15 residues. The dephosphorylation of Thr-14/Tyr-15 is necessary for the start of MPF activation and it is induced by the activation of cdc25 phosphatase. Late, to complete the activation, a small amount of active MPF induces an auto-amplification loop of MPF stimulation (MPF amplification). Dehydroleucodine (DhL) is a sesquiterpenic lactone that inhibits mammalian cell proliferation in G2. We asked whether DhL interferes with MPF activation. For this question, the effect of DhL (up to 30 μM) on the resumption of meiosis was evaluated, and visualized by germinal vesicle break down (GVBD), of Bufo arenarum oocytes induced in vitro by either: (i) removing follicle cells; (ii) progesterone stimulation; (iii) VG-content injection; or (iv) injection of mature cytoplasm. The results show that DhL induced GVBD inhibition, in a dose-dependent manner, in spontaneous and progesterone-induced oocyte maturation. Nevertheless, DhL at the doses assayed had no effect on GVBD induced by mature cytoplasm injection, but exerted an inhibitory effect on GVBD induced by GV content. On the basis of these results, we interpreted that DhL does not inhibit MPF amplification and that the target of DhL is any event in the early stages of the cdc25 activation cascade.

scholarly journals Pleiotropic Defects Caused by Loss of the Proteasome-Interacting Factors Rad23 and Rpn10 of Saccharomyces cerevisiae

Genetics ◽  
1999 ◽  
Vol 153 (1) ◽  
pp. 69-79
Author(s):  
David Lambertson ◽  
Li Chen ◽  
Kiran Madura
Keyword(s):  
Slow Growth ◽  
26S Proteasome ◽  
Cold Sensitivity ◽  
Genetic Studies ◽  
Single Mutant ◽  
M Phase ◽  
Cold Sensitive ◽  
Increased Sensitivity

Abstract Rad23 is a member of a novel class of proteins that contain unprocessed ubiquitin-like (UbL) domains. We showed recently that a small fraction of Rad23 can form an interaction with the 26S proteasome. Similarly, a small fraction of Rpn10 is a component of the proteasome. Rpn10 can bind multiubiquitin chains in vitro, but genetic studies have not clarified its role in vivo. We report here that the loss of both Rad23 and Rpn10 results in pleiotropic defects that are not observed in either single mutant. rad23Δ rpn10Δ displays slow growth, cold sensitivity, and a pronounced G2/M phase delay, implicating overlapping roles for Rad23 and Rpn10. Although rad23Δ rpn10Δ displays similar sensitivity to DNA damage as a rad23Δ single mutant, deletion of RAD23 in rpn10Δ significantly increased sensitivity to canavanine, a phenotype associated with an rpn10Δ single mutant. A mutant Rad23 that is unable to bind the proteasome (ΔUbLrad23) does not suppress the canavanine or cold-sensitive defects of rad23Δ rpn10Δ, demonstrating that Rad23/proteasome interaction is related to these effects. Finally, the accumulation of multiubiquitinated proteins and the stabilization of a specific proteolytic substrate in rad23Δ rpn10Δ suggest that proteasome function is altered.

scholarly journals Host cell factors controlling vimentin organization in the Xenopus oocyte.

1992 ◽  
Vol 119 (4) ◽  
pp. 855-866 ◽  
Author(s):  
J A Dent ◽  
R B Cary ◽  
J B Bachant ◽  
A Domingo ◽  
M W Klymkowsky
Keyword(s):  
Direct Interaction ◽  
Germinal Vesicle ◽  
Germinal Vesicle Breakdown ◽  
Head Domain ◽  
M Phase ◽  
Filament Bundles ◽  
Keratin Filament ◽  
Vimentin Filaments

To study vimentin filament organization in vivo we injected Xenopus oocytes, which have no significant vimentin system of their own, with in vitro-synthesized RNAs encoding Xenopus vimentins. Exogenous vimentins were localized primarily to the cytoplasmic surface of the nucleus and to the subplasma membrane "cortex." In the cortex of the animal hemisphere, wild-type vimentin forms punctate structures and short filaments. In contrast, long anastomosing vimentin filaments are formed in the vegetal hemisphere cortex. This asymmetry in the organization of exogenous vimentin is similar to that of the endogenous keratin system (Klymkowsky, M. W., L. A. Maynell, and A. G. Polson. 1987. Development (Camb.). 100:543-557), which suggests that the same cellular factors are responsible for both. Before germinal vesicle breakdown, in the initial stage of oocyte maturation, large vimentin and keratin filament bundles appear in the animal hemisphere. As maturation proceeds, keratin filaments fragment into soluble oligomers (Klymkowsky, M. W., L. A. Maynell, and C. Nislow. 1991. J. Cell Biol. 114:787-797), while vimentin filaments remain intact and vimentin is hyperphosphorylated. To examine the role of MPF kinase in the M-phase reorganization of vimentin we deleted the conserved proline of vimentin's single MPF-kinase site; this mutation had no apparent effect on the prophase or M-phase behavior of vimentin. In contrast, deletion of amino acids 19-68 or 18-61 of the NH2-terminal "head" domain produced proteins that formed extended filaments in the animal hemisphere of the prophase oocyte. We suggest that the animal hemisphere cortex of the prophase oocyte contains a factor that actively suppresses the formation of extended vimentin filaments through a direct interaction with vimentin's head domain. During maturation this "suppressor of extended filaments" appears to be inactivated, leading to the formation of an extended vimentin filament system.

