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Fig. 4. Pulse labeling of Ngn3-positive spermatogonia using tamoxifen-inducible Cre transgenic mice, and their stem cell activities. (A) Scheme for the tamoxifen-dependent recombination by CreER^TM, resulting in the labeling of cells with ß-gal expression. (B) The experiment schedule. (C,D) ISH on adjacent sections from a P8 Ngn3/CreER^TM transgenic mouse testis detecting Ngn3 and CreER^TM expression and indicating their overlapped expression (arrowheads). (E,F) Whole-mount X-Gal staining of a seminiferous tubule of a P8 Ngn3/CreER^TM; CAG-CAT-Z double-transgenic mouse with (E) and without (F) tamoxifen administration (tam). Inset (E) is at higher magnification. Note the tamoxifen-dependent appearance of ß-gal-positive spermatogonia. Mice with only the CAG-CAT-Z transgene do not exhibit positive staining after tamoxifen administration (data not shown). (G) Double-transgenic mice were injected with tamoxifen at P5 and P6, and their seminiferous tubules were subjected to X-gal staining at the age of 3 months. Many ß-gal-positive cells persist as distinct segments (arrowheads). (H) Cross section of a ß-gal-positive segment containing a complete set of spermatogenic cells stained with ß-gal (nuclear counterstaining in red). (I-K) Stem cell activity of Ngn3-positive spermatogonia after transplantation. Tamoxifen was administered to double-transgenic mice according to the same schedule. Their testicular cells were transplanted into the seminiferous tubules of W/W^v mice at P8 and subjected to whole-mount X-Gal staining (blue) 3 months later. A number of blue spermatogenic colonies were detected (I, arrowheads). (J) A typical section of ß-gal-positive colonies (nuclear counterstaining in red); (K) Hematoxylin and Eosin staining of the adjacent section. Scale bars: 100 µm in D,F,H,K; 1 mm in G,I.

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