710 confocal fluorescence microscope

For photos of live embryos at 30 hpf, the embryos were embedded in 2% methylcellulose (Sigma-Aldrich) in embryo medium with 0.04% tricaine (Sigma-Aldrich). For time-lapse vital microscopy, embryos were embedded in 1% low-melting agar in embryo medium with 0.04% tricaine (Sigma-Aldrich). When imaging the periderm, recording started at 28 hpf and continued for 18 h, with images acquired at 8-min intervals. A 710 laser-scanning confocal (Zeiss) and spinning disk fluorescence (Nikon) microscopes were used. When imaging the basal layer, 2-h recordings were performed between 24 and 30 hpf with images collected at 2-min intervals. A 710 confocal fluorescence microscope (Zeiss) was used. Imaging was performed with a long working distance 20× lens. All the time-lapse studies were performed at 28.5°C. Image analysis was performed using Zeiss Black or Fiji ImageJ software.

Intracellular reactive oxygen species (ROS) in living oocytes were stained using the general oxidative stress indicator (CM-H2DCFDA, C6827; Thermo Fisher Scientific, Inc.). Specifically, mature oocytes were incubated with 1 μM CM-H2DCFDA for 30 min at 37°C. After extensive washing, intracellular ROS production was assessed with a Zeiss 710 confocal fluorescence microscope at excitation and emission wavelengths of 488 and 515 nm, respectively. The average fluorescence intensity per oocyte was calculated using NIH ImageJ analysis software. A total of 60 mouse oocytes, including 30 IVM and 30 IVO oocytes, were used here.