Increased frequency of chromosome congression defects and aneuploidy in mouse oocytes cultured at lower temperature

Optimal culture conditions are essential for successful IVM of mammalian oocytes and for their further development into an embryo. In the present study we used live cell imaging microscopy to assess the effects of suboptimal culture temperature on various aspects of IVM, including duration of meiosis I, dynamics of polar body extrusion, chromosome congression, anaphase-promoting complex/cyclosome (APC/C) activation and aneuploidy. The data showed that even a small deviation from the optimal incubation temperature causes marked changes in the duration and synchronicity of meiosis, APC/C activity and the frequency of chromosome congression and segregation errors. In vitro manipulation and maturation of germ cells is widely used in both human and animal artificial reproduction techniques. Mammalian oocytes are naturally prone to chromosomal segregation errors, which are responsible for severe mental and developmental disorders. The data presented herein demonstrate that exposure of mouse oocytes to suboptimal temperature during manipulation and maturation could further increase the frequency of chromosome segregation defects in these cells.

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124. THE ROLE OF CALCIUM ACTIVATED CHLORIDE CHANNELS AT FERTILISATION

Reproduction Fertility and Development ◽

10.1071/srb10abs124 ◽

2010 ◽

Vol 22 (9) ◽

pp. 42

Author(s):

S. Dalati ◽

M. L. Day

Keyword(s):

Chloride Channels ◽

Polar Body ◽

Signal Transduction Pathway ◽

Active Role ◽

Niflumic Acid ◽

In Vitro Fertilisation ◽

Mouse Oocytes ◽

Cl Channel ◽

Polar Body Extrusion

Sperm entry into the oocyte triggers a signal transduction pathway resulting in intracellular calcium [Ca2+] oscillations that coincide with hyperpolarisations in membrane potential (Em). Ca2+ oscillations have been previously described and found to be important for embryo development, yet Em hyperpolarisations and their importance at fertilisation still remains unclear. Thimerosal, a sulfhydryl reagent, has been shown to mimic the physiological changes caused by sperm following fertilisation. It does this by direct sensitisation of the inositol 1,4,5-triphosphate receptor-1 to basal levels of inositol 1,4,5-triphopshate. Previous patch clamp analysis of unfertilised mouse oocytes has shown that thimerosal elicits simultaneous Em hyperpolarisations and Ca2+ oscillations. These results have lead us to hypothesise that hyperpolarisations in Em may be due to the activation of a Ca2+ activated Cl- channel (CaCC) present in the membrane of mouse oocytes. The present study aims to identify this CaCC and assess its role in early development following fertilisation. Hyperpolarisations induced by thimerosal were inhibited by niflumic acid, a selective blocker of CaCCs. The inhibition of Em hyperpolarisations suggests that a CaCC is present and plays an active role in initiating hyperpolarisations. To identify the function of the CaCC at fertilisation, in vitro fertilisation was performed in the presence of niflumic acid. Niflumic acid inhibited polar body extrusion and pronuclei formation; two events that are indicators of fertilisation. Furthermore, Ca2+ imaging experiments with the calcium sensitive dye fura 2-AM, demonstrated that in the presence of niflumic acid, Ca2+ oscillations induced by thimerosal are reduced in size, number and duration. Taken together these data suggest that the activation of a Ca2+ activated Cl– channel in the mouse oocyte may play an important role in the events occurring at fertilisation.

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True Nondisjunction of Whole Bivalents in Oocytes with Attachment and Congression Defects

Cytogenetic and Genome Research ◽

10.1159/000458513 ◽

2017 ◽

Vol 151 (1) ◽

pp. 10-17 ◽

Cited By ~ 3

Author(s):

Martin Sodek ◽

Kristina Kovacovicova ◽

Martin Anger

Keyword(s):

Chromosome Segregation ◽

Developmental Disorders ◽

Polar Body ◽

Direct Consequence ◽

Early Embryos ◽

First Polar Body ◽

Polar Body Extrusion ◽

Meiosis I

Chromosome segregation in mammalian oocytes is prone to errors causing aneuploidy with consequences such as precocious termination of development or severe developmental disorders. Aneuploidy also represents a serious problem in procedures utilizing mammalian gametes and early embryos in vitro. In our study, we focused on congression defects during meiosis I and observed whole nondisjoined bivalents in meiosis II as a direct consequence, together with a substantially delayed first polar body extrusion. We also show that the congression defects are accompanied by less stable attachments of the kinetochores. Our results describe a process by which congression defects directly contribute to aneuploidy.

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

Cell Division ◽

10.1186/s13008-021-00071-y ◽

2021 ◽

Vol 16 (1) ◽

Author(s):

Ming-Hong Sun ◽

Lin-Lin Hu ◽

Chao-Ying Zhao ◽

Xiang Lu ◽

Yan-Ping Ren ◽

...

