Dynamic changes in NuMA and microtubules in monkey-rabbit nuclear transfer embryos.
Previous reports have indicated that failure in cloning monkey is attributed to the removal of nuclear mitotic apparatus (NuMA) during enucleation and subsequent abnormal organization of mitotic apparatus. This study investigated the transformation and assembly of tubulin and NuMA protein during the first cell cycle of cloned monkey embryos reconstructed by using enucleated rabbit oocytes as recipients. After the oocyte fused with a fibroblast, extensive microtubule organization was observed around the introduced nucleus in most reconstructed embryos, suggesting the introduction of a somatic cell centrosome. A high proportion of fibroblast nuclei transferred into non-activated oocytes underwent premature chromosome condensation (PCC), transient spindle organization and chromosomes separation, followed by the formation of two pronucleus-like structures. In contrast, fibroblast nuclei in pre-activated ooplasm rarely underwent PCC, but formed a swollen pronucleus-like structure. Normal spindles were observed in about one third of the cloned embryos reconstructed by both methods. After transferring monkey fibroblasts into NuMA-removed enucleated rabbit oocytes, NuMA was localized in pseudo-pronuclei and gradually moved to mitotic spindle poles at the first mitotic spindle poles. NuMA antibody microinjection resulted in spindle disorganization and chromosome misalignment, but did not significantly affect early cleavage. Our findings indicate that: 1. NuMA in donor monkey fibroblast may contribute to form a normal spindle in enucleated rabbit oocyte; 2. when non-activated cytoplasts and pre-activated cytoplasts are used as recipients, the donor nuclei undergo different morphological changes, but yield similar early embryo development; 3. although abnormal spindle organization and chromosome alignment may cause low efficiency of animal cloning, these abnormalities do not significantly affect early cleavage.
Yan
Li-Ying
LY
State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100080, China.
Shi
Li-Hong
LH
Sheng
Hui-Zhen
HZ
Liu
Shu-Zhen
SZ
Huang
Jun-Cheng
JC
Zhu
Zi-Yu
ZY
OuYang
Ying-Chun
YC
Lei
Zi-Li
ZL
Song
Xiang-Fen
XF
Sun
Qing-Yuan
QY
Chen
Da-Yuan
DY
D000818:Animals; D034961:Antigens, Nuclear / Q000378:metabolism*; D018797:Cell Cycle Proteins; D002467:Cell Nucleus / Q000378:metabolism; D018385:Centrosome / Q000378:metabolism; D002843:Chromatin / Q000737:chemistry; D002875:Chromosomes / Q000378:metabolism; D005260:Female; D005347:Fibroblasts / Q000378:metabolism; D000882:Haplorhini; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008938:Mitosis; D015233:Models, Statistical; D034521:Nuclear Matrix-Associated Proteins / Q000378:metabolism*; D009865:Oocytes / Q000378:metabolism; D011485:Protein Binding; D011817:Rabbits; D008941:Spindle Apparatus / Q000378:metabolism
D034961:Antigens, Nuclear; D018797:Cell Cycle Proteins; D002843:Chromatin; C050045:NUMA1 protein, human; D034521:Nuclear Matrix-Associated Proteins