Proliferation of primordial germ cells (PGCs) in 9-day-old embryonic gonads after busulfan treatment at stage X. To detect proliferating PGCs, embryonic gonads were isolated at 9 days (4 hours after 5-ethynyl-2′-deoxyuridine (EdU) incorporation) and sectioned for staining with anti-VASA and EdU (A). Scale bars = 100 μm (common views) and 10 μm (magnified views). (B) Percentage of EdU-positive cells among VASA-positive cells in the embryonic gonads. Four embryos were evaluated for both sexes. **P <0.01 and ***P <0.001, significant difference compared with control.