Keyword(s):

Golgi Apparatus ◽

Oocyte Maturation ◽

Polar Body ◽

Mouse Oocyte ◽

Actin Dynamics ◽

Mouse Oocytes ◽

Oocyte Meiosis ◽

Ral Gtpase ◽

Polar Body Extrusion ◽

Cytoskeleton Dynamics

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

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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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Kinetochore Fibers Are Not Involved in the Formation of the First Meiotic Spindle in Mouse Oocytes, but Control the Exit from the First Meiotic M Phase

The Journal of Cell Biology ◽

10.1083/jcb.146.1.1 ◽

1999 ◽

Vol 146 (1) ◽

pp. 1-12 ◽

Cited By ~ 114

Author(s):

Stéphane Brunet ◽

Angélica Santa Maria ◽

Philippe Guillaud ◽

Denis Dujardin ◽

Jacek Z. Kubiak ◽

...

Keyword(s):

Polar Body ◽

Meiotic Spindle ◽

Homologous Chromosomes ◽

Mouse Oocytes ◽

Sister Chromatids ◽

M Phase ◽

Continuous Presence ◽

Set Up ◽

Polar Body Extrusion ◽

Reductional Division

During meiosis, two successive divisions occur without any intermediate S phase to produce haploid gametes. The first meiotic division is unique in that homologous chromosomes are segregated while the cohesion between sister chromatids is maintained, resulting in a reductional division. Moreover, the duration of the first meiotic M phase is usually prolonged when compared with mitotic M phases lasting 8 h in mouse oocytes. We investigated the spindle assembly pathway and its role in the progression of the first meiotic M phase in mouse oocytes. During the first 4 h, a bipolar spindle forms and the chromosomes congress near the equatorial plane of the spindle without stable kinetochore– microtubule end interactions. This late prometaphase spindle is then maintained for 4 h with chromosomes oscillating in the central region of the spindle. The kinetochore–microtubule end interactions are set up at the end of the first meiotic M phase (8 h after entry into M phase). This event allows the final alignment of the chromosomes and exit from metaphase. The continuous presence of the prometaphase spindle is not required for progression of the first meiotic M phase. Finally, the ability of kinetochores to interact with microtubules is acquired at the end of the first meiotic M phase and determines the timing of polar body extrusion.

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

Journal of Cell Science ◽

10.1242/jcs.102.3.457 ◽

1992 ◽

Vol 102 (3) ◽

pp. 457-467 ◽

Cited By ~ 6

Author(s):

J.Z. Kubiak ◽

M. Weber ◽

G. Geraud ◽

B. Maro

Keyword(s):

Kinase Activity ◽

Polar Body ◽

Metaphase Arrest ◽

Phosphorylated Proteins ◽

Mouse Oocytes ◽

Sequence Of Events ◽

M Phase ◽

Second Polar Body ◽

Polar Body Extrusion ◽

Centrosomal Proteins

When metaphase II-arrested mouse oocytes (M II) are activated very soon after ovulation, they respond abortively by second polar body extrusion followed by another metaphase arrest (metaphase III, M III; Kubiak, 1989). The M II/M III transition resembles the natural transition between the first and second meiotic metaphases (M I/M II). We observed that a similar sequence of events takes place during these two transitions: after anaphase, a polar body is extruded, the microtubules of the midbody disappear rapidly and a new metaphase spindle forms. The MPM-2 monoclonal antibody (which reacts with phosphorylated proteins associated with the centrosome during M-phase) stains discrete foci of peri-centriolar material only in metaphase arrested oocytes; during both transitional periods, a diffuse staining is observed, suggesting that these centrosomal proteins are dephosphorylated, as in a normal interphase. However, the chromosomes always remain condensed and an interphase network of microtubules is never observed during the transitional periods. Incorporation of 32P into proteins increases specifically during the transitional periods. Pulse-chase experiments, after labeling of the oocytes in M phase with 32P, showed that a 62 kDa phosphoprotein band disappears at the time of polar body extrusion. Histone H1 kinase activity (which reflects the activity of the maturation promoting factor) drops during both transitional periods to the level characteristic of interphase and then increases when the new spindle forms. Both the M I/M II and M II/M III transitions require protein synthesis as demonstrated by the effect of puromycin. These results suggest that the two M-phase/M-phase transitions are probably driven by the same molecular mechanism.

