331. INVESTIGATING THE ROLE OF CYCLIN A2 DURING OOCYTE MEIOSIS

2010 ◽  
Vol 22 (9) ◽  
pp. 131
Author(s):  
P. C. Jennings ◽  
K. T. Jones
Keyword(s):  
Polar Body ◽  
Germinal Vesicle ◽  
Cyclin B1 ◽  
Negative Control ◽  
Early Embryo ◽  
Female Meiosis ◽  
Germinal Vesicle Breakdown ◽  
Molecular Control ◽  
First Polar Body ◽  
Cyclin A2

Aneuploidy is often derived from chromosomal segregation errors in oocytes, leading to Down Syndrome and early embryo loss. Thus it is important to understand the molecular control of female meiosis. Cyclins and cyclin-dependent kinases (CDKs), particular those contributing to Maturation Promoting Factor (MPF, or CDK1) activity, play key roles in regulating meiosis. While cyclin B1 is classically regarded as the regulatory component of MPF, cyclin A2 can also bind and activate CDK1, and in mammalian somatic cells it is known to promote both G1/S and G2/M transitions. There is an absolute requirement for cyclin A2 during development and differentiation as its deficiency results in early embryonic lethality. As such, cyclin A2 has not been extensively studied, particularly as to its role in meiosis. To examine the role cyclin A2 plays during mammalian female meiosis, we carried out knockdown experiments. Microinjection into mouse oocytes of cyclin A2 siRNA induced a ~70% knockdown. Cyclin A2 knockdown did not inhibit germinal vesicle breakdown but did act to delay it. Extrusion of the first polar body was significantly reduced (P = 0.002) in comparison to non-injected controls and those injected with a negative control siRNA. Furthermore, microinjection of cyclin A2 can stimulate entry into meiosis. Thus it seems possible that cyclin A2/CDK can exhibit MPF activity. In conclusion, our data suggest that cyclin A2 can regulate meiotic entry in oocytes and also plays an important role in successful passage through the first meiotic division.

The Distribution and Possible Role of ERK8 in Mouse Oocyte Meiotic Maturation and Early Embryo Cleavage

2013 ◽  
Vol 19 (1) ◽  
pp. 190-200 ◽  
Author(s):  
Shang-Wu Yang ◽  
Hao Huang ◽  
Chen Gao ◽  
Lei Chen ◽  
Shu-Tao Qi ◽  
...  
Keyword(s):  
Polar Body ◽  
Germinal Vesicle ◽  
Early Embryo ◽  
Mouse Oocyte ◽  
Meiotic Maturation ◽  
Germinal Vesicle Breakdown ◽  
First Polar Body ◽  
Embryo Cleavage ◽  
Polar Body Extrusion ◽  
Oocyte Meiotic Maturation

AbstractIt is well known that extracellular signal-regulated kinase 8 (ERK8) plays pivotal roles in various mitotic events. But its physiological roles in oocyte meiotic maturation remain unclear. In this study, we found that although no specific ERK8 signal was detected in oocyte at the germinal vesicle stage, ERK8 began to migrate to the periphery of chromosomes shortly after germinal vesicle breakdown. At prometaphase I, metaphase I (MI), anaphase I, telophase I, and metaphase II (MII) stages, ERK8 was stably detected at the spindles. By taxol treatment, we clarified that the ERK8 signal was stained on the spindle fibers as well as microtubule asters in MI and MII oocytes. In fertilized eggs, the ERK8 signal was not observed in the two pronuclei stages. At prometaphase, metaphase, and anaphase of the first mitosis, ERK8 was detected on the mitotic spindle. ERK8 knock down by antibody microinjection and specific siRNA caused abnormal spindles, failed chromosome congression, and decreased first polar body extrusion. Taken together, our results suggest that ERK8 plays an important role in spindle organization during mouse oocyte meiotic maturation and early embryo cleavage.

