Maturation Promoting Factor Governs the First Polar Body Extrusion Through the ECT2-RhoA-ROCK Signaling Pathway in Mouse Oocytes.

Completion of the first meiosis in oocytes is achieved by the extrusion of the first polar body (PBI), a particular example of cell division. In mitosis, the small GTPase RhoA, which is activated by epithelial cell transforming protein 2 (ECT2), orchestrates contractile ring constriction, thus enabling cytokinesis. However, the involvement of this pathway in mammalian oocytes has not been established. To characterize the role of ECT2 in PBI emission in mouse oocytes, the small interfering RNA approach was employed. We found that ECT2 depletion significantly reduces PBI emission, induces first metaphase arrest, and generates oocytes containing two properly formed spindles of the second metaphase. Moreover, we describe, for the first time, that before PBI emission, RhoA forms a ring that is preceded by a dome-like accumulation at the oocyte cortex, next to the spindle. This unique mode of RhoA translocation failed to occur in the absence of ECT2. We further found that the Rho-dependent kinase, a main RhoA effector, is essential for PBI emission. In addition, we demonstrate herein that ECT2 is subjected to phosphorylation/dephosphorylation throughout meiosis in oocytes and further reveal that PBI emission is temporally associated with ECT2 dephosphorylation. Our data provide the first demonstration that an active cyclin-dependent kinase 1, the catalytic subunit of the maturation-promoting factor, phosphorylates ECT2 during the first meiotic metaphase and that cyclin-dependent kinase 1 inactivation at anaphase allows ECT2 dephosphorylation. In conclusion, our study demonstrates the indispensable role of the maturation-promoting factor/ECT2/RhoA pathway in PBI extrusion in mouse oocytes.

Download Full-text

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

Development ◽

10.1242/dev.122.7.1995 ◽

1996 ◽

Vol 122 (7) ◽

pp. 1995-2003 ◽

Cited By ~ 7

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.

Download Full-text

Mad2 is required for inhibiting securin and cyclin B degradation following spindle depolymerisation in meiosis I mouse oocytes

Reproduction ◽

10.1530/rep.1.00856 ◽

2005 ◽

Vol 130 (6) ◽

pp. 829-843 ◽

Cited By ~ 75

Author(s):

Hayden A Homer ◽

Alex McDougall ◽

Mark Levasseur ◽

Alison P Murdoch ◽

Mary Herbert

Keyword(s):

Spindle Assembly Checkpoint ◽

Polar Body ◽

Cyclin B ◽

Mutant Form ◽

Anaphase Promoting Complex ◽

Mouse Oocytes ◽

First Polar Body ◽

Polar Body Extrusion ◽

Meiosis I

Mad2 is a pivotal component of the spindle assembly checkpoint (SAC) which inhibits anaphase promoting complex/cyclo-some (APC/C) activity by sequestering Cdc20 thereby regulating the destruction of securin and cyclin B. During mitosis, spindle depolymerisation induces a robust Mad2-dependent arrest due to inhibition of securin and cyclin B destruction. In contrast to mitosis, the molecular details underpinning the meiosis I arrest experienced by mouse oocytes exposed to spindle depolymerisation remain incompletely characterised. Notably, the role of Mad2 and the fate of the anaphase-marker, securin, are unexplored. As shown previously, we find that spindle depolymerisation by nocodazole inhibits first polar body extrusion (PBE) and stabilises cyclin B and cyclin-dependent kinase 1 activity in mouse oocytes. Here we show that stabilisation of cyclin B in nocodazole can be sustained for several hours and is associated with stabilisation of securin. These effects are SAC-mediated as, in oocytes depleted of the majority of Mad2 by morpholino antisense, securin and cyclin B are destabilised and 15% of oocytes undergo PBE. This reflects premature APC/C activation as a mutant form of cyclin B lacking its APC/C degradation signal is stable in Mad2-depleted oocytes. Moreover, homologues do not disjoin during the prolonged meiosis I arrest (> 18 h) induced by nocodaozole indicating that a non-cleavage mechanism is insufficient on its own for resolution of arm cohesion in mammalian oocytes. In conclusion, when all kinetochores lack attachment and tension, mouse oocytes mount a robust Mad2-dependent meiosis I arrest which inhibits the destruction of securin and cyclin B.

