Spindle assembly in the absence of chromosomes in mouse oocytes

State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Chaoyang, Beijing 100101, People's Republic of China.
Reproduction (Cambridge, England) (Impact Factor: 3.17). 01/2008; 134(6):731-8. DOI: 10.1530/REP-07-0149
Source: PubMed


This study was carried out to investigate the contributions of chromosomes to spindle assembly in mouse oocytes. We generated two groups of cytoplasts (holo- and hemi-cytoplasts) by enucleation of germinal vesicle (GV), metaphase I (MI), and metaphase II (MII) oocytes using micromanipulation technology. After in vitro culture for 18 h, spindles with different shapes (bi-, mono-, or multipolar) formed in most of these cytoplasts except in hemi-GV cytoplasts. Two or more spindles were observed in most of holo-GV, holo-MI, and holo-MII cytoplasts (76.1, 77.0, and 83.7% respectively). However, the proportions of hemi-MI and hemi-MII cytoplasts with multiple sets of spindles decreased to 17.6 and 20.7% respectively. A single bipolar spindle was observed in each sham-operated oocyte generated by removing different volumes of cytoplasm from the oocytes and keeping nuclei intact. Localization of gamma-tubulin showed that microtubule organizing centers (MTOCs) were dispersed at each pole of the multiple sets of spindles formed in holo-cytoplasts. However, most of the MTOCs aggregated at the two poles of the bipolar spindle in sham-operated oocytes. Our results demonstrate that chromosomes are not essential for initiating spindle assembly but for directing distinct MTOCs to aggregate to form a bipolar spindle. Some factors of undetermined nature may pre-exist in an inactive form in GV-stage ooplasm, serving as initiators of spindle assembly upon their activation. Moreover, GV materials released into the cytoplasm may facilitate spindle assembly in normal meiotic maturation.