Pleiotropic effect of okadaic acid on maturing mouse oocytes

Development ◽  
1991 ◽  
Vol 112 (4) ◽  
pp. 971-980 ◽  
Author(s):  
H. Alexandre ◽  
A. Van Cauwenberge ◽  
Y. Tsukitani ◽  
J. Mulnard
Keyword(s):  
Protein Kinase ◽  
Okadaic Acid ◽  
Protein Phosphatases ◽  
Chromatin Condensation ◽  
Polar Body ◽  
Germinal Vesicle ◽  
Inhibitory Effect ◽  
Negative Control ◽  
Germinal Vesicle Breakdown ◽  
Mouse Oocytes

Okadaic acid (OA), a potent inhibitor of types 1 and 2A protein phosphatases, was shown recently to induce chromatin condensation and germinal vesicle breakdown (GVBD) in mouse oocytes arrested at the dictyate stage by dibutyryl cAMP (dbcAMP), isobutyl methylxanthine (IBMX) and 12,13-phorbol dibutyrate (PDBu). We confirm these results using IBMX and another phorbol diester, 12-O-tetradecanoylphorbol-13-acetate (TPA) and show that OA also bypasses the inhibitory effect of 6-dimethylaminopurine (6-DMAP). It has been concluded that protein phosphatases 1 and/or 2A (PP1, 2A), involved in the negative control of MPF activation, are thus operating downstream from both the protein kinase A and protein kinase C catalysed phosphorylation steps that prevent the breakdown of GV. Similar enzymatic activities are also able to counteract the general inhibition of protein phosphorylation. However, PP1 and/or PP2A are positively involved in the activation of pericentriolar material (PCM) into microtubule organizing centres (MTOCs). This explains the inhibitory effect of OA on spindle assembly. Finally, OA interferes with the integrity and/or function of actomyosin filaments. This results in a dramatic ruffling of the plasma membrane leading to the internalization of large vacuoles, the inhibition of chromosome centrifugal displacement and, consequently, the prevention of polar body extrusion.

Involvement of GABAA receptor in Bufo arenarum oocyte maturation

Zygote ◽  
2008 ◽  
Vol 16 (2) ◽  
pp. 135-144
Author(s):  
G. Sánchez Toranzo ◽  
L. Zelarayán ◽  
F. Bonilla ◽  
J. Oterino ◽  
M.I. Bühler
Keyword(s):  
Receptor Agonist ◽  
Polar Body ◽  
Germinal Vesicle ◽  
Dependent Manner ◽  
Germinal Vesicle Breakdown ◽  
First Polar Body ◽  
Bufo Arenarum ◽  
Turn Over ◽  
Pkc Activation

SummaryAmphibian oocytes meiotic arrest is released under the stimulus of progesterone; this hormone interacts with the oocyte surface and starts a cascade of events leading to the activation of a cytoplasmic maturation promoting factor (MPF) that induces germinal vesicle breakdown (GVBD), chromosome condensation and extrusion of the first polar body.The aim of this work was to determine whether the activation of a GABAA receptor is able to induce GVBD in fully grown denuded oocytes of Bufo arenarum and to analyse its possible participation in progesterone-induced maturation. We also evaluated the role of purines and phospholipids in the maturation process induced by a GABAA receptor agonist such as muscimol.Our results indicated that the activation of the GABAA receptor by muscimol induces maturation in a dose- and time-dependent manner and that this activation is a genuine maturation that enables oocytes to form pronuclei. Assays with a receptor antagonist, picrotoxine, showed that the maturation induced by muscimol was inhibited. Treatment with picrotoxine, however, shows that the participation of GABAA receptor in progesterone-induced maturation is not significant.In addition, our results indicate that high intracellular levels of purines obtained by the use of db-AMPc and theophylline or the inhibition of the phosphatidylinositol 4,5-bisphosphate (PIP2 hydrolysis by neomycin and PIP2 turn over by LiCl, respectively, inhibited the maturation induced by muscimol. Treatment with H-7 indicated, however, that PKC activation is not necessary for GVBD induced by the GABAA receptor agonist. Results suggest that the transduction pathway used by the GABAA receptor to induce maturation is different from those used by progesterone.