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The effect of osmolarity on mouse parthenogenesis

Development ◽

10.1242/dev.31.2.513 ◽

1974 ◽

Vol 31 (2) ◽

pp. 513-526

Author(s):

M. H. Kaufman ◽

M. A. H. Surani

Keyword(s):

Protein Synthesis ◽

Culture Medium ◽

Culture Media ◽

Polar Body ◽

Amino Acid Mixture ◽

Culture Conditions ◽

Follicle Cells ◽

Acid Mixture ◽

Second Polar Body

Eggs from (C57B1 × A2G)F1 mice were activated by treatment with hyaluronidase, which removed the follicle cells, and cultured in vitro. Observations were made 6–8 h after hyaluronidase treatment to determine the frequency of activation and the types of parthenogenones induced. Cumulus-free eggs resulting from hyaluronidase treatment were incubated for 2¼ h in culture media of various osmolarities. The frequency of activation was found to be dependent on the postovulatory age of oocytes, while the types of parthenogenones induced were dependent on the osmolarity of the in vitro culture medium and their postovulatory age. Culture in low osmolar medium suppressed the extrusion of the second polar body (2PB). This decreased the incidence of haploid eggs with a single pronucleus and 2PB and immediately cleaved eggs from 97·5% to 42·3% of the activated population. Where 2PB extrusion had been suppressed, 97·4% of parthenogenones contained two haploid pronuclei. Very few were observed with a single and presumably diploid pronucleus. Serial observations from 11 to 18 h after hyaluronidase treatment were made on populations of activated eggs as they entered the first cleavage mitosis after 2¼ h incubation in medium either of normal (0·287 osmol) or low (0·168 osmol) osmolarity. A delay in the time of entry into the first cleavage mitosis similar to the duration of incubation in low osmolar medium was observed. Further, eggs were incubated in control and low osmolar culture media containing uniformly labelled [U-14C]amino acid mixture to examine the extent of protein synthesis in recently activated eggs subjected to these culture conditions. An hypothesis is presented to explain the effect of incubation in low osmolar culture medium in delaying the first cleavage mitosis.

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

Journal of Cell Science ◽

10.1242/jcs.104.3.861 ◽

1993 ◽

Vol 104 (3) ◽

pp. 861-872 ◽

Cited By ~ 7

Author(s):

M.S. Szollosi ◽

J.Z. Kubiak ◽

P. Debey ◽

H. de Pennart ◽

D. Szollosi ◽

...

Keyword(s):

Protein Phosphorylation ◽

Kinase Inhibitor ◽

Polar Body ◽

Histone H1 ◽

Sperm Chromatin ◽

Oocyte Activation ◽

Mouse Oocytes ◽

Spindle Rotation ◽

Polar Body Extrusion ◽

Pronuclear Formation

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

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Effect of Melatonin on the In Vitro Maturation of Porcine Oocytes, Development of Parthenogenetically Activated Embryos, and Expression of Genes Related to the Oocyte Developmental Capability

Animals ◽

10.3390/ani10020209 ◽

2020 ◽

Vol 10 (2) ◽

pp. 209 ◽

Cited By ~ 8

Author(s):

Ling Yang ◽

Qingkai Wang ◽

Maosheng Cui ◽

Qianjun Li ◽

Shuqin Mu ◽

...

Keyword(s):

In Vitro Maturation ◽

Polar Body ◽

Melatonin Receptors ◽

Melatonin Treatment ◽

In Vitro Development ◽

First Polar Body ◽

Mitochondrial Distribution ◽

Polar Body Extrusion ◽

Vitro Development

Melatonin treatment can improve quality and in vitro development of porcine oocytes, but the mechanism of improving quality and developmental competence is not fully understood. In this study, porcine cumulus–oocyte complexes were cultured in TCM199 medium with non-treated (control), 10−5 M luzindole (melatonin receptor antagonist), 10−5 M melatonin, and melatonin + luzindole during in vitro maturation, and parthenogenetically activated (PA) embryos were treated with nothing (control), or 10−5 M melatonin. Cumulus oophorus expansion, oocyte survival rate, first polar body extrusion rate, mitochondrial distribution, and intracellular levels of reactive oxygen species (ROS) and glutathione of oocytes, and cleavage rate and blastocyst rate of the PA embryos were assessed. In addition, expression of growth differentiation factor 9 (GDF9), tumor protein p53 (P53), BCL2 associated X protein (BAX), catalase (CAT), and bone morphogenetic protein 15 (BMP15) were analyzed by real-time quantitative PCR. The results revealed that melatonin treatment not only improved the first polar body extrusion rate and cumulus expansion of oocytes via melatonin receptors, but also enhanced the rates of cleavage and blastocyst formation of PA embryos. Additionally, melatonin treatment significantly increased intraooplasmic level of glutathione independently of melatonin receptors. Furthermore, melatonin supplementation not only significantly enhanced mitochondrial distribution and relative abundances of BMP15 and CAT mRNA, but also decreased intracellular level of ROS and relative abundances of P53 and BAX mRNA of the oocytes. In conclusion, melatonin enhanced the quality and in vitro development of porcine oocytes, which may be related to antioxidant and anti-apoptotic mechanisms.

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