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: 3.56). 01/2008; 134(6):731-8. DOI: 10.1530/REP-07-0149
Source: PubMed

ABSTRACT 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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    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.
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May 27, 2014