scholarly journals 011.Waking up the egg. How the sperm regulates exit out of the meiotic cell cycle

2004 ◽  
Vol 16 (9) ◽  
pp. 11
Author(s):  
K. T. Jones
Keyword(s):  
Cell Cycle ◽  
Polar Body ◽  
Cyclin B1 ◽  
26S Proteasome ◽  
Calcium Signal ◽  
Calcium Spikes ◽  
M Phase ◽  
Second Polar Body ◽  
Polar Body Extrusion ◽  
Mammalian Eggs

A series of calcium spikes are induced in the mammalian egg cytoplasm at fertilisation. These calcium spikes, which last for several hours, are the necessary and sufficient signal that stimulates the egg to escape from arrest at metaphase of the second meiotic division. Metaphase arrest is achieved by preventing the destruction of cyclin B1, the regulatory component of Maturation (M-Phase) Promoting Factor, and securin, which prevents segregation of sister chromatids. Both these proteins are destroyed by tagging with ubiquitin, using an E3 ligase the Anaphase-Promoting Complex (APC). Ubiquitination tags them for proteolysis by the 26S proteasome. Work from my lab has demonstrated that the sperm calcium signal works through activating the APC, not the 26S proteasome. Although we do not know which APC component is affected by calcium, this activation appears specific to a metaphase-arrested cell cycle state. More recently we have found that the APC is differently regulated at specific points during exit from meiosis II. Before extrusion of the second polar body it is the APC activator cdc20 that regulates APC activity. However, following extrusion of the second polar body cdh1 appears the major regulator. It is probable, therefore, that the calcium spiking affects the activity of both APCcdc20 and APCcdh1. This swap in APC activator at the time of second polar body extrusion has not been reported in eggs of other species, in fact non-mammalian eggs all lack cdh1. Since APCcdc20 and APCcdh1 have different substrate specificities, the function of APCcdh1 in mammalian eggs warrants further investigation.

scholarly journals Cell cycle modification during the transitions between meiotic M-phases in mouse oocytes

1992 ◽  
Vol 102 (3) ◽  
pp. 457-467 ◽  
Author(s):  
J.Z. Kubiak ◽  
M. Weber ◽  
G. Geraud ◽  
B. Maro
Keyword(s):  
Kinase Activity ◽  
Polar Body ◽  
Metaphase Arrest ◽  
Phosphorylated Proteins ◽  
Mouse Oocytes ◽  
Sequence Of Events ◽  
M Phase ◽  
Second Polar Body ◽  
Polar Body Extrusion ◽  
Centrosomal Proteins

When metaphase II-arrested mouse oocytes (M II) are activated very soon after ovulation, they respond abortively by second polar body extrusion followed by another metaphase arrest (metaphase III, M III; Kubiak, 1989). The M II/M III transition resembles the natural transition between the first and second meiotic metaphases (M I/M II). We observed that a similar sequence of events takes place during these two transitions: after anaphase, a polar body is extruded, the microtubules of the midbody disappear rapidly and a new metaphase spindle forms. The MPM-2 monoclonal antibody (which reacts with phosphorylated proteins associated with the centrosome during M-phase) stains discrete foci of peri-centriolar material only in metaphase arrested oocytes; during both transitional periods, a diffuse staining is observed, suggesting that these centrosomal proteins are dephosphorylated, as in a normal interphase. However, the chromosomes always remain condensed and an interphase network of microtubules is never observed during the transitional periods. Incorporation of 32P into proteins increases specifically during the transitional periods. Pulse-chase experiments, after labeling of the oocytes in M phase with 32P, showed that a 62 kDa phosphoprotein band disappears at the time of polar body extrusion. Histone H1 kinase activity (which reflects the activity of the maturation promoting factor) drops during both transitional periods to the level characteristic of interphase and then increases when the new spindle forms. Both the M I/M II and M II/M III transitions require protein synthesis as demonstrated by the effect of puromycin. These results suggest that the two M-phase/M-phase transitions are probably driven by the same molecular mechanism.

