Aberrant spindle assembly checkpoint in bovine somatic cell nuclear transfer oocytes.
Nuclear, microtubular dynamics and spindle assembly checkpoint (SAC) in bovine somatic cell nuclear transfer (SCNT) oocytes receiving G1/0 or M phase somatic cell nuclei were studied. SCNT oocytes assembled microtubules, however, the spindles were structurally abnormal, including bi-, tri-polar or elongated spindles with scattered premature chromosome condensation (PCC) in G1/0 phase nuclei, and some miniature spindles with unaligned chromosomes in M phase nuclei. In contrast, demecolcine-treated SCNT oocytes formed chromosome clusters with membrane protrusion and significantly induced maturation-promoting factor (MPF) activity elevation (up to 177%) for 3 hours, indicating that first SAC at second metaphase (MII) is established upon spindle disruption in SCNT oocytes. After parthenogenetic stimuli, unlike MII oocytes which prevent exit from MII arrest with high MPF activity upon spindle disruption by second SAC, demecolcine-treated SCNT oocytes could not prevent exit from MII arrest with inactivation of MPF activities, whereas MG132-treated SCNT oocytes could persist at MII arrest, indicating that SCNT oocytes lack the ability for second SAC establishment, however, two G1/0 phase nuclei in an ooplasm restored second SAC establishment upon spindle disruption. Furthermore, the developmental potential of demecolcine-treated SCNT oocytes receiving G1/0 phase nuclei to blastocyst stage was not significantly different than untreated SCNT oocytes (29% vs 31%). These results indicate that unlike MII oocytes, SCNT oocytes have aberrant spindle morphology and SAC at MII due to insufficient SAC signals from somatic cell nuclei, thus aberrant remodeling has started immediately after somatic cell nuclear transfer and may be responsible for chromosome instability in SCNT embryos as well as the low successful efficiency of cloning.
Tani
Tetsuya
T
Laboratory of Animal Reproduction, College of Agriculture, Kinki University, 3327-204, Nakamachi, Nara, 631-8505, Japan.
Kato
Yoko
Y
Tsunoda
Yukio
Y
D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D016200:Maturation-Promoting Factor / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D053652:Nuclear Transfer Techniques; D009865:Oocytes / Q000201:enzymology / Q000648:ultrastructure*; D008941:Spindle Apparatus / Q000648:ultrastructure*
D016200:Maturation-Promoting Factor; D020928:Mitogen-Activated Protein Kinases