231 STAGE-DEPENDENT CHANGES OF IP3R1 PHOSPHORYLATION DURING IN VITRO MATURATION AND DEVELOPMENT IN PIG OOCYTES

2008 ◽  
Vol 20 (1) ◽  
pp. 195
Author(s):  
J. Ito ◽  
C. Suzukamo ◽  
T. Mochida ◽  
A. Furugaichi ◽  
N. Nakajima ◽  
...  
Keyword(s):  
Cell Cycle ◽  
In Vitro Maturation ◽  
Mammalian Species ◽  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Cell Stage ◽  
Mouse Oocytes ◽  
M Phase ◽  
Pn Stage

During fertilization in mammalian species, a sperm-induced intracellular Ca2+ signal [Ca2+] is well suited to mediate the highly specialized spatiotemporal patterns of [Ca2+]i responses that underlie fertilization. Recently, we demonstrated that the expression pattern of inositol 1,4,5- triphosphate receptor type 1 (IP3R1) did not change during in vitro maturation and parthenogenetic activation in mouse oocytes; however, the phosphorylation status of IP3R1 depended on the cell cycle during meiosis. Moreover, it was shown that IP3R1 phosphorylation played a crucial role in the induction of [Ca2+]i oscillations (Lee et al. 2006 Development 133, 4355–4365). In other species, expression of IP3R1, especially phosphorylation levels of IP3R1 during meiosis, has not been examined. The aim of this study was to examine the kinetics of IP3R1 expression and phosphorylation during in vitro maturation and activation in pig oocytes. Immature oocytes at the germinal vesicle (GV) stage were collected from ovaries and cultured in modified NCSU37 up to 48 h. After culture, cumulus cells were removed and oocytes were parthenogenetically activated by 25 µm Ca2+ ionophore for 3 min and 2 mm 6-DMAP for 6 h. After activation, oocytes were further cultured up to the 2-cell stage. Groups of 30 oocytes were collected at each culture period for detection of IP3R1. According to our previous report in the mouse, IP3R1s were detected by western blotting using MPM-2 and Rbt03 antibody for detecting IP3R1 phosphorylation and total IP3R1 expression, respectively (Lee et al. 2006). In pig oocytes, IP3R1 was abundantly expressed at the GV stage. The total level of IP3R1 expression did not change during in vitro maturation or after activation. However, phosphorylated IP3R1 levels increased by 24 h although they were undetectable at the start of culture. Phosphorylation of IP3R1 reached maximal levels at 36 h. After activation, phosphorylation levels decreased progressively until the pronuclear (PN) stage. Phosphorylation of IP3R1 was observed at mitosis I to some extent. From these results, we detected for the first time IP3R1 expression and phosphorylation in pig oocytes. Moreover, our data suggest that phosphorylation of IP3R1 is dependent on cell cycle at least during meiosis, especially M-phase, as already shown for mouse oocytes. In vitro kinase assays for p34cdc2 kinase and MAPK will be carried out to clarify the relationship between IP3R1 phosphorylation and M-phase kinase(s).

scholarly journals Proteomic analysis of porcine oocytes during in vitro maturation reveals essential role for the ubiquitin C-terminal hydrolase-L1

Reproduction ◽  
2007 ◽  
Vol 134 (4) ◽  
pp. 559-568 ◽  
Author(s):  
Andrej Susor ◽  
Zdenka Ellederova ◽  
Lucie Jelinkova ◽  
Petr Halada ◽  
Daniel Kavan ◽  
...  
Keyword(s):  
Proteomic Analysis ◽  
Essential Role ◽  
Specific Inhibitor ◽  
Germinal Vesicle ◽  
Cyclin B1 ◽  
Cyclin Dependent Kinase ◽  
Proteomic Approach ◽  
Metaphase I

