Dp 71

Mitochondrial mass was evaluated by Mitotracker green (MTG, Invitrogen/Molecular Probes, Eugene, OR) that integrates into the mitochondrial lipid compartment regardless of the membrane potential, reporting on the whole mitochondrial mass^{51 (link),52 (link)}. Briefly, zona free oocytes were kept in PBS/PVA containing 500 nM MTG for 1 h at 38.5 °C. Then, oocytes were washed several times in PBS/PVA medium and placed individually in drops of PBS/PVA. The fluorescence intensity of MTG (Excitation/Emission: 490/516 nm) was determined for each oocyte individually using an inverted fluorescence microscopy (Olympus BX51, Tokyo, Japan). A digital image of each oocyte was taken through a highly sensitive camera (DP‐71 Olympus). The Fluorescence intensity of MTG was measured using Image J software. The mitochondrial mass measurement was performed in triplicates.

In order to assess the maturation rate following IVM, cumulus cells were removed from COCs by vortexing with 300 IU/ml hyaluronidase. The denuded oocytes were washed 3 times in phosphate buffer solution without calcium and magnesium (PBS-B) containing 1 mg/ml polyvinyl alcohol (PBS/PVA), afterward fixed for 20 min in 4% paraformaldehyde. Then, they were stained with Hoechst 33342 (10 µg/ml) for 5 min. After mounting the stained oocytes on glass slide, the image of the stained oocyte was captured and assessed by a high-resolution digital camera (DP-71 Olympus) using DP2‐BSW software (provided by the instrument manufacturer).

Mitochondrial membrane potential (MMP) was assessed using JC‐1 (JC‐1 fluorochrome; Cat. No. M34152; Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. Briefly, zona free oocytes were transferred to HTCM medium with JC1 (20 µg/ml) and cultured for 1 h in a 5% CO₂ incubator at 38.5 °C. After 1 h, oocytes were washed several times in PBS/PVA medium. Then, oocytes were placed in drops of PBS/PVA individually and fluorescence intensity of JC1 aggregates and monomer was determined for each oocyte individually using an inverted fluorescence microscopy (Olympus BX51, Tokyo, Japan). A digital image of each oocyte was taken through a highly sensitive camera (DP‐71 Olympus). Fluorescence intensities of aggregates and monomer were measured using Image J software. The ratio of aggregates and monomer was calculated as the average intensity of aggregates divided by the average intensity of monomer. The JC1 measurement was performed in triplicates.