Meiotic spindle position and second polar body orientation enable the prediction of embryonic developmental potential and embryo euploidy

Polar body extrusion during oocyte maturation is critically dependent on asymmetric positioning of the meiotic spindle, which is established through migration of the meiosis I (MI) spindle/chromosomes from the oocyte interior to a subcortical location. In this study, we show that MI chromosome migration is biphasic and driven by consecutive actin-based pushing forces regulated by two actin nucleators, Fmn2, a formin family protein, and the Arp2/3 complex. Fmn2 was recruited to endoplasmic reticulum structures surrounding the MI spindle, where it nucleated actin filaments to initiate an initially slow and poorly directed motion of the spindle away from the cell center. A fast and highly directed second migration phase was driven by actin-mediated cytoplasmic streaming and occurred as the chromosomes reach a sufficient proximity to the cortex to activate the Arp2/3 complex. We propose that decisive symmetry breaking in mouse oocytes results from Fmn2-mediated perturbation of spindle position and the positive feedback loop between chromosome signal-induced Arp2/3 activation and Arp2/3-orchestrated cytoplasmic streaming that transports the chromosomes.

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Participation of the ubiquitin-proteasome pathway in rat oocyte activation

Zygote ◽

10.1017/s0967199405003114 ◽

2005 ◽

Vol 13 (1) ◽

pp. 87-95 ◽

Cited By ~ 2

Author(s):

Xin Tan ◽

An Peng ◽

Yong-Chao Wang ◽

Yue Wang ◽

Qing-Yuan Sun

Keyword(s):

Meiotic Division ◽

Polar Body ◽

Meiotic Spindle ◽

Oocyte Activation ◽

Parthenogenetic Activation ◽

Ubiquitin Proteasome Pathway ◽

Ubiquitin Proteasome ◽

Proteasome Pathway ◽

Second Polar Body

The role of the ubiquitin-proteasome pathway (UPP) in mitosis is well known. However, its role in meiotic division is still poorly documented, especially in the activation of mammalian oocytes. In this study, the role of proteasome in the spontaneous and parthenogenetic activation of rat oocytes was investigated. We found that ALLN, an inhibitor of proteasome, when applied to metaphase II oocytes, inhibited spontaneous activation, blocked extrusion of the second polar body (PB) and caused the withdrawal of the partially extruded second PB. ALLN also inhibited the parthenogenetic activation induced by cycloheximide, but had no effect on the formation of pronuclei in activated eggs. In metaphase and anaphase, ubiquitin and proteasome localized to the meiotic spindle, concentrating on both sides of the oocyte–second PB boundary during PB extrusion. This pattern of cellular distribution suggests that UPP may have a role in regulating nuclear division and cytokinesis. Ubiquitin was seen to form a ring around the pronucleus, whereas proteasome was evenly distributed in the pronuclear region. Taken together, our results indicate that (1) UPP is required for the transitions of oocytes from metaphase II to anaphase II and from anaphase II to the end of meiosis; and (2) the UPP plays a role in cytokinesis of the second meiotic division.

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Deficiencies in extrusion of the second polar body due to high calcium concentrations during in vitro fertilization in inbred C3H/He mice

Zygote ◽

10.1017/s096719941500060x ◽

2015 ◽

Vol 24 (4) ◽

pp. 603-616 ◽

Cited By ~ 3

Author(s):

Yuki Ohta ◽

Yoshikazu Nagao ◽

Naojiro Minami ◽

Satoshi Tsukamoto ◽

Seiji Kito

Keyword(s):

Calcium Concentration ◽

Polar Body ◽

Cell Mass ◽

Meiotic Spindle ◽

Dependent Manner ◽

Cytoskeletal Organization ◽

High Calcium ◽

High Calcium Concentration ◽

Second Polar Body

SummarySuccessful in vitro fertilization (IVF) of all inbred strains of laboratory mice has not yet been accomplished. We have previously shown that a high calcium concentration improved IVF in various inbred mice. However, we also found that in cumulus-free ova of C3H/He mice such IVF conditions significantly increased the deficiency of extrusion of the second polar body (PBII) in a dose-dependent manner (2% at 1.71 mM and 29% at 6.84 mM, P < 0.05) and that PBII extrusion was affected by high calcium levels at 2–3 h post-insemination. While developmental competence of ova without PBII extrusion to blastocysts after 96 h culture was not affected, a significant reduction in the nuclear number of the inner cell mass was observed in blastocyst fertilized under high calcium condition. We also examined how high calcium concentration during IVF affects PBII extrusion in C3H/He mice. Cumulus cells cultured under high calcium conditions showed a significantly alleviated deficient PBII extrusion. This phenomenon is likely to be specific to C3H/He ova because deficient PBII extrusion in reciprocal fertilization between C3H and BDF1 gametes was observed only in C3H/He ova. Sperm factor(s) was still involved in deficient PBII extrusion due to high calcium concentrations, as this phenomenon was not observed in ova activated by ethanol. The cytoskeletal organization of ova without PBII extrusion showed disturbed spindle rotation, incomplete formation of contractile ring and disturbed localization of actin, suggesting that high calcium levels affect the anchoring machinery of the meiotic spindle. These results indicate that in C3H/He mice high calcium levels induce abnormal fertilization, i.e. deficient PBII extrusion by affecting the cytoskeletal organization, resulting in disturbed cytokinesis during the second meiotic division. Thus, use of high calcium media for IVF should be avoided for this strain.