scholarly journals Glycogen synthase kinase 3B in bovine oocytes and granulosa cells: possible involvement in meiosis during in vitro maturation

Reproduction ◽  
2009 ◽  
Vol 138 (2) ◽  
pp. 235-246 ◽  
Author(s):  
Svetlana Uzbekova ◽  
Mohamad Salhab ◽  
Christine Perreau ◽  
Pascal Mermillod ◽  
Joëlle Dupont
Keyword(s):  
Granulosa Cells ◽  
Map Kinases ◽  
Glycogen Synthase ◽  
Polar Body ◽  
Germinal Vesicle ◽  
Glycogen Synthase Kinase ◽  
Aurora A Kinase ◽  
Germinal Vesicle Breakdown ◽  
First Polar Body

Glycogen synthase kinase 3 (GSK3) regulates cellular metabolism and cell cycle via different signalling pathways. In response to insulin and growth factors GSK3 is serine-phosphorylated and inactivated. We analysed GSK3B expression and activation in bovine cumulus cells (CC) and oocytes at different meiotic stagesin vitroin parallel with MAP kinases ERK (MAPK3/MAPK1) and p38 (MAPK14). GSK3B localised to cytoplasm in granulosa cells and in oocytes throughout folliculogenesis. In mature metaphase-II (MII) oocytes, GSK3B was concentrated to the region of midzone between the oocyte and the first polar body, as well as active phospho-Thr Aurora A kinase (AURKA). Duringin vitromaturation (IVM), in oocytes, phospho-Ser9-GSK3B level increased as well as phospho-MAPK3/MAPK1, while phospho-MAPK14 decreased. In CC, phospho-MAPK14 increased upon germinal vesicle breakdown (GVBD)/metaphase-I (MI) and then decreased during transition to MII. Administration of inhibitors of GSK3 activity (lithium chloride or 2′Z,3′E -6-bromoindirubin-3′-oxime) rapidly increased phospho-Ser9-GSK3B, and led to transient decrease of phospho-MAPK3/MAPK1 and to durable enhancing of phospho-MAPK14 in granulosa primary cell culture. GSK3 inhibitors during IVM diminished cumulus expansion and delayed meiotic progression. In cumulus, phospho-MAPK14 level was significantly higher in the presence of inhibitors, comparing with control, through the time of MI/MII transition. In oocytes, phospho-GSK3B was increased and phospho-MAPK3/MAPK1 was decreased before GVBD and oocytes were mainly arrested at MI. Therefore, GSK3B might regulate oocyte meiosis, notably MI/MII transition being the part of MAPK3/1 and MAPK14 pathways in oocytes and CC. GSK3B might be also involved in the local activation of AURKA that controls this transition.

scholarly journals NAT10-Mediated N4-Acetylcytidine of RNA Contributes to Post-transcriptional Regulation of Mouse Oocyte Maturation in vitro

2021 ◽  
Vol 9 ◽  
Author(s):  
Yuting Xiang ◽  
Chuanchuan Zhou ◽  
Yanyan Zeng ◽  
Qi Guo ◽  
Jiana Huang ◽  
...  
Keyword(s):  
Oocyte Maturation ◽  
High Throughput Sequencing ◽  
Epigenetic Modification ◽  
Binding Protein ◽  
Polar Body ◽  
Germinal Vesicle ◽  
Translation Efficiency ◽  
Germinal Vesicle Breakdown ◽  
First Polar Body

