Table 1.
Summary of adult mouse experiment.
Fig 1.
Stereomicroscopic view of cultured adult mouse testis tissues.
(A) Six-week-old Acr-GFP mouse testis tissue fragments on culture days 0, 3, and 10. Upper row is GFP excitation view and lower row is bright view. (B) Six-week-old Acr-GFP mouse testis tissue fragments on culture days 0, 15, and 82. Weak GFP expression was maintained. (C-E) Immunohistochemistry of cultured tissues with antibodies to BrdU and GFP, counterstained with Hoechst. Testis tissue fragments of 6-week-old Acr-GFP (C and D) and 24-week-old Gsg2-GFP (E) mice were cultured, and then analyzed on days 42 (C), 85 (D), and 48 (E), respectively. The dashed rectangular area in the left picture of C is enlarged on the right. Arrows indicate BrdU-positive round spermatids in C and elongating spermatids in E. Scale bars, 1 mm (A, B); 10 μm (C, E); 50 μm (D).
Fig 2.
(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).
Table 2.
Summary of VAD experiment.
Fig 3.
Extent of GFP expression in adult and pup testis tissues.
(A) GFP expression images of testis tissues, from 2.5 dppp and seven-week-old Acr-GFP mice, cultured for 5 weeks. (B) PAS staining images of testis tissues, from 2.5 dppp and seven-week-old Acr-GFP mice, cultured for 5 weeks. The dotted rectangular areas are enlarged on the right. (C) Adults and pups were compared on the basis of the extent of Acr-GFP expression scored (judged at culture week 5). Testis tissue fragments of Acr-GFP mice, 2.5–4.5 dpp and 7–8 weeks old, were cultured for five weeks. Adult and pup testis tissues were examined in a total of 34 and 39 tissue pieces in three culture experiments, respectively (means±s.d., P<0.025 (Student’s t-test)). (D) The frequency of seminiferous tubules containing differentiated germ cells. Adult and pup testis tissues were examined in a total of 32 and 36 tissue pieces in three culture experiments, respectively. Sections stained with periodic acid Schiff were examined and the number of seminiferous tubules containing differentiated germ cells (spermatocytes, round spermatids, and/or elongating spermatids) asa well as seminiferous tubules not containing them were counted in each tissue to calculate the percentage of spermatogenesis-positive seminiferous tubule (means±s.d., P<0.002 (Student’s t-test)). Scale bars, 1 mm (A); 100 μm (B).
Table 3.
Summary of adult Sl/Sld mouse experiment, chronologically arranged.
Fig 4.
Sl/Sld mutant adult mouse experiments.
(A) Six-week-old Sl/Sld mouse testis tissues, cultured for 45 days without KitL nor CSF-1 (control) showed no spermatogenesis. (B) The testis tissue of the same mouse showed differentiated germ cell accumulation in the seminiferous tubules when cultured with KitL (100 ng/mL). (C, D) When cultured for 40 days with KitL (100 ng/mL) plus CSF-1 (20 ng/mL), meiotic cells in the seminiferous tubules were observed. The rectanglar area in C is enlarged in D. (E, F) Culturing with KitL (500 ng/mL) for 38 days induced spermatogenesis up to round spermatids. The rectanglar area in E is shown in F, demonstrating several round spermatids with PAS-stained acrosomal caps or dots. Scale bars, 50 μm (A-C, E); 20 μm (D, F).
Table 4.
Summary of adult Sl/Sld mouse experiment.