Oocyte maturation in rabbits: effects of calmodulin inhibitors

Zygote ◽  
1997 ◽  
Vol 5 (3) ◽  
pp. 255-260 ◽  
Author(s):  
Michael A. Henry ◽  
Richard G. Rawlins ◽  
Ewa Radwanska ◽  
Mary M. Fahy
Keyword(s):  
Oocyte Maturation ◽  
Calcium Binding ◽  
Kinase Inhibitor ◽  
Polar Body ◽  
Germinal Vesicle ◽  
Germinal Vesicle Breakdown ◽  
Polar Bodies ◽  
Downstream Effectors ◽  
First Polar Body ◽  
Metaphase I

SummaryOocyte maturation in mammals follows a highly conserved pattern of release from arrest through to the extrusion of the first polar body and formation of the second metaphase spindle. Oscillations in cytoplasmic calcium concentration precede the events of maturation in many species. These calcium ions interact with and activate calcium-binding proteins, including calmodulin, within the cell. Thus, it was of interest to us to examine whether calcium acted through calmodulin in the initial stages of maturation in rabbit oocytes or whether calmodulin was required for continuation through metaphase I on to metaphase II. Using the calmodulin inhibitor W-7 we found a significant (p < 0.05) decrease in the percentage of oocytes that underwent germinal vesicle breakdown. Calmidazolium did not prevent germinal vesicle breakdown; however, it caused a significant (p < 0.05) decrease in the proportion of oocytes with fully elaborated spindles and taxol-induced cytoplasmic asters. Both inhibitors caused a significant (p < 0.05) reduction in the proportion of oocytes that extruded their first polar bodies. The kinase inhibitor 6-DMAP caused a significant reduction in the proportion of oocytes with spindles and condensed chromatin, indicating the necessity for phosphorylation events in the resumption of meiosis. In rabbit oocytes calmodulin may play a role in the release from prophase arrest, and it is necessary for spindle preservation and continuation through metaphase I to metaphase II. The varying effects of the two inhibitor stems from their different binding sites on the calmodulin molecule thus causing a differential effect on its downstream effectors.

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 &lt; 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.

Heparin effect on in vitro nuclear maturation of bovine oocytes

Zygote ◽  
2008 ◽  
Vol 16 (1) ◽  
pp. 1-8 ◽  
Author(s):  
Juan Carlos Flores-Alonso ◽  
Leticia Lezama-Monfil ◽  
María Luisa Sánchez-Vázquez ◽  
Rosalina Reyes ◽  
Néstor M. Delgado
Keyword(s):  
Oocyte Maturation ◽  
Morphological Changes ◽  
Polar Body ◽  
Germinal Vesicle ◽  
Salt Medium ◽  
Germinal Vesicle Breakdown ◽  
Maturation Factor ◽  
First Polar Body ◽  
Bovine Oocytes

SummaryOocytes undergo numerous biochemical and morphological changes during their development from preantral to preovulatory phases. In vitro studies have suggested several compounds that might induce oocyte maturation. Heparin is a natural component of ooplasm, follicular fluid and uterine fluid and previous studies indicated that it might act as a chromatin maturation factor in bovine oocytes. We tested this hypothesis in vitro by timing germinal vesicle breakdown (GVBD) and first polar body (PB) formation without any other natural or introduced factors that might influence the rate of oocyte maturation. We also determined if these oocytes could be fertilized.Bovine oocytes were incubated in a salt medium and TCM 199 supplemented with different concentrations of heparin for 24 h at 37.5 °C in a humidified atmosphere of 5% CO2. With 1.0 and 6.5 mg/ml heparin, the time of GVBD was reduced from 4.7 ± 1.1 h to about 1.5 h and the time of first PB formation was reduced from 22.0 ± 1.1 h to 9.0–11.0 h in salt medium. In TCM 199, only 6.5 mg/ml heparin significantly reduced the time of PB formation. In both incubation media, 1.0 and 6.5 mg/ml heparin induced GVBD, extrusion of the first PB and formation of the metaphase II nucleus. Moreover, heparin did not interfere with the fertilization of oocytes matured in TCM 199. Based on the results, we propose that heparin plays an important role in the rearrangement of the oocyte chromatin and acts as an oocyte maturation factor.

Antioxidants Stimulate Germinal Vesicle Breakdown, But Inhibit Chromosome Formation And Spindle Assembly In Porcine Oocytes

2000 ◽  
Vol 6 (S2) ◽  
pp. 964-965
Author(s):  
Qing-Yuan Sun ◽  
Randall S. Prather ◽  
Heide Schatten
Keyword(s):  
Oocyte Maturation ◽  
Germinal Vesicle ◽  
Spindle Assembly ◽  
Mouse Oocyte ◽  
Germinal Vesicle Breakdown ◽  
Oocyte Meiosis ◽  
First Polar Body ◽  
Porcine Oocyte ◽  
Chromosome Formation

Mammalian oocytes are arrested at the diplotene stage of the first meiotic division. Release of oocytes from their follicles induces meiotic resumption characterized by germinal vesicle breakdown (GVBD), followed by the chromosome formation and metaphase I spindle organization and finally the extrusion the first polar body. Recently it was shown that cellpermeant antioxidants significantly inhibit spontaneous resumption of meiosis in mouse oocytes, which may indicate a role of oxygen radicals in oocyte maturation. The regulation of mouse oocyte meiosis resumption is different from that of large domestic animals in that GVBD is independent of Ca2+ and protein synthesis. The present study investigated the influence of two cell-permeant antioxidants, 2(3)-ter-butyl-4-hydroxyanisole (BHA) and nordihydroguaiaretic acid (NDGA), on porcine oocyte meiosis resumption, chromatin behavior and spindle assembly. Our findings revealed a different role of antioxidants in porcine oocyte meiosis resumption than in mouse oocyte maturation.

