Involvement of GABAA receptor in Bufo arenarum oocyte maturation

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.

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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.

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

Microscopy and Microanalysis ◽

10.1017/s1431927600037314 ◽

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.

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The role of calcium in the nuclear maturation of Bufo arenarum oocytes

Zygote ◽

10.1017/s096719940400259x ◽

2004 ◽

Vol 12 (1) ◽

pp. 49-56 ◽

Cited By ~ 4

Author(s):

Liliana I. Zelarayán ◽

Graciela Sánchez Toranzo ◽

Julia M. Oterino ◽

Marta I. Bühler

Keyword(s):

Intracellular Calcium ◽

Germinal Vesicle ◽

Reproductive Period ◽

Protein Kinase Activity ◽

Channel Blockers ◽

Germinal Vesicle Breakdown ◽

Nuclear Maturation ◽

Bufo Arenarum ◽

Hormonal Stimulus

In Bufo arenarum, progesterone is the physiological maturation inducer. However, in this species, oocytes reinitiate meiosis with no need of an exogenous hormonal stimulus when deprived of their enveloping cell, a phenomenon called spontaneous maturation. We demonstrated that in Bufo arenarum spontaneous maturation occurs only in oocytes obtained during the reproductive period, which can be considered competent to mature spontaneously, in contrast to those in the non-reproductive period, which are incompetent. Interestingly, full-grown Bufo arenarum oocytes always respond to progesterone regardless of the season in which they are obtained. There is a general consensus that both a transient increase in intracellular calcium and a decrease in cAMP-dependent protein kinase activity are the first steps in the mechanisms by which progesterone induces maturation in amphibians. In the present work we analysed the role of calcium in the spontaneous and progesterone-induced maturation of Bufo arenarum oocytes. Results demonstrated that the absence of calcium in the incubation medium or the prevention of Ca2+ influx by channel blockers such as CdCl2 or NiCl2 did not prevent meiosis reinitiation in either type of maturation. The inhibition of the Ca2+-calmodulin complex in no case affected the maturation of the treated oocytes. However, when the oocytes were deprived of calcium by incubation in Ca2+-free AR + A23187, meiosis resumption was inhibited. In brief, we demonstrated that in Bufo arenarum the reinitiation of meiosis is a process independent of extracellular calcium at any period of the year and that oocytes require adequate levels of intracellular calcium for germinal vesicle breakdown to occur.

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

Reproduction ◽

10.1530/rep-09-0136 ◽

2009 ◽

Vol 138 (2) ◽

pp. 235-246 ◽

Cited By ~ 25

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.

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

Toxicology and Industrial Health ◽

10.1177/0748233708095862 ◽

2008 ◽

Vol 24 (4) ◽

pp. 227-234 ◽

Cited By ~ 8

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.

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

Reproduction ◽

10.1530/rep.1.00336 ◽

2005 ◽

Vol 129 (2) ◽

pp. 229-234 ◽

Cited By ~ 13

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.

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In vitro steroid-induced meiosis in Rhinella arenarum oocytes: role of pre-MPF activation

Zygote ◽

10.1017/s0967199415000143 ◽

2015 ◽

Vol 24 (2) ◽

pp. 252-258 ◽

Cited By ~ 4

Author(s):

Ana Josefina Arias Torres ◽

Marta Inés Bühler ◽

Liliana Isabel Zelarayán

Keyword(s):

Germinal Vesicle ◽

Reproductive Period ◽

Time Dependent ◽

Dependent Manner ◽

Germinal Vesicle Breakdown ◽

The Relationship ◽

Follicle Maturation ◽

T Testosterone

SummaryIn this work we showed the relationship between seasonal periods and the response of R. arenarum follicles and oocytes to different steroids. Using in vitro germinal vesicle breakdown (GVBD) assays, we demonstrated that P4 is the main steroid capable of inducing maturation in R. arenarum oocytes and follicles. In the second part of this work we showed that androgens can activate pre-maturation promoting factors (pre-MPFs) such as P4, by cytoplasm microinjection experiments. The results indicated that the steroids assayed induced oocyte and follicle maturation in a dose- and time-dependent manner. In oocytes, P4 was the most efficient steroid as a maturation inducer (EC50 of the reproductive period, 6 nM, EC50 of the non-reproductive period ≅ 30 nM). Androgens (DHEA, dehydroepiandrosterone; T, testosterone; and AD, androstenedione) were less efficient maturation inducers than P4 (EC50 reproductive period ≅ 50, 120 and 600 nM respectively). Similar results were obtained with intact follicles in both seasonal periods. Although the response of follicles to the different androgens was variable, in no case was it above the above the response induced by P4. Independently of the season, oocytes and follicles incubated in P4, P5 and T underwent GVBD after 6–10 h while oocytes and follicles incubated in DHEA and AD matured more slowly. Furthermore, we demonstrated that microinjection of mature cytoplasm from androgen-treated oocytes is sufficient to promote GVBD in immature recipient oocytes (DHEA, 57 ± 12%; AD, 60 ± 8%; T, 56 ± 13%). Thus, androgens such as DHEA, T and AD are as competent as P4 to activate pre-MPF.

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

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2020.580876 ◽

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

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.704341 ◽

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.

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

Journal of Cell Science ◽

10.1242/jcs.22.3.531 ◽

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.

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