scholarly journals Mammalian egg activation: from Ca2+ spiking to cell cycle progression

Reproduction ◽  
2005 ◽  
Vol 130 (6) ◽  
pp. 813-823 ◽  
Author(s):  
Keith T Jones
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Polar Body ◽  
Signal Transduction Pathway ◽  
Cyclin B1 ◽  
Egg Activation ◽  
Anaphase Promoting Complex ◽  
Sister Chromatids ◽  
Dependent Protein ◽  
Second Polar Body

Mammalian eggs arrest at metaphase of the second meiotic division (MetII). Sperm break this arrest by inducing a series of Ca2+spikes that last for several hours. During this time cell cycle resumption is induced, sister chromatids undergo anaphase and the second polar body is extruded. This is followed by decondensation of the chromatin and the formation of pronuclei. Ca2+spiking is both the necessary and solely sufficient sperm signal to induce full egg activation. How MetII arrest is established, how the Ca2+spiking is induced and how the signal is transduced into cell cycle resumption are the topics of this review. Although the roles of most components of the signal transduction pathway remain to be fully investigated, here I present a model in which a sperm-specific phospholipase C (PLCζ) generates Ca2+spikes to activate calmodulin-dependent protein kinase II and so switch on the Anaphase-Promoting Complex/Cyclosome (APC/C). APC/C activation leads to securin and cyclin B1 degradation and in so doing allows sister chromatids to be segregated and to decondense.

301 ZINC IS INVOLVED IN PORCINE OOCYTE MEIOTIC MATURATION

2015 ◽  
Vol 27 (1) ◽  
pp. 239
Author(s):  
M.-H. Zhao ◽  
T. Kim ◽  
N.-H. Kim ◽  
X.-S. Cui
Keyword(s):  
Technology Development ◽  
Polar Body ◽  
Development Program ◽  
Germinal Vesicle ◽  
Cyclin B1 ◽  
Republic Of Korea ◽  
Metaphase Arrest ◽  
Second Polar Body ◽  
Polar Body Extrusion

Zinc is an extremely important trace element that plays important roles in several biological processes. In this study, we investigated the role of zinc during meiotic resumption and metaphase arrest in in vitro-matured porcine oocytes. Oocytes at either germinal vesicle (GV) or MII stage were treated with TPEN, a Zn2+ chelator. Meiotic resumption and activation were assayed. Effect of PMA, a PKC activator, on GV breakdown (GVBD) and oocytes activation after TPEN treatment were checked. Results showed that depletion of zinc with 3 µM TPEN-blocked oocytes at GV stage (60.85 ± 5.15 v. 15.60 ± 0.20%; P < 0.05) after 25 h of maturation. The 10-µM TPEN treatment at MII stage significantly (P < 0.05) increased pronucleus formation (90.61 ± 9.10 v. 5.56 ± 9.62%; P < 0.05) and the second polar body extrusion (93.64 ± 5.53 v. 8.59 ± 8.34%; P < 0.05). The p34cdc2 activity was decreased in both MII and GVBD oocytes that were treated with TPEN as compared to control. Phosphorylated MAPK measured by Western blot was also decreased in GVBD oocytes when zinc was depleted. This might be explained by the low expression of C-mos Cyclin B1 and Cdc2 at this stage. Treatment of the oocytes with PKC agonist PMA (100 nM) rescued the meiotic resumption arrest observed after TPEN treatment (GV stage: 26.91 ± 3.10 v. 83.89 ± 11.94%; P < 0.05). The level of phosphorylation of MAPK and p34cdc2 activity were rescued when PMA were used. Treatment oocytes with 100 nM PMA in the GV stage also increased the signal of zinc indicator, fluozin-3-a.m., by about 4-fold in cytoplasm (P < 0.05). These results showed that zinc regulates meiotic resumption probably through PKC. However, although the TPEN treatment reduced phosphorylation of PKC substrates in both meiotic resumption and the MII stage, rescue of PKC substrates phosphorylation with PMA did not prevent the activation of oocytes caused by zinc depletion. These data demonstrate that zinc regulates meiotic resumption via a PKC-dependent pathway, but independent of that in maintaining of metaphase arrest in porcine oocytes.This work was supported by the Bio-industry Technology Development Program, Ministry of Agriculture, Food and Rural Affairs, Republic of Korea, and by a grant from the Next-Generation BioGreen 21 Program (No. PJ009601 and PJ009098), Rural Development Administration, Republic of Korea.

