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

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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Specific deletion of Cdc42 does not affect meiotic spindle organization/migration and homologous chromosome segregation but disrupts polarity establishment and cytokinesis in mouse oocytes

Molecular Biology of the Cell ◽

10.1091/mbc.e13-03-0123 ◽

2013 ◽

Vol 24 (24) ◽

pp. 3832-3841 ◽

Cited By ~ 31

Author(s):

Zhen-Bo Wang ◽

Zong-Zhe Jiang ◽

Qing-Hua Zhang ◽

Meng-Wen Hu ◽

Lin Huang ◽

...

Keyword(s):

Chromosome Segregation ◽

Homologous Chromosome ◽

Polar Body ◽

Conditional Knockout ◽

Meiotic Spindle ◽

Spindle Positioning ◽

Mouse Oocytes ◽

Polar Bodies ◽

And Migration ◽

Specific Deletion

Mammalian oocyte maturation is distinguished by highly asymmetric meiotic divisions during which a haploid female gamete is produced and almost all the cytoplasm is maintained in the egg for embryo development. Actin-dependent meiosis I spindle positioning to the cortex induces the formation of a polarized actin cap and oocyte polarity, and it determines asymmetric divisions resulting in two polar bodies. Here we investigate the functions of Cdc42 in oocyte meiotic maturation by oocyte-specific deletion of Cdc42 through Cre-loxP conditional knockout technology. We find that Cdc42 deletion causes female infertility in mice. Cdc42 deletion has little effect on meiotic spindle organization and migration to the cortex but inhibits polar body emission, although homologous chromosome segregation occurs. The failure of cytokinesis is due to the loss of polarized Arp2/3 accumulation and actin cap formation; thus the defective contract ring. In addition, we correlate active Cdc42 dynamics with its function during polar body emission and find a relationship between Cdc42 and polarity, as well as polar body emission, 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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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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The localization of LAP2β during pronuclear formation in bovine oocytes after fertilization or activation

Zygote ◽

10.1017/s0967199406003613 ◽

2006 ◽

Vol 14 (2) ◽

pp. 157-167 ◽

Cited By ~ 2

Author(s):

Mamiko Isaji ◽

Hisataka Iwata ◽

Hiroshi Harayama ◽

Masashi Miyake

Keyword(s):

Chromatin Condensation ◽

Polar Body ◽

Somatic Cells ◽

Histone H3 ◽

Cumulus Cells ◽

Polar Bodies ◽

Second Polar Body ◽

Pronuclear Formation ◽

Bovine Oocytes

SummaryWe have shown that the assembly of lamin-associated polypeptide (LAP) 2β was detected surrounding the chromatin mass around the time of extrusion of the second polar body (PB) in some fertilized oocytes, but not in most activated oocytes, by using A23187 and cycloheximide (CaA + CH). Here, we immunohistologically analysed the correlation between LAP2β assembly and chromatin condensation in fertilized and activated oocytes during the second meiosis. In bovine cumulus cells, the onset of LAP2β assembly was observed around anaphase chromosomes with strongly phosphorylated histone H3. No LAP2β assembled around the chromosomes in the first and second polar bodies and the alternative oocyte chromatin (oCh) if histone H3 was phosphorylated. Only histone H3 of oCh was completely dephosphorylated during the telophase II/G1 transition (Tel II/G1), and then LAP2β assembled around only the oCh without phosphorylated histone H3. In the oocytes activated by CaA + CH, LAP2β did not assemble around the condensed oCh during the Tel II/G1 transition, although their histone H3 dephosphorylation occurred rather rapidly compared with that of the fertilized oocytes. The patterns of histone H3 dephosphorylation and LAP2β assembly in oocytes activated by CaA alone showed greater similarity to those in fertilized oocytes than to those in oocytes activated by CaA + CH. These results show that LAP2β assembles around only oCh after complete dephosphorylation of histone H3 after fertilization and activation using CaA alone, and that the timing of histone H3 dephosphorylation and LAP2β assembly in these oocytes is different from that of somatic cells. The results also indicate that CH treatment inhibits LAP2β assembly around oCh but not histone H3 dephosphorylation.

