Oocytes matured in vitro for 14 hours in CZB medium supplemented with l -glutamine under mineral oil were transferred to CZB containing 100 μM monastrol (MilliporeSigma, no. M8515) and incubated an extra 2 hours; monastrol is an Eg5-kinesin inhibitor, which induces monopolar spindle formation and thus results in a rosette of chromosome distribution (33 (link), 34 (link)). After treatment with monastrol, oocytes at metaphase II stage were fixed in a freshly prepared solution of 2.5% paraformaldehyde (MilliporeSigma, no. P6148) and stained with CREST autoimmune serum (Antibodies Incorporated, no. 15-234; 1:25) to label the kinetochores. DAPI was used to label the DNA. Fluorescence was observed using a 40× oil objective using a Leica DMI8 microscope, and oocytes were imaged using 0.5-μm Z-intervals to observe all kinetochores. Oocytes were analyzed individually and were scored either as euploid (containing 40 kinetochores) or as aneuploid (containing greater or less than 40 kinetochores).
Protocol detail
Assessing Oocyte Aneuploidy via Monastrol-Induced Monopolar Spindles
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After treatment
Aneuploid
Autoimmune antibodies
Chromosome
Crest
Dapi
Fluorescence
Glutamine
Kinesin
Kinetochores
Metaphase
Microscope
Mineral oil
Monastrol
Oocytes
Paraformaldehyde
Serum
Corresponding Organization : University of Missouri
Other organizations : National Heart Lung and Blood Institute, National Institutes of Health
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Variable analysis
independent variables
- Incubation of oocytes in CZB medium containing 100 μM monastrol (Eg5-kinesin inhibitor)
- Chromosome distribution (rosette-like pattern)
- Number of kinetochores in metaphase II oocytes (euploid: 40 kinetochores, aneuploid: greater or less than 40 kinetochores)
- Maturation of oocytes in CZB medium supplemented with L-glutamine under mineral oil for 14 hours
- Positive control: Not specified
- Negative control: Not specified
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