FIG. 2.

RA stimulates DNA replication in the male germ cells. Male germ cells isolated at 13.5 dpc (A), 14.5 dpc (B), and 15.5 dpc (C) were cultured with or without 1 μM RA for 1, 3, and 5 days. On each of these days, the germ cells were incubated with EdU for 5 h. After incubation, the germ cells were categorized into three groups—single cells without RA (RA [−] Single), single cells with RA (RA [+] Single), and clumped cells with RA (RA [+] Clumped)—and examined for EdU-positive cell numbers. Without RA, the cells did not perform DNA replication in all stages. In the presence of RA, the germ cells isolated at 13.5 dpc increased DNA replication. Especially in the clumped cells, more than 50% of them were EdU positive after 3 days of culture. In the germ cells isolated at 14.5 dpc, the EdU-positive cell number decreased. The male germ cells isolated at 15.5 dpc did not perform DNA replication even in the presence of RA. Male germ cells isolated at 13.5 dpc (D), 14.5 dpc (E), and 15.5 dpc (F) were also cultured with 10 μM RA (RA 10×) for 1, 3, and 5 days. The ratios of DNA replication were the same as the original concentration of RA in all stages. Each column represents the mean of three replicate experiments. Error bars show the SEM. Asterisks indicate statistically significant differences (*P < 0.05, **P < 0.01, ***P < 0.001) between the RA negative control (RA [−] Single) and RA-treated samples or between RA-treated single cells and clumped cells in each culture day.