Maturation promoting factor in ascidian oocytes is regulated by different intracellular signals at meiosis I and II

Development ◽  
1996 ◽  
Vol 122 (7) ◽  
pp. 1995-2003 ◽  
Author(s):  
G.L. Russo ◽  
K. Kyozuka ◽  
L. Antonazzo ◽  
E. Tosti ◽  
B. Dale
Keyword(s):  
Kinase Activity ◽  
Phase 1 ◽  
Polar Body ◽  
Calcium Transients ◽  
Phase 2 ◽  
Phase 3 ◽  
First Polar Body ◽  
Second Polar Body ◽  
Polar Body Extrusion ◽  
Maturation Promoting Factor

Using the fluorescent dye Calcium Green-dextran, we measured intracellular Ca2+ in oocytes of the ascidian Ciona intestinalis at fertilization and during progression through meiosis. The relative fluorescence intensity increased shortly after insemination in a single transient, the activation peak, and this was followed by several smaller oscillations that lasted for approximately 5 minutes (phase 1). The first polar body was extruded after the completion of the phase 1 transients, about 9 minutes after insemination, and then the intracellular calcium level remained at baseline for a period of 5 minutes (phase 2). At 14 minutes postinsemination a second series of oscillations was initiated that lasted 11 minutes (phase 3) and terminated at the time of second polar body extrusion. Phases 1 and 3 were inhibited by preloading oocytes with 5 mM heparin. Simultaneous measurements of membrane currents, in the whole-cell clamp configuration, showed that the 1–2 nA inward fertilization current correlated temporally with the activation peak, while a series of smaller oscillations of 0.1-0.3 nA amplitude were generated at the time of the phase 3 oscillations. Biochemical characterization of Maturation Promoting Factor (MPF) in ascidian oocytes led to the identification of a Cdc2-like kinase activity. Using p13suc1-sepharose as a reagent to precipitate the MPF complex, a 67 kDa (67 × 10(3) Mr) protein was identified as cyclin B. Histone H1 kinase activity was high at metaphase I and decreased within 5 minutes of insemination reaching a minimum level during phase 2, corresponding to telophase I. During phase 3, H1 kinase activity increased and then decayed again during telophase II. Oocytes preloaded with BAPTA and subsequently inseminated did not generate any calcium transients, nonetheless H1 kinase activity decreased 5 minutes after insemination, as in the controls, and remained low for at least 30 minutes. Injection of BAPTA during phase 2 suppressed the phase 3 calcium transients, and inhibited both the increase in H1 kinase activity normally encountered at metaphase II and second polar body extrusion.

scholarly journals Kinetochore Fibers Are Not Involved in the Formation of the First Meiotic Spindle in Mouse Oocytes, but Control the Exit from the First Meiotic M Phase

1999 ◽  
Vol 146 (1) ◽  
pp. 1-12 ◽  
Author(s):  
Stéphane Brunet ◽  
Angélica Santa Maria ◽  
Philippe Guillaud ◽  
Denis Dujardin ◽  
Jacek Z. Kubiak ◽  
...  
Keyword(s):  
Polar Body ◽  
Meiotic Spindle ◽  
Homologous Chromosomes ◽  
Mouse Oocytes ◽  
Sister Chromatids ◽  
M Phase ◽  
Continuous Presence ◽  
Set Up ◽  
Polar Body Extrusion ◽  
Reductional Division