Download Full-text

From ubiquitin‐proteasomal degradation to CDK1 inactivation: requirements for the first polar body extrusion in mouse oocytes

The FASEB Journal ◽

10.1096/fj.12-209866 ◽

2012 ◽

Vol 26 (11) ◽

pp. 4495-4505 ◽

Cited By ~ 8

Author(s):

Yael Pomerantz ◽

Judith Elbaz ◽

Inbal Ben‐Eliezer ◽

Yitzhak Reizel ◽

Yael David ◽

...

Keyword(s):

Polar Body ◽

Proteasomal Degradation ◽

Mouse Oocytes ◽

First Polar Body ◽

Polar Body Extrusion

Download Full-text

Txndc9 Is Required for Meiotic Maturation of Mouse Oocytes

BioMed Research International ◽

10.1155/2017/6265890 ◽

2017 ◽

Vol 2017 ◽

pp. 1-9

Author(s):

Fanhua Ma ◽

Liming Hou ◽

Liguo Yang

Keyword(s):

Polar Body ◽

Germinal Vesicle ◽

Redox Balance ◽

Redox Status ◽

Mouse Oocyte ◽

Meiotic Maturation ◽

Mouse Oocytes ◽

Oocyte Meiosis ◽

First Polar Body ◽

Polar Body Extrusion

Txndc9 (thioredoxin domain containing protein 9) has been shown to be involved in mammalian mitosis; however, its function in mammalian oocyte meiosis remains unclear. In this study, we initially found that Txndc9 is expressed during meiotic maturation of mouse oocytes and higher expression of Txndc9 mRNA and protein occurred in germinal vesicle (GV) stage. By using confocal scanning, we observed that Txndc9 localized at both nucleus and cytoplasm, especially at spindle microtubules. Specific depletion of Txndc9 by siRNA in mouse oocyte resulted in decreasing the rate of first polar body extrusion and increasing abnormal spindle assemble. Moreover, knockdown of Txndc9 in germinal vesicle (GV) stage oocytes led to higher level of reactive oxygen species (ROS) and lower level of antioxidant glutathione (GSH) as compared with control oocytes, which indicated that Txndc9 may be involved in mediating the redox balance. In summary, our results demonstrated that Txndc9 is crucial for mouse oocyte maturation by regulating spindle assembly, polar body extrusion, and redox status.

Download Full-text

Ral GTPase is essential for actin dynamics and Golgi apparatus distribution in mouse oocyte maturation

Cell Division ◽

10.1186/s13008-021-00071-y ◽

2021 ◽

Vol 16 (1) ◽

Author(s):

Ming-Hong Sun ◽

Lin-Lin Hu ◽

Chao-Ying Zhao ◽

Xiang Lu ◽

Yan-Ping Ren ◽

...

Keyword(s):

Golgi Apparatus ◽

Oocyte Maturation ◽

Polar Body ◽

Mouse Oocyte ◽

Actin Dynamics ◽

Mouse Oocytes ◽

Oocyte Meiosis ◽

Ral Gtpase ◽

Polar Body Extrusion ◽

Cytoskeleton Dynamics

Abstract Background Ral family is a member of Ras-like GTPase superfamily, which includes RalA and RalB. RalA/B play important roles in many cell biological functions, including cytoskeleton dynamics, cell division, membrane transport, gene expression and signal transduction. However, whether RalA/B involve into the mammalian oocyte meiosis is still unclear. This study aimed to explore the roles of RalA/B during mouse oocyte maturation. Results Our results showed that RalA/B expressed at all stages of oocyte maturation, and they were enriched at the spindle periphery area after meiosis resumption. The injection of RalA/B siRNAs into the oocytes significantly disturbed the polar body extrusion, indicating the essential roles of RalA/B for oocyte maturation. We observed that in the RalA/B knockdown oocytes the actin filament fluorescence intensity was significantly increased at the both cortex and cytoplasm, and the chromosomes were failed to locate near the cortex, indicating that RalA/B regulate actin dynamics for spindle migration in mouse oocytes. Moreover, we also found that the Golgi apparatus distribution at the spindle periphery was disturbed after RalA/B depletion. Conclusions In summary, our results indicated that RalA/B affect actin dynamics for chromosome positioning and Golgi apparatus distribution in mouse oocytes.

Download Full-text

Triphenyltin chloride induces spindle microtubule depolymerisation and inhibits meiotic maturation in mouse oocytes

Reproduction Fertility and Development ◽

10.1071/rd12332 ◽

2014 ◽

Vol 26 (8) ◽

pp. 1084 ◽

Cited By ~ 2

Author(s):

Yu-Ting Shen ◽

Yue-Qiang Song ◽

Xiao-Qin He ◽

Fei Zhang ◽

Xin Huang ◽

...

