Onset and Endpoint of S Phase and the Endpoint of M Phase of Mouse Zygotes

The onset and endpoint of S phase was determined using the BrdU incorporation experiment described previously (Zhang et al., 2016). The experiment was conducted over two time periods, 8 to 11 hpi and 16 to 19 hpi. During those times, BrdU incorporation was measured every other hour by incubating the zygotes in 1 mM BrdU-containing embryo culture medium for 30 min. Then, zygotes were fixed in 2.5% paraformaldehyde for 15 min, placed on polylysine slides, incubated in 1 mM HCl for 30 min, and then washed with 0.1 mM borate buffer solution for 20 min. Cells were permeabilized by washing three times with PBS containing 10% FBS and 0.2% Triton X-100, then blocked in the same solution for 30 min at 37°C. Zygotes were subsequently incubated with 6 μg/mL anti-BrdU antibody (Sigma, B8434) for 1 h at 37°C, rinsed thrice with PBS, and treated with secondary Cy3-conjugated goat anti-mouse IgG antibody (1:400, Abcam, ab97035) for 1 h. The nuclei were then stained with 4′, 6-diamidino-2-phenylindole (DAPI; Biosharp, Beijing, China) for 20 min. The number of total zygotes and BrdU-positive zygotes were counted to assess the frequency of BrdU-positive zygotes (number of BrdU-positive zygotes/total number of zygotes scored). The time point at which the frequency of BrdU-positive ≥ 10% was accepted as the starting time of S phase, and the time at which 90% BrdU-positive disappeared was accepted as the end of S phase. The time point when 95% of the zygotic embryos were cleaved represented the endpoint of M phase.