N4-acetylcytidine (ac4C), a newly identified epigenetic modification within mRNA, has been characterized as a crucial regulator of mRNA stability and translation efficiency. However, the role of ac4C during oocyte maturation, the process mainly controlled via post-transcriptional mechanisms, has not been explored. N-acetyltransferase 10 (NAT10) is the only known enzyme responsible for ac4C production in mammals and ac4C-binding proteins have not been reported yet. In this study, we have documented decreasing trends of both ac4C and NAT10 expression from immature to mature mouse oocytes. With NAT10 knockdown mediated by small interfering RNA (siRNA) in germinal vesicle (GV)-stage oocytes, ac4C modification was reduced and meiotic maturation in vitro was significantly retarded. Specifically, the rate of first polar body extrusion was significantly decreased with NAT10 knockdown (34.6%) compared to control oocytes without transfection (74.6%) and oocytes transfected with negative control siRNA (72.6%) (p < 0.001), while rates of germinal vesicle breakdown (GVBD) were not significantly different (p = 0.6531). RNA immunoprecipitation and high-throughput sequencing using HEK293T cells revealed that the modulated genes were enriched in biological processes associated with nucleosome assembly, chromatin silencing, chromatin modification and cytoskeletal anchoring. In addition, we identified TBL3 as a potential ac4C-binding protein by a bioinformatics algorithm and RNA pulldown with HEK293T cells, which may mediate downstream cellular activities. Taken together, our results suggest that NAT10-mediated ac4C modification is an important regulatory factor during oocyte maturation in vitro and TBL3 is a potential ac4C-binding protein.

Thioglycolic acid inhibits mouse oocyte maturation and affects chromosomal arrangement and spindle configuration

2008 ◽  
Vol 24 (4) ◽  
pp. 227-234 ◽  
Author(s):  
SY Hou ◽  
L Zhang ◽  
K Wu ◽  
L Xia
Keyword(s):  
Thioglycolic Acid ◽  
Polar Body ◽  
Germinal Vesicle ◽  
Germinal Vesicle Breakdown ◽  
Chromosomal Arrangement ◽  
First Polar Body ◽  
Inverted Microscope ◽  
Polar Body Formation ◽  
Spindle Elongation

Previous studies have shown that thioglycolic acid (TGA) leads to potential reproductive toxicology. To clarify the exact effects of this compound on reproduction, mice oocytes were treated with different TGA doses. At the end of the culture period, the nuclear status of mice oocytes was assessed under an inverted microscope. After immunofluorescence staining, the chromosomal arrangement and spindle configuration of oocytes were evaluated. The results indicated that TGA decreases the percentage of first polar body formation but does not influence that of germinal vesicle breakdown. TGA induces abnormal chromosomal arrangement and spindle elongation. In conclusion, TGA inhibits in-vitro maturation of mice oocytes and affects chromosomal arrangement and spindle configuration. Furthermore, it probably interferes with biochemical changes that occur during meiosis, resulting in aberrant development.

scholarly journals Translocation of phospho-protein kinase Cs implies their roles in meiotic-spindle organization, polar-body emission and nuclear activity in mouse eggs

Reproduction ◽  
2005 ◽  
Vol 129 (2) ◽  
pp. 229-234 ◽  
Author(s):  
Zhen-Yu Zheng ◽  
Qing-Zhang Li ◽  
Da-Yuan Chen ◽  
Heide Schatten ◽  
Qing-Yuan Sun
Keyword(s):  
Protein Kinase ◽  
Polar Body ◽  
Germinal Vesicle ◽  
Mouse Oocyte ◽  
Meiotic Spindle ◽  
Germinal Vesicle Breakdown ◽  
Nuclear Activity ◽  
Spindle Poles ◽  
Oocyte Meiosis ◽  
First Polar Body

The protein kinase Cs (PKCs) are a family of Ser/Thr protein kinases categorized into three subfamilies: classical, novel, and atypical. The phosphorylation of PKC in germ cells is not well defined. In this study, we described the subcellular localization of phopho-PKC in the process of mouse oocyte maturation, fertilization, and early embryonic mitosis. Confocal microscopy revealed that phospho-PKC (pan) was distributed abundantly in the nucleus at the germinal vesicle stage. After germinal vesicle breakdown, phospho-PKC was localized in the vicinity of the condensed chromosomes, distributed in the whole meiotic spindle, and concentrated at the spindle poles. After metaphase I, phospho-PKC was translocated gradually to the spindle mid-zone during emission of the first polar body. After sperm penetration and electrical activation, the distribution of phospho-PKC was moved from the spindle poles to the spindle mid-zone. After the extrusion of the second polar body (PB2) phospho-PKC was localized in the area between the oocyte and the PB2. In fertilized eggs, phospho-PKC was concentrated in the pronuclei except for the nucleolus. Phospho-PKC was dispersed after pronuclear envelope breakdown, but distributed on the entire spindle at mitotic metaphase. The results suggest that PKC activation may play important roles in regulating spindle organization and stabilization, polar-body extrusion, and nuclear activity during mouse oocyte meiosis, fertilization, and early embryonic mitosis.