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56 THE EFFECT OF VITRIFICATION FOR SHEEP OOCYTES VIABILITY

Reproduction Fertility and Development ◽

10.1071/rdv27n1ab56 ◽

2015 ◽

Vol 27 (1) ◽

pp. 121 ◽

Cited By ~ 1

Author(s):

Y. M. Toishibekov ◽

R. K. Tursunova ◽

M. Sh. Yermekova

Keyword(s):

Polar Body ◽

Control Group ◽

Polar Bodies ◽

Maturation Medium ◽

Chi Squared ◽

Fetal Bovine ◽

Second Polar Body ◽

Cryopreservation Techniques ◽

Humidified Air

Advances in reproduction technologies, such as in vitro maturation, IVF, and in vitro culture, stimulated research for efficient cryopreservation techniques for mammalian oocytes. It is well known that the oocyte is the largest cell of an animal's body and as such, is full of water and, in many species, fat, making it difficult to cryopreserve. The objective of this work was to study the effect of vitrification for cryopreservation of the metaphase II plate (MPII) of sheep oocytes. Ovaries from 20 ewes of Kazakh Arkharo-Merino breed were acquired after slaughter and maintained at 37°C in TCM-199. The maturation medium was TCM-199, containing 1 mM of glutamine, 10% FBS, 5 μg mL–1 FSH, 5 μg mL–1 LH, 1 μg mL–1 oestradiol, 0.3 mM sodium pyruvate, and 100 mM cysteamine. The oocytes were incubated in 400 μL of medium in 4-well dishes covered with mineral oil. The IVM conditions were 5% CO2 in humidified air at 39°C for 24 h. Then they were placed for 10 min in a media with Hoechst 33342 (3 μg mL–1) and cytochalasin B (7 μg mL–1) to facilitate the enucleation of the MPII with a minimum volume of ooplasm. The MPII plates were divided into 2 groups: the vitrification group was exposed to vitrification media containing 1.12 M ethylene glycol (ET) + 0.87 M ME2SO for 5 min and was exposed in vitrification media containing 2.24 M ET + 1.75 M ME2SO for 5 min, and then in vitrification solution containing 4.48 M ET + 40% ME2SO + 0.25 M sucrose for 30 s. Oocytes were loaded into cryoloop and plunged into liquid nitrogen (LN2). Oocytes were thawed in a 25°C water bath and then placed in TCM-199 at 20% fetal bovine serum. After 15 min of incubation the oocytes were activated for extrusion of the second polar body in 1 mg mL–1 Ca ionophore for 5 min and washed for 5 min followed by 4 h in 6-DMAP (0.12 mM) + cycloheximide (0.6 μg mL–1). After activation the MPII were washed and cultured for 20 h. The control group received the same treatment, but they were not vitrified. Differences between the experimental groups were tested using Chi-squared test. Our research showed the expulsion of the second polar body after activation was observed in more than 62.2% of the MPII that were not vitrified (control group), whereas 40.5% of vitrified plates had expulsion of polar bodies (P < 0.05). These preliminary studies showed that it is possible to vitrify MPII plates. On the other hand, the drastic reduction of the volume of the sheep oocytes might make cryopreservation possible with greater efficiency.

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The structured core of human β tubulin confers isotype-specific polymerization properties

The Journal of Cell Biology ◽

10.1083/jcb.201603050 ◽

2016 ◽

Vol 213 (4) ◽

pp. 425-433 ◽

Cited By ~ 52

Author(s):

Melissa C. Pamula ◽

Shih-Chieh Ti ◽

Tarun M. Kapoor

Keyword(s):

Dynamic Instability ◽

Sequence Divergence ◽

Regulatory Proteins ◽

Microtubule Dynamic ◽

Microtubule Associated Proteins ◽

Cytoskeleton Organization ◽

Wide Range ◽

Associated Proteins ◽

And Function ◽

Β Tubulin

Diversity in cytoskeleton organization and function may be achieved through variations in primary sequence of tubulin isotypes. Recently, isotype functional diversity has been linked to a “tubulin code” in which the C-terminal tail, a region of substantial sequence divergence between isotypes, specifies interactions with microtubule-associated proteins. However, it is not known whether residue changes in this region alter microtubule dynamic instability. Here, we examine recombinant tubulin with human β isotype IIB and characterize polymerization dynamics. Microtubules with βIIB have catastrophe frequencies approximately threefold lower than those with isotype βIII, a suppression similar to that achieved by regulatory proteins. Further, we generate chimeric β tubulins with native tail sequences swapped between isotypes. These chimeras have catastrophe frequencies similar to that of the corresponding full-length construct with the same core sequence. Together, our data indicate that residue changes within the conserved β tubulin core are largely responsible for the observed isotype-specific changes in dynamic instability parameters and tune tubulin’s polymerization properties across a wide range.