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Available from: Yi-liang Miao, Oct 10, 2015
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    • "These nuclei have a different epigenetic status from germ cells and must undergo reprogramming in the recipient ooplasm. Actually, abnormal global DNA methylation and histone modification patterns have been found in SCNT embryos (Yang X. et al., 2007). Moreover, although SCNT embryos show normal genomic imprinting pattern (Inoue et al., 2002), altered X-inactivation (Inoue et al., 2010) and abnormal Oct3/4 expression (Boiani et al., 2002; Kishigami et al., 2006a) in SCNT embryos have been also reported. "
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    ABSTRACT: To clarify the causes of the poor success rate of somatic cell nuclear transfer (SCNT), we addressed the impact of abnormalities observed at early cleavage stages of development on further full-term development using 'less-damage' imaging technology. To visualize the cellular and nuclear division processes, SCNT embryos were injected with a mixture of mRNAs encoding enhanced green fluorescent protein coupled with α-tubulin (EGFP-α-tubulin) and monomeric red fluorescent protein 1 coupled with histone H2B (H2B-mRFP1) and monitored until the morula/blastocyst stage three-dimensionally. First, the rate of development of SCNT embryos and its effect on the full-term developmental ability were analyzed. The speed of development was retarded and varied in SCNT embryos. Despite the rate of development, SCNT morulae having more than eight cells at 70h after activation could develop to term. Next, chromosomal segregation was investigated in SCNT embryos during early embryogenesis. To our surprise, more than 90% of SCNT embryos showed abnormal chromosomal segregation (ACS) before they developed to morula stage. Importantly, ACS per se did not affect the rate of development, morphology or cellular differentiation in preimplantation development. However, ACS occurring before the 8-cell stage severely inhibited postimplantation development. Thus, the morphology and/or rate of development are not significant predictive markers for the full-term development of SCNT embryos. Moreover, the low efficiency of animal cloning may be caused primarily by genetic abnormalities such as ACS, in addition to the epigenetic errors described previously.
    Developmental Biology 04/2012; 364(1):56-65. DOI:10.1016/j.ydbio.2012.01.001 · 3.55 Impact Factor
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    • "These results demonstrated that meiotic spindle assembly in mouse oocytes is driven by selforganization of MT arrays into bipolar structures (Brunet et al. 1999), similar to spindle assembly in other systems (Karsenti and Vernos 2001). Because the enucleation or bisection of oocytes (Brunet et al. 1999; Yang et al. 2007) could not remove all Ran and RCC1, RanGTP likely supported the observed MT selforganization . It is possible that X. laevis egg extracts are depleted of components responsible for spontaneous MT formation. "
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    ABSTRACT: The maturation of vertebrate oocyte into haploid gamete, the egg, consists of two specialized asymmetric cell divisions with no intervening S-phase. Ran GTPase has an essential role in relaying the active role of chromosomes in their own segregation by the meiotic process. In addition to its conserved role as a key regulator of macromolecular transport between nucleus and cytoplasm, Ran has important functions during cell division, including in mitotic spindle assembly and in the assembly of nuclear envelope at the exit from mitosis. The cellular functions of Ran are mediated by RanGTP interactions with nuclear transport receptors (NTRs) related to importin β and depend on the existence of chromosome-centered RanGTP gradient. Live imaging with FRET biosensors indeed revealed the existence of RanGTP gradient throughout mouse oocyte maturation. NTR-dependent transport of cell cycle regulators including cyclin B1, Wee2, and Cdc25B between the oocyte cytoplasm and germinal vesicle (GV) is required for normal resumption of meiosis. After GVBD in mouse oocytes, RanGTP gradient is required for timely meiosis I (MI) spindle assembly and provides long-range signal directing egg cortex differentiation. However, RanGTP gradient is not required for MI spindle migration and may be dispensable for MI spindle function in chromosome segregation. In contrast, MII spindle assembly and function in maturing mouse and Xenopus laevis eggs depend on RanGTP gradient, similar to X. laevis MII-derived egg extracts.
    Results and problems in cell differentiation 01/2011; 53:235-67. DOI:10.1007/978-3-642-19065-0_12
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    • "The formation of a stable bipolar meiotic spindle is crucial for chromosome disjunction in which chromosome attachment, alignment and pulling toward spindle poles are critical steps for accurate segregation. Although chromatin interaction with microtubules appears to influence microtubule dynamics, a bipolar spindle can still form in the absence of chromatin, as is the case in enucleated mouse oocytes [45], [46]. Our studies for the first time showed that phosphorylated MAPKAPK 2 (p-MK2) is localized to bipolar spindle minus ends and chromosomes during metaphase I and metaphase II in meiosis. "
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    ABSTRACT: MAPK-activated protein kinase 2 (MK2), a direct substrate of p38 MAPK, plays key roles in multiple physiological functions in mitosis. Here, we show for the first time the unique distribution pattern of MK2 in meiosis. Phospho-MK2 was localized on bipolar spindle minus ends and along the interstitial axes of homologous chromosomes extending over centromere regions and arm regions at metaphase of first meiosis (MI stage) in mouse oocytes. At metaphase of second meiosis (MII stage), p-MK2 was localized on the bipolar spindle minus ends and at the inner centromere region of sister chromatids as dots. Knockdown or inhibition of MK2 resulted in spindle defects. Spindles were surrounded by irregular nondisjunction chromosomes, which were arranged in an amphitelic or syntelic/monotelic manner, or chromosomes detached from the spindles. Kinetochore-microtubule attachments were impaired in MK2-deficient oocytes because spindle microtubules became unstable in response to cold treatment. In addition, homologous chromosome segregation and meiosis progression were inhibited in these oocytes. Our data suggest that MK2 may be essential for functional meiotic bipolar spindle formation, chromosome segregation and proper kinetochore-microtubule attachments.
    PLoS ONE 06/2010; 5(6):e11247. DOI:10.1371/journal.pone.0011247 · 3.23 Impact Factor
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