Aberrant spindle assembly checkpoint in bovine somatic cell nuclear transfer oocytes

Area of Science:

• Reproductive Biology
• Cellular Dynamics
• Developmental Biology

Background:

• Somatic cell nuclear transfer (SCNT) is a key technology in assisted reproduction and cloning.
• Aberrant nuclear and microtubular dynamics in SCNT oocytes can lead to developmental errors.
• The spindle assembly checkpoint (SAC) plays a critical role in ensuring accurate chromosome segregation.

Purpose of the Study:

• To investigate nuclear and microtubular dynamics in bovine SCNT oocytes.
• To analyze the function of the spindle assembly checkpoint (SAC) in SCNT.
• To understand the reasons for low cloning efficiency in SCNT.

Main Methods:

• SCNT was performed using G1/0 or M phase somatic cell nuclei.
• Microtubule dynamics were visualized using immunofluorescence.
• Spindle assembly checkpoint (SAC) function was assessed by inhibiting microtubule polymerization (demecolcine) and proteasome activity (MG132).
• Maturation-promoting factor (MPF) activity was measured.
• Developmental potential to blastocyst stage was evaluated.

Main Results:

• SCNT oocytes exhibited abnormal spindle structures (bi-, tri-polar, elongated, or miniature) and premature chromosome condensation.
• Demecolcine treatment induced SAC activation in SCNT oocytes, indicated by elevated MPF activity.
• Unlike MII oocytes, demecolcine-treated SCNT oocytes failed to maintain MII arrest, suggesting a deficient second SAC.
• However, two G1/0 nuclei in an ooplasm could restore the second SAC.
• Developmental potential to blastocyst stage was not significantly affected by demecolcine treatment.

Conclusions:

• Bovine SCNT oocytes possess abnormal spindle morphology and a defective SAC at the MII stage.
• Insufficient SAC signals from somatic cell nuclei contribute to these defects.
• Aberrant remodeling and chromosome instability in SCNT embryos may result from these early defects.
• The findings highlight a critical barrier to improving SCNT efficiency.