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Mitochondrial activity and cytoskeleton organization in three pronuclei oocytes after intracytoplasmic sperm injection

Zygote ◽

10.1017/s0967199418000278 ◽

2018 ◽

Vol 26 (4) ◽

pp. 319-325

Author(s):

Tuğba Kotil ◽

M. Ertan Kervancıoğlu ◽

Gülçin Ekter Kanten ◽

Gülden Tunalı ◽

Seyhun Solakoğlu

Keyword(s):

Membrane Potential ◽

Mitochondrial Membrane Potential ◽

Intracytoplasmic Sperm Injection ◽

Mitochondrial Membrane ◽

Polar Body ◽

Meiotic Spindle ◽

Mitochondrial Activity ◽

Electron Microscopic ◽

Second Polar Body ◽

Polar Body Extrusion

SummaryDigyny, the presence of a third pronucleus due to the failure of second polar body extrusion, is problematic after intracytoplasmic sperm injection (ICSI) practices. Mitochondria have critical roles such as production of adenosine triphosphate (ATP) and regulation of Ca2+ homeostasis during oocyte maturation, fertilization and the following development, while the regulation of meiotic spindle formation, chromosome segregation, pronuclear apposition and cytokinesis is closely associated with the cytoskeleton. In this study, mitochondrial membrane potential, distribution of F-actin and γ-tubulin, and the ultrastructure of three pronuclear (3PN) oocytes were investigated. 3PN oocytes after ICSI procedure were taken from patients who were enrolled in assisted reproduction programmes. For mitochondrial membrane potential analysis, fresh oocytes stained with the mitochondrial membrane potential probe JC-1, were evaluated under fluorescence microscopy. The mitochondrial membrane potential of three pronuclear oocytes showed similar results to normal zygotes. γ-Tubulin was stained strongly at the subplasmalemmal domain and microfilaments were localized at the cortical, but not the perinuclear, area. Cytoplasmic halos were moderately or not detected by electron microscopy; lipofuscin granules, degenerated mitochondria, and multilamellated bodies were seen in the ooplasm. Immunohistochemistry and electron microscopic findings suggested that mitochondrial membrane potential has no direct effect on second polar body extrusion. This abnormality can be associated with an altered cytoskeleton due to poor oocyte quality.

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Comparison of embryo developmental potential of in vitro matured metaphase i (IVM-MI) oocytes according to meiotic spindle position

Fertility and Sterility ◽

10.1016/j.fertnstert.2014.07.1159 ◽

2014 ◽

Vol 102 (3) ◽

pp. e342-e343

Author(s):

E. Garcia-Cerrudo ◽

J.H. Moon ◽

A. Mahfoudh ◽

S. Henderson ◽

H. Holzer ◽

...

Keyword(s):

Meiotic Spindle ◽

Developmental Potential ◽

Spindle Position ◽

Metaphase I

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Differential Expression and Functions of Cortical Myosin IIA and IIB Isotypes during Meiotic Maturation, Fertilization, and Mitosis in Mouse Oocytes and Embryos

Molecular Biology of the Cell ◽

10.1091/mbc.9.9.2509 ◽

1998 ◽

Vol 9 (9) ◽

pp. 2509-2525 ◽

Cited By ~ 92

Author(s):

Calvin Simerly ◽

Grzegorz Nowak ◽

Primal de Lanerolle ◽

Gerald Schatten

Keyword(s):

Differential Expression ◽

Polar Body ◽

Myosin Ii ◽

Meiotic Spindle ◽

Meiotic Maturation ◽

Myosin Isoforms ◽

Cleavage Furrow ◽

Myosin Iia ◽

Second Polar Body

To explore the role of nonmuscle myosin II isoforms during mouse gametogenesis, fertilization, and early development, localization and microinjection studies were performed using monospecific antibodies to myosin IIA and IIB isotypes. Each myosin II antibody recognizes a 205-kDa protein in oocytes, but not mature sperm. Myosin IIA and IIB demonstrate differential expression during meiotic maturation and following fertilization: only the IIA isoform detects metaphase spindles or accumulates in the mitotic cleavage furrow. In the unfertilized oocyte, both myosin isoforms are polarized in the cortex directly overlying the metaphase-arrested second meiotic spindle. Cortical polarization is altered after spindle disassembly with Colcemid: the scattered meiotic chromosomes initiate myosin IIA and microfilament assemble in the vicinity of each chromosome mass. During sperm incorporation, both myosin II isotypes concentrate in the second polar body cleavage furrow and the sperm incorporation cone. In functional experiments, the microinjection of myosin IIA antibody disrupts meiotic maturation to metaphase II arrest, probably through depletion of spindle-associated myosin IIA protein and antibody binding to chromosome surfaces. Conversely, the microinjection of myosin IIB antibody blocks microfilament-directed chromosome scattering in Colcemid-treated mature oocytes, suggesting a role in mediating chromosome–cortical actomyosin interactions. Neither myosin II antibody, alone or coinjected, blocks second polar body formation, in vitro fertilization, or cytokinesis. Finally, microinjection of a nonphosphorylatable 20-kDa regulatory myosin light chain specifically blocks sperm incorporation cone disassembly and impedes cell cycle progression, suggesting that interference with myosin II phosphorylation influences fertilization. Thus, conventional myosins break cortical symmetry in oocytes by participating in eccentric meiotic spindle positioning, sperm incorporation cone dynamics, and cytokinesis. Although murine sperm do not express myosin II, different myosin II isotypes may have distinct roles during early embryonic development.