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.

233 DEHIDROLEUCODINE INDUCES PARTHENOGENETIC ACTIVATION OF BOVINE OOCYTES

2009 ◽  
Vol 21 (1) ◽  
pp. 214
Author(s):  
N. Canel ◽  
D. Salamone
Keyword(s):  
Polar Body ◽  
Metaphase Plate ◽  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Control Group ◽  
Germinal Vesicle Breakdown ◽  
Parthenogenetic Activation ◽  
Polar Bodies ◽  
Bovine Oocytes ◽  
Full Activation

Dehydroleucodine (DhL) is a sesquiterpene lactone that inhibits germinal vesicle breakdown in Bufo arenarum oocytes. Its action takes place over early stages of the cdc25 activation cascade (Bühler MI et al. 2007 Zygote 15, 183–187). The aim of this study was to evaluate the potential of DhL to induce parthenogenetic activation by observing nuclear dynamics and second polar body (2PB) extrusion of bovine oocytes, in the presence or absence of Cytochalasin B (CB), comparing these treatments with 6-Dimethylaminopurine (DMAP), an activation agent widely used. Cumulus–oocyte complexes were collected from cow ovaries obtained from a slaughterhouse. They were matured in TCM 199, supplemented with 5% FCS, 10 UI mL–1 penicillin, 10 μg mL–1 FSH, 100 μM cysteamine, 0.3 mm sodium pyruvate and 2 mm glutamine, at 39°C under 6% CO2 in air for 24 h. After removal of cumulus cells, metaphase II (MII) oocytes were selected and treated with 5 μm ionomycin (Io) for 4 min. Afterwards, oocytes were randomly allocated into one of the following treatments: a) incubation with 2 mm DMAP for 3 h (DMAP); b) incubation with 5 μm DhL for 3 h (DhL); and c) incubation with 5 μm DhL and 5 μg mL–1 CB, for 3 h (DhL-CB). A control group was only treated with Io. Activated oocytes were cultured in the maturation medium during 4, 11 or 17 h (Io exposure = 0 h), stained with Hoechst 33342 and analyzed under fluorescence microscope to evaluate nuclear stage and 2PB extrusion. Activation data are presented in Table 1. Oocytes with two extruded polar bodies and a metaphase plate were considered as partially activated (PA) and those exhibiting one pronucleus (PN) or already cleaved, as fully activated (FA). Oocytes that remained arrested at MII were not included in the table. Rates of 2PB emission were 98.3, 4.9, 83.6 and 61.5% for Io, DMAP, DhL and DhL-CB, respectively. These percentages were determined over total number of activated oocytes (PA and FA) within each group, including results from all evaluation times because no differences were found between them. Nuclear evaluation suggests that DhL is as effective as DMAP to induce full activation when combined with CB, and its use does not induce the early PN formation observed with DMAP at 4 h post Io. Most of the oocytes activated with DhL extruded a 2PB; these results were statistically different from those observed for other groups. These results indicate that DhL might be a useful agent to induce parthenogenesis, allowing 2PB extrusion and avoiding early PN formation in bovine oocytes. Table 1.Partial and full activation of bovine oocytes at 4, 11 and 17 h post treatments

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.

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 Potential upstream regulators and downstream targets of AMP-activated kinase signaling during oocyte maturation in a marine worm

Reproduction ◽  
2011 ◽  
Vol 142 (1) ◽  
pp. 29-39 ◽  
Author(s):  
Stephen A Stricker
Keyword(s):  
Oocyte Maturation ◽  
Kinase Inhibitor ◽  
Germinal Vesicle ◽  
Ampk Activation ◽  
Cyclin Dependent Kinase ◽  
Germinal Vesicle Breakdown ◽  
Mapk Activation ◽  
Ampk Signaling ◽  
Cyclin Dependent Kinase Inhibitor ◽  
Upstream Regulators

Unlike in mice, where the onset of oocyte maturation (germinal vesicle breakdown, GVBD) is blocked by cAMP and triggered by AMP-activated kinase (AMPK), oocytes of the marine nemertean wormCerebratulusundergo GVBD in response to cAMP elevations and AMPK deactivation. Since the pathways underlying AMPK's effects on mammalian or nemertean GVBD have not been fully defined, follicle-free nemertean oocytes were treated with pharmacological modulators and subsequently analyzed via immunoblotting methods using phospho-specific antibodies to potential regulators and targets of AMPK. Based on such phosphorylation patterns, immature oocytes possessed an active LKB1-like kinase that phosphorylated AMPK's T172 site to activate AMPK, whereas during oocyte maturation, AMPK and LKB1-like activities declined. In addition, given that MAPK can deactivate AMPK in somatic cells, oocytes were treated with inhibitors of ERK1/2 MAPK activation. However, these assays indicated that T172 dephosphorylation during maturation-associated AMPK deactivation did not require MAPK and that an observed inhibition of GVBD elicited by the MAPK kinase blocker U0126 was actually due to ectopic AMPK activation rather than MAPK inactivation. Similarly, based on tests using an inhibitor of maturation-promoting factor (MPF), T172 dephosphorylation occurred upstream to, and independently of, MPF activation. Alternatively, active MPF and MAPK were necessary for fully phosphorylating a presumably inhibitory S485/491 site on AMPK. Furthermore, in assessing signals possibly linking AMPK deactivation to MPF activation, evidence was obtained for maturing oocytes upregulating target-of-rapamycin activity and downregulating the cyclin-dependent kinase inhibitor Kip1. Collectively, these findings are discussed relative to multiple pathways potentially mediating AMPK signaling during GVBD.

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.

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