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.

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

2014 ◽  
Vol 26 (8) ◽  
pp. 1084 ◽  
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.

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.

Meiotic arrest maintained by cAMP during the initiation of maturation enhances meiotic potential and developmental competence and reduces polyspermy of IVM/IVF porcine oocytes

Zygote ◽  
2003 ◽  
Vol 11 (3) ◽  
pp. 199-206 ◽  
Author(s):  
Tamás Somfai ◽  
Kazuhiro Kikuchi ◽  
Akira Onishi ◽  
Masaki Iwamoto ◽  
Dai-ichiro Fuchimoto ◽  
...  
Keyword(s):  
Cyclic Amp ◽  
Germinal Vesicle ◽  
Developmental Competence ◽  
Meiotic Maturation ◽  
Intracellular Level ◽  
Dibutyryl Cyclic Amp ◽  
Oocyte Collection ◽  
Porcine Oocyte ◽  
Blastocyst Rate

We investigated effects of invasive adenylate cyclase (iAC), 3-isobutyl-1-methylxanthine (IBMX) and dibutyryl cyclic AMP (dbcAMP) on porcine oocyte in vitro maturation (IVM), in vitro fertilisation (IVF) and subsequent embryonic development. Porcine oocytes were collected in Hepes-buffered NCSU-37 supplemented with or without 0.1 μg/ml iAC and 0.5 mM IBMX. IVM was performed in a modified NCSU-37 supplemented with or without 1 mM dbcAMP for 22 h and then without dbcAMP for an additional 24 h. After IVF, oocytes were cultured in vitro for 6 days. After 12 h of IVM, no difference in nuclear status was observed irrespective of supplementation with these chemicals during collection and IVM. At 22 h, most (95%) of the oocytes cultured with dbcAMP remained at the germinal vesicle (GV) stage, whereas 44.3% of the oocytes cultured without dbcAMP underwent GV breakdown. At 36 h, oocytes cultured with dbcAMP had progressed to prometaphase I or metaphase I (MI) (32.6% and 49.3%, respectively), whereas non-treated oocytes had progressed further to anaphase I, telophase I or metaphase II (MII) (13.6%, 14.3% and 38.0%, respectively). At 46 h, the rate of matured oocytes at MII was higher in oocytes cultured with dbcAMP (81%) than without dbcAMP (57%), while the proportion of oocytes arrested at MI was lower when cultured with dbcAMP (15%) than without dbcAMP (31%). The rate of monospermic fertilisation was higher when oocytes were cultured with dbcAMP (21%) than without dbcAMP (9%), with no difference in total penetration rates (58% and 52%, respectively). The blastocyst rate was higher in oocytes cultured with dbcAMP (32%) than without dbcAMP (19%). These results suggest that a change in intracellular level of cAMP during oocyte collection does not affect maturational and developmental competence of porcine oocytes and that synchronisation of meiotic maturation using dbcAMP enhances the meiotic potential of oocytes by promoting the MI to MII transition and results in high developmental competence by monospermic fertilisation.

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.

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.

Capacity of mouse oocytes to become activated depends on completion of cytoplasmic but not nuclear meiotic maturation

Zygote ◽  
1995 ◽  
Vol 3 (1) ◽  
pp. 45-55 ◽  
Author(s):  
Joise M.L. McConnell ◽  
Liz Campbell ◽  
Caroline Vincent
Keyword(s):  
Chromosomal Rearrangements ◽  
Polar Body ◽  
Calcium Ionophore ◽  
Germinal Vesicle ◽  
Calcium Ionophore A23187 ◽  
Meiotic Maturation ◽  
Ionophore A23187 ◽  
Germinal Vesicle Breakdown ◽  
Mouse Oocytes ◽  
Polar Body Extrusion

SummaryThe ability of mouse oocytes to become activated after exposure too the calcium ionophore A23187 has been investigated at different stages of meiotic maturation. The potential to respond to ionophore has been studied in relation to the time since resumption of meiotic maturation, the chromosomal conformation of the DNA within each cell and the protein synthetic profile of the maturing oocyte. Our studies demonstrate that when maturing oocytes from an MF1 strain of mice were treated with A23187 activation occured only in oocytes which had reached second meiotic metaphase (MII). However, development of the ability to respond to ionophore was not dependent on an orderly progression through normal chromosomal rearrangements such as separation at metaphase I (MI) and subsequent polar body extrusion, since there process could be prevented and the capacity to be activated became apparent in such oocytes at a time when control cells had reached MII. These data suggest that the ability to respond to ionophore depends on the development of a cytoplasmic or complex capable of monitoring the time since initiation of germinal vesicle breakdown. Metabolic radiolabelling of oocytes which were able to respond to calcium ionophore, even though they had been prevented from undergoing normal chromosomal rearrangements, showed them to be synthesising a group of proteins known as the 35 kDa complex.

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.

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.

Meiotic maturation of bovine oocytes in vitro: improvement of meiotic competence by dibutyryl cyclic adenosine 3′,5′-monophosphate.

1990 ◽  
Vol 68 (4) ◽  
pp. 1182-1187 ◽  
Author(s):  
E. Sato ◽  
M. Matsuo ◽  
H. Miyamoto
Keyword(s):  
Polar Body ◽  
Calf Serum ◽  
Germinal Vesicle ◽  
Razor Blade ◽  
Germinal Vesicle Breakdown ◽  
First Polar Body ◽  
Polar Body Formation ◽  
Bovine Oocytes ◽  
Meiotic Competence

Abstract The present study was undertaken to determine the precise stage of growth at which the ability to resume meiosis is acquired in bovine oocytes. Oocytes of various sizes were isolated from ovaries by mechanical dissection using an 18-gauge needle followed by a razor blade. This method yielded an average of 26.2 ± 7.4 growing and fully grown oocytes from an ovary. Cumulus-enclosed oocytes were cultured in vitro in tissue culture medium 199 containing 10% fetal calf serum. Oocytes ≤ 90 µm in diameter did not resume meiosis. However, germinal vesicle breakdown was observed in oocytes whose diameters exceeded 91 µm. Polar body formation was observed in oocytes with diameters exceeding 101 µm. About 80% of the oocytes with diameters ≥ 121 µm were able to extrude the polar body. The percentage of large oocytes (101 to 120 µm) with first polar body increased when incubated in medium containing dibutyryl cyclic adenosine 3′,5′-monophosphate; however, oocytes 90 to 101 µm did not extrude the first polar body even when cultured in a medium containing dibutyryl cyclic adenosine 3′,5′-monophosphate. These observations indicate that the capability to resume meiosis is acquired gradually during development of oocytes and that dibutyryl cyclic adenosine 3′,5′-monophosphate can improve the meiotic competence of bovine oocytes in culture.

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