BrdU incorporation assay
To detect DNA replication, zygotes were labeled at 22 h after hCG injection (reaching the PN3 stage when the DNA replication began actively) with 1 mmol/l BrdU in KSOM for 2 h. Then the embryos were fixed in 4% PFA for 20 min and permeabilized for 20 min in 0.5% Triton X-100. Then BrdU incorporation into DNA was detected using the primary rat anti-BrdU (Abcam, ab6326, 1:200) antibody with a corresponding secondary antibody conjugated with Alexa FITC goat anti-rat antibody (ZSGB-Bio, ZF-0315, 1:1000).