During meiosis, two successive divisions occur without any intermediate S phase to produce haploid gametes. The first meiotic division is unique in that homologous chromosomes are segregated while the cohesion between sister chromatids is maintained, resulting in a reductional division. Moreover, the duration of the first meiotic M phase is usually prolonged when compared with mitotic M phases lasting 8 h in mouse oocytes. We investigated the spindle assembly pathway and its role in the progression of the first meiotic M phase in mouse oocytes. During the first 4 h, a bipolar spindle forms and the chromosomes congress near the equatorial plane of the spindle without stable kinetochore– microtubule end interactions. This late prometaphase spindle is then maintained for 4 h with chromosomes oscillating in the central region of the spindle. The kinetochore–microtubule end interactions are set up at the end of the first meiotic M phase (8 h after entry into M phase). This event allows the final alignment of the chromosomes and exit from metaphase. The continuous presence of the prometaphase spindle is not required for progression of the first meiotic M phase. Finally, the ability of kinetochores to interact with microtubules is acquired at the end of the first meiotic M phase and determines the timing of polar body extrusion.

scholarly journals Palmitic Acid Methyl Ester Induces G2/M Arrest in Human Bone Marrow-Derived Mesenchymal Stem Cells via the p53/p21 Pathway

2019 ◽  
Vol 2019 ◽  
pp. 1-15
Author(s):  
Jian-Hong Lin ◽  
Pei-Ching Ting ◽  
Wen-Sen Lee ◽  
Hung-Wen Chiu ◽  
Chun-An Chien ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
P53 Protein ◽  
Acid Methyl Ester ◽  
Cyclin B1 ◽  
Cycle Arrest ◽  
Acid Methyl ◽  
M Phase ◽  
Intracellular Ca ◽  
Palmitic Acid Methyl Ester

Bone marrow-derived mesenchymal cells (BM-MSCs) are able to differentiate into adipocytes, which can secrete adipokines to affect BM-MSC proliferation and differentiation. Recent evidences indicated that adipocytes can secrete fatty acid metabolites, such as palmitic acid methyl ester (PAME), which is able to cause vasorelaxation and exerts anti-inflammatory effects. However, effects of PAME on BM-MSC proliferation remain unclear. The aim of this study was to investigate the effect of PAME on human BM-MSC (hBM-MSC) proliferation and its underlying molecular mechanisms. hBM-MSCs were treated with PAME for 48 h and then subjected to various analyses. The results from the present study show that PAME significantly reduced the levels of G2/M phase regulatory proteins, cyclin-dependent kinase 1 (Cdk1), and cyclin B1 and inhibited proliferation in hBM-MSCs. Moreover, the level of Mdm2 protein decreased, while the levels of p21 and p53 protein increased in the PAME-treated hBM-MSCs. However, PAME treatment did not significantly affect apoptosis/necrosis, ROS generation, and the level of Cdc25C protein. PAME also induced intracellular acidosis and increased intracellular Ca2+ levels. Cotreatment with PAME and Na+/H+ exchanger inhibitors together further reduced the intracellular pH but did not affect the PAME-induced decreases of cell proliferation and increases of the cell population at the G2/M phase. Cotreatment with PAME and a calcium chelator together inhibited the PAME-increased intracellular Ca2+ levels but did not affect the PAME-induced cell proliferation inhibition and G2/M cell cycle arrest. Moreover, the half-life of p53 protein was prolonged in the PAME-treated hBM-MSCs. Taken together, these results suggest that PAME induced p53 stabilization, which in turn increased the levels of p53/p21 proteins and decreased the levels of Cdk1/cyclin B1 proteins, thereby preventing the activation of Cdk1, and eventually caused cell cycle arrest at the G2/M phase. The findings from the present study might help get insight into the physiological roles of PAME in regulating hBM-MSC proliferation.

scholarly journals TLC388 Induces DNA Damage and G2 Phase Cell Cycle Arrest in Human Non-Small Cell Lung Cancer Cells