scholarly journals Mitochondrial Ca2+ Overload Leads to Mitochondrial Oxidative Stress and Delayed Meiotic Resumption in Mouse Oocytes

2020 ◽  
Vol 8 ◽  
Author(s):  
Luyao Zhang ◽  
Zichuan Wang ◽  
Tengfei Lu ◽  
Lin Meng ◽  
Yan Luo ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Mitochondrial Function ◽  
Maternal Obesity ◽  
Polar Body ◽  
Germinal Vesicle ◽  
Oocyte Quality ◽  
Obese Women ◽  
Germinal Vesicle Breakdown ◽  
Mouse Oocytes ◽  
First Polar Body

Overweight or obese women seeking pregnancy is becoming increasingly common. Human maternal obesity gives rise to detrimental effects during reproduction. Emerging evidence has shown that these abnormities are likely attributed to oocyte quality. Oxidative stress induces poor oocyte conditions, but whether mitochondrial calcium homeostasis plays a key role in oocyte status remains unresolved. Here, we established a mitochondrial Ca2+ overload model in mouse oocytes. Knockdown gatekeepers of the mitochondrial Ca2+ uniporters Micu1 and Micu2 as well as the mitochondrial sodium calcium exchanger NCLX in oocytes both increased oocytes mitochondrial Ca2+ concentration. The overload of mitochondria Ca2+ in oocytes impaired mitochondrial function, leaded to oxidative stress, and changed protein kinase A (PKA) signaling associated gene expression as well as delayed meiotic resumption. Using this model, we aimed to determine the mechanism of delayed meiosis caused by mitochondrial Ca2+ overload, and whether oocyte-specific inhibition of mitochondrial Ca2+ influx could improve the reproductive abnormalities seen within obesity. Germinal vesicle breakdown stage (GVBD) and extrusion of first polar body (PB1) are two indicators of meiosis maturation. As expected, the percentage of oocytes that successfully progress to the germinal vesicle breakdown stage and extrude the first polar body during in vitro culture was increased significantly, and the expression of PKA signaling genes and mitochondrial function recovered after appropriate mitochondrial Ca2+ regulation. Additionally, some indicators of mitochondrial performance—such as adenosine triphosphate (ATP) and reactive oxygen species (ROS) levels and mitochondrial membrane potential—recovered to normal. These results suggest that the regulation of mitochondrial Ca2+ uptake in mouse oocytes has a significant role during oocyte maturation as well as PKA signaling and that proper mitochondrial Ca2+ reductions in obese oocytes can recover mitochondrial performance and improve obesity-associated oocyte quality.

Meiotic maturation of mouse oocytes in vitro: inhibition of maturation at specific stages of nuclear progression

1976 ◽  
Vol 22 (3) ◽  
pp. 531-545
Author(s):  
P.M. Wassarman ◽  
W.J. Josefowicz ◽  
G.E. Letourneau
Keyword(s):  
Cyclic Amp ◽  
Cytochalasin B ◽  
Polar Body ◽  
Germinal Vesicle ◽  
Meiotic Maturation ◽  
Dibutyryl Cyclic Amp ◽  
Germinal Vesicle Breakdown ◽  
Mouse Oocytes ◽  
First Polar Body

