Mitochondrial Distribution and ROS Analysis

Oocytes were observed using a Nikon C1/TE2000-U laser scanning confocal microscope (Nikon Instruments, Firenze, Italy) equipped with the objective Nikon Plan Apo 60×/NA 1.40 in oil immersion. A 543 nm helium/neon laser and a G-2A filter were used to detect the MitoTracker Orange CMTM Ros (551 nm excitation and 576 nm emission). A 488 nm argon ion laser and a B-2A filter were used to detect dichlofluorescein (DCF) (495 nm excitation and 519 nm emission). To allow 3D distribution analysis, oocytes were observed in 25 optical sections with a step size of 0.45 µm. The mitochondrial distribution pattern was evaluated using previously reported criteria: (1) finely granular, typical of immature oocytes; (2) perinuclear and subcortical (P/S), indicating cytoplasmic maturity; and (3) abnormal, displaying irregular distribution of mitochondria [18 (link)]. Oocytes with intracellular ROS diffused throughout the cytoplasm, together with areas/sites of mitochondria/ROS overlapping, were considered viable.