2020 ◽  
Vol 27 (1) ◽  
pp. 107327481989797
Author(s):  
Kun-Ming Wu ◽  
Chih-Wen Chi ◽  
Jerry Cheng-Yen Lai ◽  
Yu-Jen Chen ◽  
Yu Ru Kou
Keyword(s):  
Lung Cancer ◽  
Cell Cycle ◽  
Cyclin B1 ◽  
Small Cell ◽  
Phase Cell ◽  
Small Cell Lung ◽  
Cycle Arrest ◽  
M Phase ◽  
G2 Phase ◽  
Induced Apoptosis

TLC388, a camptothecin-derivative targeting topoisomerase I, is a potential anticancer drug. In this study, its effect on A549 and H838 human non-small cell lung cancer (NSCLC) cells was investigated. Cell viability and proliferation were determined by thiazolyl blue tetrazolium bromide and clonogenic assays, respectively, and cell cycle analysis and detection of phosphorylated histone H3 (Ser10) were performed by flow cytometry. γ-H2AX protein; G2/M phase-associated molecules ataxia-telangiectasia mutated (ATM), CHK1, CHK2, CDC25C, CDC2, and cyclin B1; and apoptosis were assessed with immunofluorescence staining, immunoblotting, and an annexin V assay, respectively. The effect of co-treatment with CHIR124 (a checkpoint kinase 1 [CHK1] inhibitor) was also studied. TLC388 decreased the viability and proliferation of cells of both NSCLC lines in a dose-dependent manner. TLC388 inhibited the viability of NSCLC cell lines with an estimated concentration of 50% inhibition (IC50), which was 4.4 and 4.1 μM for A549 and H838 cells, respectively, after 24 hours. Moreover, it resulted in the accumulation of cells at the G2/M phase and increased γ-H2AX levels in A549 cells. Levels of the G2 phase–related molecules phosphorylated ATM, CHK1, CHK2, CDC25C, and cyclin B1 were increased in TLC388-treated cells. CHIR124 enhanced the cytotoxicity of TLC388 toward A549 and H838 cells and induced apoptosis of the former. TLC388 inhibits NSCLC cell growth by inflicting DNA damage and activating G2/M checkpoint proteins that trigger G2 phase cell cycle arrest to enable DNA repair. CHIR124 enhanced the cytotoxic effect of TLC388 and induced apoptosis.

scholarly journals In VitroAntiproliferative Effect ofArthrocnemum indicumExtracts on Caco-2 Cancer Cells through Cell Cycle Control and Related Phenol LC-TOF-MS Identification

2013 ◽  
Vol 2013 ◽  
pp. 1-11 ◽  
Author(s):  
Mondher Boulaaba ◽  
Khaoula Mkadmini ◽  
Soninkhishig Tsolmon ◽  
Junkyu Han ◽  
Abderrazak Smaoui ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cancer Cells ◽  
Antioxidant Activities ◽  
Cyclin B1 ◽  
Antiproliferative Effect ◽  
Cell Cycle Distribution ◽  
Dapi Staining ◽  
Human Colon Cancer ◽  
M Phase ◽  
Tof Ms

This study aimed to determinate phenolic contents and antioxidant activities of the halophyteArthrocnemum indicumshoot extracts. Moreover, the anticancer effect of this plant on human colon cancer cells and the likely underlying mechanisms were also investigated, and the major phenols were identified by LC-ESI-TOF-MS. Results showed that shoot extracts had an antiproliferative effect of about 55% as compared to the control and were characterised by substantial total polyphenol content (19 mg GAE/g DW) and high antioxidant activity (IC50=40 μg/mL for DPPH test). DAPI staining revealed that these extracts decrease DNA synthesis and reduce the proliferation of Caco-2 cells which were stopped at the G2/M phase. The changes in the cell-cycle-associated proteins (cyclin B1, p38, Erk1/2, Chk1, and Chk2) correlate with the changes in cell cycle distribution. Eight phenolic compounds were also identified. In conclusion,A. indicumshowed interesting antioxidant capacities associated with a significant antiproliferative effect explained by a cell cycle blocking at the G2/M phase. Taken together, these data suggest thatA. indicumcould be a promising candidate species as a source of anticancer molecules.