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A casein kinase 1 prevents expulsion of the oocyte meiotic spindle into a polar body by regulating cortical contractility

Molecular Biology of the Cell ◽

10.1091/mbc.e17-01-0056 ◽

2017 ◽

Vol 28 (18) ◽

pp. 2410-2419 ◽

Cited By ~ 3

Author(s):

Jonathan R. Flynn ◽

Francis J. McNally

Keyword(s):

Polar Body ◽

Spindle Pole ◽

Casein Kinase ◽

Meiotic Spindle ◽

Contractile Ring ◽

Direct Inhibition ◽

Casein Kinase 1 ◽

Asymmetric Cell Divisions ◽

Polar Bodies ◽

Cortical Contractility

During female meiosis, haploid eggs are generated from diploid oocytes. This reduction in chromosome number occurs through two highly asymmetric cell divisions, resulting in one large egg and two small polar bodies. Unlike mitosis, where an actomyosin contractile ring forms between the sets of segregating chromosomes, the meiotic contractile ring forms on the cortex adjacent to one spindle pole, then ingresses down the length of the spindle to position itself at the exact midpoint between the two sets of segregating chromosomes. Depletion of casein kinase 1 gamma (CSNK-1) in Caenorhabditis elegans led to the formation of large polar bodies that contain all maternal DNA, because the contractile ring ingressed past the spindle midpoint. Depletion of CSNK-1 also resulted in the formation of deep membrane invaginations during meiosis, suggesting an effect on cortical myosin. Both myosin and anillin assemble into dynamic rho-dependent cortical patches that rapidly disassemble in wild-type embryos. CSNK-1 was required for disassembly of both myosin patches and anillin patches. Disassembly of anillin patches was myosin independent, suggesting that CSNK-1 prevents expulsion of the entire meiotic spindle into a polar body by negatively regulating the rho pathway rather than through direct inhibition of myosin.

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Review of the longevity of the second polar body in the mouse

Zygote ◽

10.1017/s0967199403001047 ◽

2003 ◽

Vol 11 (1) ◽

pp. 23-34 ◽

Cited By ~ 2

Author(s):

Roland Bartholomeusz

Keyword(s):

Single Gene ◽

Polar Body ◽

Research Model ◽

Polar Bodies ◽

Single Gene Disorders ◽

First Polar Body ◽

Excellent Research ◽

Second Polar Body ◽

Normal Offspring ◽

Potential Use

The polar bodies are derived from meiotic divisions during oogenesis and are contained together with the oocyte within the zona pellucida. Fertilisation triggers the second meiotic division, at which time the second polar body (PB2) is formed (Hogan et al., 1986; Schatten et al., 1988; Johnson & Everitt, 1995) There is no clear evidence on the fate of the polar bodies in any mammal including the mouse, which is the commonly used research model. However, the polar bodies are generally considered as waste material, and therefore not essential to embryo development. In recent years the polar bodies have gained prominence as they have been used in humans for pre-implantation genetic diagnostic purposes (PGD), of single gene disorders, such as determining whether an embryo may have inherited the cystic fibrosis allele from its mother (Munne et al., 1995; Strom et al., 1998; Rechitsky et al., 2000). PB2 also has a potential use in cloning, for the harvesting of stem cells. Wakayama et al. (1997) have shown that PB2 has the same genetic potential as the female pronuclei and can be used for the production of normal offspring in mice. The successful use of PB2 for these purposes is dependent on its age, for its longevity, rate and nature of degeneration has yet to be determined. While there is little doubt that the first polar body (PB1) experiences a necrotic fate, the same cannot be said for PB2, which may experience an apoptotic fate. Furthermore if PB2 experiences an apoptotic fate rather than a necrotic one, it would not only be the earliest evidence of apoptosis in a mammal but also provide an excellent research model for the study of apoptosis.

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