Chromatin status, spindle conformation and chromosomal alignment

Chromatin status at GV stage (as an indicator for transcriptional activity and developmental competence) was analyzed in fixed and DAPI-stained in vitro-grown oocytes at day 12 or 16 as described previously [9].

Meiotic spindle and chromosomal alignment were analyzed after preovulatory aging in MII oocytes grown in vitro. Oocytes were fixed (2% w/v paraformaldehyde, 0.1% v/v Triton X100, 1 μM taxol, 0.01% w/v aprotinine, 0.1 M Pipes, pH 6.9, 5 mM MgCl₂ and 2.5 mM EGTA) for 45 min at 37°C and blocked (0.2% w/v sodium azide, 2% v/v normal goat serum, 0.2% w/v powdered milk, 0.1 M v/v glycine, 0.01% v/v Triton X100 and 1% w/v BSA in PBS) for 60 min at 37°C. For immunostaining monoclonal mouse anti-α-tubulin antibody and FITC-conjugated secondary anti-mouse antibody (both Sigma-Aldrich) were used. Counterstaining of chromosomes was conducted with DAPI. Spindle morphology and chromosome alignment were analyzed by z-axial scanning with sequential scan mode using a Leica LCSSP2 confocal laser scanning microscope. A spindle abnormality was defined as a spindle showing at least one of the following criteria: 1) Spindle is not bipolar, 2) asymmetric or 3) very small. Chromosomes alignment was assessed using the following 3 categories: 1) aligned, 2) scattered, 3) displaced.