(A) Testes of VAD treated Gsg2-GFP mice at, 5, 9 and 13 weeks old, showing decreasing GFP expression. Histology shows a corresponding extent of spermatogenesis on HE staining. At 13 weeks old, the GFP had completely disappeared and differentiating germ cells were totally removed. (B) Testis tissues of a VAD-treated Acr-GFP mouse, 24 weeks old, upper two images, and a VAD-treated Gsg2-GFP mouse, 13 weeks old, lower two images, regained GFP expression during culturing. Images were obtained on culture days 23 (upper two) and 70 (left two), respectively. (C) Immunohistochemistry of cultured testis tissue, on culture day 45, derived from a VAD-treated Acr-GFP 23-week-old mouse. Antibodies used were against GFP and PNA. Counterstaining with Hoechst was applied. The rectangular area in the left upper image is enlarged in the right upper. GFP (green) and PNA (red) channels are independently shown in the lower images. (D) Haploid cells were observed in the dissociated sample of cultured testis tissues from the VAD-treated Gsg2-GFP mouse, 13 weeks old, on culture day 73. Dissociated cells were stained with antibodies against GFP (green) and SP56 (red), a sperm-specific protein that localizes to the sperm surface and in the acrosomal matrix. Nuclei were stained with Hoechst. (E) A sperm was found in a dissociated sample of cultured tissue from a VAD-treated Acr-GFP mouse, 23 weeks old, on culture day 50. It was stained with anti-GFP antibody along with nuclear staining by Hoechst. Scale bars, 1 mm (B); 50 μm (A, C); 10 μm (D, E).