In this study, we performed proteomic analysis of porcine oocytes during in vitro maturation. Comparison of oocytes at the initial and final stages of meiotic division characterized candidate proteins that were differentially synthesized during in vitro maturation. While the biosynthesis of many of these proteins was significantly decreased, we found four proteins with increased biosynthetic rate, which are supposed to play an essential role in meiosis. Among them, the ubiquitin C-terminal hydrolase-L1 (UCH-L1) was identified by mass spectrometry. To study the regulatory role of UCH-L1 in the process of meiosis in pig model, we used a specific inhibitor of this enzyme, marked C30, belonging to the class of isatin O-acyl oximes. When germinal vesicle (GV) stage cumulus-enclosed oocytes were treated with C30, GV breakdown was inhibited after 28 h of culture, and most of the oocytes were arrested at the first meiosis after 44 h. The block of metaphase I–anaphase transition was not completely reversible. In addition, the inhibition of UCH-L1 resulted in elevated histone H1 kinase activity, corresponding to cyclin–dependent kinase(CDK1)–cyclin B1 complex, and a low level of monoubiquitin. These results supported the hypothesis that UCH-L1 might play a role in metaphase I–anaphase transition by regulating ubiquitin-dependent proteasome mechanisms. In summary, a proteomic approach coupled with protein verification study revealed an essential role of UCH-L1 in the completion of the first meiosis and its transition to anaphase.

scholarly journals Cycling of intracellular pH during cell division of Xenopus embryos is a cytoplasmic activity depending on protein synthesis and phosphorylation.

1990 ◽  
Vol 111 (2) ◽  
pp. 523-532 ◽  
Author(s):  
N Grandin ◽  
M Charbonneau
Keyword(s):  
Cell Division ◽  
Intracellular Ph ◽  
Germinal Vesicle ◽  
Egg Activation ◽  
Xenopus Embryos ◽  
M Phase ◽  
The Difference ◽  
Two Peaks

In Xenopus embryos, the successive and rapid cell divisions that follow fertilization are accompanied by periodic oscillations of intracellular pH (pHi). Cycling of pHi occurs in phase with several other oscillatory activities, namely nuclear divisions, M phase-promoting factor (MPF) activity, and surface contraction waves (SCWs). We report that treatments that abolish cycling of MPF activity and the SCWs also suppress the pHi oscillations, whereas those that block cell division without affecting neither MPF activity nor the SCWs do not suppress the pHi oscillations. Experiments on enucleated oocytes, matured in vitro and activated, demonstrated that the activity governing the rhythmicity of the pHi oscillations resided in the cytoplasm of the oocyte. In this respect, the activity responsible for the pHi oscillations was different from that which drives the SCWs, which necessitated the presence of the oocyte germinal vesicle (Ohsumi et al., 1986), but more closely resembled MPF activity that did not require the presence of the oocyte germinal vesicle (Dabauvalle et al., 1988). In mature eggs enucleated at the time of egg activation, the pHi oscillations were similar to those in control nucleated eggs, whereas the period between two peaks of SCWs was 35-60 min vs. 20-35 min in nucleated control eggs. Previous studies had shown that the periodicity of SCWs was larger in anucleate egg fragments than in their nucleate counterparts (Sakai and Kubota, 1981), the difference being on the order of 6-15 min (Shinagawa, 1983). However, in these previous studies, enucleation was performed 30-50 min after fertilization. Our results clearly demonstrate that the periodicity of the SCWs is lengthened when the interval between egg activation and enucleation is shortened, thereby providing an easier way to assess the nuclear dependency of the SCWs. Finally, the various possibilities concerning the role of pHi cycling during cell division are discussed.

Meiotic inhibition with different cyclin-dependent kinase inhibitors in bovine oocytes and its effects on maturation and embryo development

Zygote ◽  
2004 ◽  
Vol 12 (3) ◽  
pp. 197-204 ◽  
Author(s):  
Paulo Roberto Adona ◽  
Cláudia Lima Verde Leal
Keyword(s):  
Embryo Development ◽  
Kinase Inhibitors ◽  
Germinal Vesicle ◽  
Oocyte Activation ◽  
Cyclin Dependent Kinase ◽  
Nuclear Maturation ◽  
Inhibition Control ◽  
Inhibition Experiment ◽  
Bovine Oocytes

