Spindle assembly in the absence of chromosomes in mouse oocytes.
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.
State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Chaoyang, Beijing 100101, People's Republic of China.
Ji-Wen
JW
Yang
Zi-Li
ZL
Lei
Yi-Liang
YL
Miao
Jun-Cheng
JC
Huang
Li-Hong
LH
Shi
Ying-Chun
YC
OuYang
Qing-Yuan
QY
Sun
Da-Yuan
DY
Chen
D000818:Animals; D015415:Biomarkers / Q000032:analysis; D002467:Cell Nucleus; D033481:Chromosomes, Mammalian / Q000502:physiology; D003593:Cytoplasm / Q000737:chemistry / Q000648:ultrastructure; D005260:Female; D008540:Meiosis / Q000502:physiology*; D008677:Metaphase; D051379:Mice; D008846:Micromanipulation; D022101:Microtubule-Organizing Center / Q000648:ultrastructure; D009865:Oocytes / Q000737:chemistry / Q000166:cytology*; D008941:Spindle Apparatus / Q000502:physiology* / Q000648:ultrastructure; D013194:Staining and Labeling; D014404:Tubulin / Q000032:analysis
D015415:Biomarkers; D014404:Tubulin