c-Myb Plays a Role InG2/M Cell Cycle Transition by Direct Regulation of Cyclin B1 Expression in Hematopoietic Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1355-1355 ◽  
Author(s):  
Yuji Nakata ◽  
Susan Shetzline ◽  
Chizuko Sakashita ◽  
Anna Kalota ◽  
Andrzej Ptasznik ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Hematopoietic Cells ◽  
Cyclin B1 ◽  
Leukemia Cells ◽  
Human Leukemia ◽  
Human Leukemia Cells ◽  
M Phase ◽  
Cell Cycle Transition ◽  
Myb Protein

Abstract Myb family transcription factors are found throughout the phyla, and recent studies have demonstrated that Drosophila myb, as well as plant and yeast c-myb-like transcription factors, play an important role in regulating transition though the G1/S and G2/M phases of the cell cycle. Myb’s ability to regulate passage through G2/M is due at least in part to its ability to induce Cyclin B1 expression. A recent study in human T98G ganglioblastoma cells revealed that E2F, together with B-Myb, regulated cyclin B1 expression. Though c-myb was expressed in these cells, it was not found in immunoprecipitated E2F-B-Myb protein complexes and for this reason was felt not to participate in cyclin B1 expression in these cells. Since c-myb plays such a critical role in regulating hematopoietic cell proliferation, and its role in regulating G2/M in blood cells has not previously been explored, we investigated whether c-myb was important is regulating this phase of the cell cycle using K562 and Mo7e cells, as well as PHA stimulated human T lymphocytes. In distinct contrast to findings reported for T98G cells, we now report that in normal and malignant human hematopoietic cells, c-Myb directly upregulates cyclin B1 expression. Several lines of evidence support this claim. First, cyclin B1 expression decreased in Mo7e human leukemia cells in which c-myb had been silenced with siRNA. siRNA targeted to B-myb also decreased cyclin B1 expression, while neither siRNA species decreased cdc2 or cyclin A in these cells. As expected, siRNA targeted against c-myb or B-myb impaired Mo7e cell proliferation. Simultaneous exposure to both siRNA blocked proliferation completely. Second, using an alternative strategy, an inducible dominant negative c-Myb protein also decreased cyclin B1 expression in K562 human leukemia cells. The expected consequence of this, accelerated exit from the M phase, was also observed. Third, we examined c-Myb expression in human T cells by western and Real Time PCR, pre and post PHA stimulation. c-Myb expression began to gradually increase in the G1 phase of cell cycle, continued to increase after S phase, with the maximal protein level being found in G2/M phase, and concordant with cyclin B1 expression. These results indicated a correlation between c-Myb and cyclin B1 expression but did not indicate if c-Myb regulated cyclin B1 expression directly. To address this question, several additional experiments were carried out. A CAT assay showed that overexpressing c-Myb protein could increase activity when driven by a cyclin B1 promoter construct ~5X compared to K562 control cells. Next, examination of the cyclin B1 promoter showed eight potential c-Myb binding sites. Two were canonical [5′-pyrimidine AACG/TG-3′] and located upstream of 6 others which were [5′-AACNG-3′] in type. An in vitro c-Myb binding assay revealed that c-Myb bound the canonical sites. We then performed a Chromatin Immunoprecipitation (ChIP) Assay with anti-c-Myb antibody and specifically enriched cyclin B1 promoter DNA sequences which strongly suggested that c-Myb bound the cyclin B1 promoter in vivo. A control antibody was inactive. Finally, a conditionally active c-Myb restored cyclin B1 mRNA expression in K562 human leukemia cells in presence of cycloheximide within 6 hours. Therefore, in addition to its role in regulating G1/S cell cycle transition, c-Myb also regulates cyclin B1 expression and therefore transition through the G2/M phase in human hematopoietic cells.

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