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Relationship between meiotic spindle location with regard to the polar body position and oocyte developmental potential after ICSI

Human Reproduction ◽

10.1093/humrep/deg274 ◽

2003 ◽

Vol 18 (6) ◽

pp. 1289-1293 ◽

Cited By ~ 121

Author(s):

L. Rienzi

Keyword(s):

Body Position ◽

Polar Body ◽

Meiotic Spindle ◽

Developmental Potential

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Modifications of chemically induced-enucleated nuclear transfer technique by reverse-order nuclear transfer in mouse

Zygote ◽

10.1017/s0967199409005346 ◽

2009 ◽

Vol 17 (3) ◽

pp. 261-268 ◽

Cited By ~ 2

Author(s):

Yongsheng Wang ◽

Jun Liu ◽

Shuang Tang ◽

Zhixing An ◽

Zhilin Guo ◽

...

Keyword(s):

Nuclear Transfer ◽

Polar Body ◽

Developmental Rate ◽

Cell Number ◽

Reverse Order ◽

Cleavage Rate ◽

Simple Method ◽

Developmental Potential ◽

Chemically Induced ◽

Second Polar Body

SummaryTo improve the developmental potential of somatic cell cloned embryos derived from demecolcine (DC) induced-enucleated nuclear transfer (INT), we modified the INT procedures by transferring donor nuclei into recipient cytoplasts prior to the induced enucleation of the recipient cytoplasts, and we called this modified INT technique as reverse-order and induced-enucleated nuclear transfer (RINT). Standard nuclear transfer (SNT) and INT were performed as controls. The dynamic changes of maternal and transferred donor nuclei in the RINT oocytes were monitored to evaluate the feasibility of this new nuclear transfer (NT) technique by timed immunofluorescence. Timed immunofluorescence showed that RINT is feasible because none of the transferred donor nuclei were expelled with the second polar body (Pb) in the RINT oocytes, while 42.2% of the oocytes showed extrusion of all maternal chromosome and spindles with the second Pb at 60 min after activation and DC treatment. Although there was no difference in cleavage rate (86.6% vs. 82.1%), the rates of successful enucleation and blastocyst formation were significantly increased in RINT compared with INT (44.1% vs. 27.5% and 43.3% vs. 12.8%, respectively; p < 0.01). Compared with SNT, there was no difference in cleavage rate (86.6% vs. 78.4%), but the blastocyst developmental rate was significantly increased in the RINT group (43.3% vs. 25.3%; p < 0.01). Blastocysts derived from RINT had a higher total cell number than those from SNT (45.1 ± 3 vs. 37.6 ± 4; p < 0.05). Our results provide evidence that RINT is feasible and may provide a more efficient and simple method for NT than INT.

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PAR-1 and the microtubule-associated proteins CLASP2 and dynactin-p50 have specific localisation on mouse meiotic and first mitotic spindles

Reproduction ◽

10.1530/rep.1.00651 ◽

2005 ◽

Vol 130 (3) ◽

pp. 311-320 ◽

Cited By ~ 8

Author(s):

Catherine A Moore ◽

Magdalena Zernicka-Goetz

Keyword(s):

Polar Body ◽

Model Systems ◽

Regulatory Proteins ◽

Meiotic Spindle ◽

Mitotic Spindles ◽

Microtubule Associated Proteins ◽

Mouse Oocytes ◽

Polar Bodies ◽

Associated Proteins ◽

Second Polar Body

The site of second meiotic division, marked by the second polar body, is an important reference point in the early mouse embryo. To study its formation, we look at the highly asymmetric meiotic divisions. For extrusion of the small polar bodies during meiosis, the spindles must be located cortically. The positioning of meiotic spindles is known to involve the actin cytoskeleton, but whether microtubules are also involved is not clear. In this study we investigated the patterns of localisation of microtubule regulatory proteins in mouse oocytes. PAR-1 is a member of the PAR (partitioning-defective) family with known roles in regulation of microtubule stability and spindle positioning in other model systems. Here we show its specific localisation on mouse meiotic and first mitotic spindles. In addition, the microtubule-associated proteins CLASP2 (a CLIP associating protein) and dynactin-p50 are found on kinetochores and a subset of microtubule-organising centres. Thus we show specific localisation of microtubule regulatory proteins in mouse oocytes, which could indicate roles in meiotic spindle organisation.

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