Cyclin-dependent kinase inhibitors (CDKIs) such as butyrolactone I (BL-I) and roscovitine (ROS) maintain bovine oocytes blocked at the germinal vesicle (GV) stage. Bohemine (BOH), another CDKI, has been used for oocyte activation. The objective of this study was to determine whether BOH blocks meiosis and to compare its efficiency with other CDKIs (ROS and BL-I). Oocytes were cultured for 24 h in 0, 50, 100 and 150 μM BOH to determine the best concentration for blocking meiosis (experiment 1). GV rates were 3.3%, 64.5%, 83.3% and 88.9% (0, 50, 100 and 150 μM, respectively). Experiment 2 compared meiotic inhibition efficiency of BOH (100 μM), ROS (25 μM) and BL-I (100 μM). BL-I presented the highest GV rates (97.5%). BOH and ROS were similar to each other (85.4% and 79.9%, respectively). To assess the reversibility of meiotic inhibition (experiment 3), oocytes underwent in vitro maturation (IVM) for 18 h after the 24 h inhibition. Control oocytes were submitted to IVM for 18 h (C18) or 24 h (C24). Maturation rates were either similar to (ROS and BL-I: 96.0% and 93.6%, respectively) or superior to (BOH, 96.9%) C24 (91.0%). All groups were superior to C18 (82.5%). In experiment 4, oocytes were treated as in experiment 3 and then in vitro fertilized and cultured for 8 days. Blastocyst rates for BL-I (32.3%) were similar to C24 (35.0%), while those for BOH (20.2%) and ROS (24.2%) were inferior. All groups were inferior to C18 (43.4%). The results show that: (a) BOH inhibits meiosis resumption; (b) BL-I is the most effective of the CDKIs tested for blocking meiosis; (c) culture of oocytes with meiosis inhibitors is fully reversible in terms of nuclear maturation but they may either decrease (BOH and ROS) or maintain (BL-I) embryo development rates.

Inhibition of Platelet Aggregation by Ethanol in Vitro Shows Specificity for Aggregating Agent Used and Is Influenced by Platelet Lipid Composition

1982 ◽  
Vol 48 (01) ◽  
pp. 049-053 ◽  
Author(s):  
C G Fenn ◽  
J M Littleton
Keyword(s):  
Platelet Aggregation ◽  
Lipid Composition ◽  
Saturated Fatty Acids ◽  
Calcium Ionophore ◽  
Cholesterol Content ◽  
Membrane Lipid ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
Increased Sensitivity

SummaryEthanol at physiologically tolerable concentrations inhibited platelet aggregation in vitro in a relatively specific way, which may be influenced by platelet membrane lipid composition. Aggregation to collagen, calcium ionophore A23187 and thrombin (low doses) were often markedly inhibited by ethanol, adrenaline and ADP responses were little affected, and aggregation to exogenous arachidonic acid was actually potentiated by ethanol. Aggregation to collagen, thrombin and A23187 was inhibited more by ethanol in platelets enriched with saturated fatty acids than in those enriched with unsaturated fats. Platelets enriched with cholesterol showed increased sensitivity to ADP, arachidonate and adrenaline but this increase in cholesterol content did not appear to influence the inhibition by ethanol of platelet responses. The results suggest that ethanol may inhibit aggregation by an effect on membrane fluidity and/or calcium mobilization resulting in decreased activity of a membrane-bound phospholipase.

a Novel Combinational Nanodrug Delivery System Induces Synergistic Inhibition of Lung Adenocarcinoma Cells in vitro

2020 ◽  
Vol 17 ◽  
Author(s):  
Mingliang Fan ◽  
Jiping Li
Keyword(s):  
Lung Adenocarcinoma ◽  
Delivery System ◽  
The Body ◽  
Anticancer Efficacy ◽  
Nanodrug Delivery ◽  
Drug Molecules ◽  
Cellular Assays ◽  
Lung Adenocarcinoma Cell Line ◽  
M Phase

Background: The combination of two or more therapeutic drugs is an attractive approach to improve the treatment of experimental tumors. Leveraging nanocarriers for combinational drug delivery can allow a control over drug biological fate and promote co-localization in the same area of the body. However, there are certain concerns regarding the biodegradability and potential long-term toxicity arising from these synthetic nanoscale carriers. Objective: Our aim was to develop a combinational nanodrug delivery system formed by self-assembling of amphiphilic drug molecules，minimizing potential toxicities associated with using additional synthetic nanocarriers. Methods: A novel prodrug chlorambucil gemcitabine conjugate was synthesized, this prodrug was used for the encapsulation of an additional hydrophobic anticancer drug paclitaxel, taking the form of combinational nanodrugs. Particle size and zeta potential were evaluated, cytotoxicity assay and apoptosis/cell cycle analysis were also performed to validate the anticancer efficacy of the combinational nanodrugs. Results: The combinational nanodrugs were acquired by means of nanoprecipitation. In A549 lung adenocarcinoma cell line, cellular assays revealed that co-delivery of low dosage paclitaxel with chlorambucil gemcitabine conjugate can act synergistically to inhibit cell growth and induce accumulation of cells in the G2/M phase with a concomitant decrease in G0/G1 compartment. Conclusion: Chlorambucil gemcitabine conjugate and paclitaxel can co-assemble into composite nanoparticles by a nanoprecipitation process and the resulting combinational nanodrugs showed synergistic anticancer effect. This synthetic nanocarrier-free approach might broaden the nanodrug concept and have potential in cancer therapy.

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