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. 2015 Dec 1;5:17594. doi: 10.1038/srep17594

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Copyright © 2015, Macmillan Publishers Limited

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(a) The experimental schedule. Id4-CreERT2-tdTomato;ROSA26-flox-stop-lacZ double transgenic mice were TM pulsed at 5-6 weeks of age. Busulfan (10 mg/kg) was injected to induce regeneration after 2 days. Two months later, the contribution of the pulse-labeled Id4⁺ spermatogonia to the regenerating spermatogenesis was evaluated after X-gal staining, and then compared with controls that were treated in the same manner, but without busulfan injection. (b). Whole-mount seminiferous tubule X-gal staining of busulfan-treated (Treated) and untreated Id4-CreERT2-tdTomato;ROSA26-lacZ mice (Control). Scale bar, 1 mm. (c) Top panel, Clones of LacZ-positive cells with (Treated, right panel) or without (Control, left panel) busulfan treatment. Scale bar, 1 mm. Bottom panel, Cross-sections of the clones with (Treated, right panel) or without (Control, left panel) busulfan treatment. X-gal-stained (blue) sections counterstained with neutral red (pink). Scale bar, 50 μm. (d) Left panel, Number of LacZ⁺, labeled cell-derived clones with (Treated, n = 6 mice) or without (Control, n = 6 mice) busulfan treatment. The clone number is decreased in testes treated with busulfan. Right panel, Length of LacZ⁺, labeled cell-derived clones with (Treated, n = 6) or without (Control, n = 6) busulfan treatment. The clone length is increased in testes treated with busulfan treatment compared with untreated mice. Average numbers and length of clones per testis are shown. *P = 0.003605204, **P = 5.63421E-07 (two-tailed Student’s t-test).