In vitro studies of meiotic maturation of mouse oocytes have been carried out in the presence of several drugs. The individual steps of nuclear progression, including dissolution of the nuclear (germinal vesicle) membrane, condensation of dictyate chromatin into compact bivalents, formation of the first metaphase spindle, and extrusion of the first polar body, are each susceptible to one or more of these drugs. Germinal vesicle breakdown, the initial morphological feature characteristic of meiotic maturation, is inhibited by dibutyryl cyclic AMP. However, even in the presence of dibutyryl cyclic AMP, the nuclear membrane becomes extremely convoluted and condensation of chromatin is initiated but aborts at a stage short of compact bivalents. Germinal vesicle breakdown and chromatin condensation take place in an apparently normal manner in the presence of puromycin, Colcemid, or cytochalasin B. Nuclear progression is blocked at the circular bivalent stage when oocytes are cultured continuously in the presence of puromycin or Colcemid, whereas oocytes cultured in the presence of cytochalasin B proceed to the first meiotic metaphase, form an apparently normal spindle, and arrest. Emission of a polar body is inhibited by all of these drugs. The inhibitory effects of these drugs on meiotic maturation are reversible to varying degrees dependent upon the duration of exposure to the drug and upon the nature of the drug. These studies suggest that dissolution of the mouse oocyte's germinal vesicle and condensation of chromatin are not dependent upon concomitant protein synthesis or upon microtubules. On the other hand, the complete condensation of chromatin into compact bivalents apparently requires breakdown of the germinal vesicle. Failure of homologous chromosomes to separate after normal alignment on the meiotic spindle in the presence of cytochalasin B suggest that microfilaments may be involved in nuclear progression at this stage of maturation. Cytokinesis, in the form of polar body formation, is blocked when any one of the earlier events of maturation fails to take place.

Transcriptional activity associated with meiotic competence in fully grown mouse GV oocytes

Zygote ◽  
2002 ◽  
Vol 10 (4) ◽  
pp. 327-332 ◽  
Author(s):  
Honglin Liu ◽  
Fugaku Aoki
Keyword(s):  
Transcriptional Activity ◽  
Polar Body ◽  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Meiotic Maturation ◽  
Germinal Vesicle Breakdown ◽  
First Polar Body ◽  
Active Transcription ◽  
Subsequent Activation ◽  
Meiotic Competence

The involvement of cumulus cells and chromatin organisation in transcriptional activity was investigated. In addition, the relationship between transcriptional activity and meiotic competence in fully grown mouse oocytes was surveyed. Transcriptional activity was detected in fully grown oocytes in which chromatin did not surround the nucleolus in the germinal vesicle (NSN-type oocytes), but not in oocytes in which chromatin surrounded the nucleolus (SN-type oocytes). Cumulus cells seemed to downregulate transcriptional activity in NSN-type oocytes, since transcriptional activity was 3 times greater in the denuded NSN-type oocytes free of cumulus cells (DO oocytes) than in NSN-type oocytes enclosed in cumulus cells (COC oocytes). Higher transcriptional activity corresponded to lower germinal vesicle breakdown (GVB) competence of fully grown oocytes in culture. Although GVB occurred in nearly all (99%) the SN-type oocytes, it occurred in 88% of COC/NSN-type oocytes (cumulus-oocyte complex with SN-type configuration) and in 61% of DO/NSN-type oocytes (denuded oocytes with NSN-type configuration). There was a negative correlation between transcriptional activity and the capacity of a cell to complete the progression to the second metaphase (MII). In GVB oocytes, the percentage of first polar body (PBI) extrusion differed among COC/NSN-type (81%), DO/SN-type (66%), COC/NSN-type (47%) and DO/NSN-type (29%) oocytes. After activation with 10 mM Sr2+, the frequency of parthenogenetic activation was greater in SN-type oocytes (46.9%) than in transcriptionally active NSN-type oocytes (27.5%). These results suggest that transcriptional activity has a detrimental effect on the competence of meiotic maturation and subsequent activation in fully grown GV oocytes. Alternatively, active transcription in the fully grown oocytes suggests that they are still in the process of synthesising substances required for meiotic maturation and are not yet competent for these processes.

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