Keyword(s):

Polar Body ◽

Meiotic Maturation ◽

Dependent Manner ◽

Germinal Vesicle Breakdown ◽

Mouse Oocytes ◽

First Polar Body ◽

Triphenyltin Chloride ◽

Dose Dependent

Meiosis produces haploid gametes for sexual reproduction. Triphenyltin chloride (TPTCL) is a highly bioaccumulated and toxic environmental oestrogen; however, its effect on oocyte meiosis remains unknown. We examined the effect of TPTCL on mouse oocyte meiotic maturation in vitro and in vivo. In vitro, TPTCL inhibited germinal vesicle breakdown (GVBD) and first polar body extrusion (PBE) in a dose-dependent manner. The spindle microtubules completely disassembled and the chromosomes condensed after oocytes were exposed to 5 or 10 μg mL–1 TPTCL. γ-Tubulin protein was abnormally localised near chromosomes rather than on the spindle poles. In vivo, mice received TPTCL by oral gavage for 10 days. The general condition of the mice deteriorated and the ovary coefficient was reduced (P < 0.05). The number of secondary and mature ovarian follicles was significantly reduced by 10 mg kg–1 TPTCL (P < 0.05). GVBD decreased in a non-significant, dose-dependent manner (P > 0.05). PBE was inhibited with 10 mg kg–1 TPTCL (P < 0.05). The spindles of in vitro and in vivo metaphase II oocytes were disassembled with 10 mg kg–1 TPTCL. These results suggest that TPTCL seriously affects meiotic maturation by disturbing cell-cycle progression, disturbing the microtubule cytoskeleton and inhibiting follicle development in mouse oocytes.

Download Full-text

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

The Journal of Cell Biology ◽

10.1083/jcb.146.1.1 ◽

1999 ◽

Vol 146 (1) ◽

pp. 1-12 ◽

Cited By ~ 114

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.

Download Full-text

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

Journal of Cell Science ◽

10.1242/jcs.102.3.457 ◽

1992 ◽

Vol 102 (3) ◽

pp. 457-467 ◽

Cited By ~ 6

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.

Download Full-text

Inhibition of protein kinases by 6-dimethylaminopurine accelerates the transition to interphase in activated mouse oocytes

Journal of Cell Science ◽

10.1242/jcs.104.3.861 ◽

1993 ◽

Vol 104 (3) ◽

pp. 861-872 ◽

Cited By ~ 7

Author(s):

M.S. Szollosi ◽

J.Z. Kubiak ◽

P. Debey ◽

H. de Pennart ◽

D. Szollosi ◽

...

Keyword(s):

Protein Phosphorylation ◽

Kinase Inhibitor ◽

Polar Body ◽

Histone H1 ◽

Sperm Chromatin ◽

Oocyte Activation ◽

Mouse Oocytes ◽

Spindle Rotation ◽

Polar Body Extrusion ◽

Pronuclear Formation

Mouse oocyte activation is followed by a peculiar period during which the interphase network of microtubules does not form and the chromosomes remain condensed despite the inactivation of MPF. To evaluate the role of protein phosphorylation during this period, we studied the effects of the protein kinase inhibitor 6-dimethylaminopurine (6-DMAP) on fertilization and/or parthenogenetic activation of metaphase II-arrested mouse oocytes. 6-DMAP by itself does not induce the inactivation of histone H1 kinase in metaphase II-arrested oocytes, and does not influence the dynamics of histone H1 kinase inactivation during oocyte activation. However, 6-DMAP inhibits protein phosphorylation after oocyte activation. In addition, the phosphorylated form of some proteins disappear earlier in oocytes activated in the presence of 6-DMAP than in the activated control oocytes. This is correlated with the acceleration of some post-fertilization morphological events, such as sperm chromatin decondensation and its transient recondensation, formation of the interphase network of microtubules and pronuclear formation. In addition, numerous abnormalities could be observed: (1) the spindle rotation and polar body extrusion are inhibited; (2) the exchange of protamines into histones seems to be impaired, as judged by the morphology of DNA fibrils by electron microscopy; (3) the formation of a new nuclear envelope around the sperm chromatin proceeds prematurely, while recondensation is not yet completed. These observations suggest that the 6-DMAP-sensitive kinase(s) is (are) involved in the control of post-fertilization events such as the formation of the interphase network of microtubules, the remodelling of sperm chromatin and pronucleus formation.

Download Full-text