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First published online 15 March 2006
doi: 10.1242/dev.02316

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The first round of mouse spermatogenesis is a distinctive program that lacks the self-renewing spermatogonia stage

¹ Department of Pathology and Tumor Biology, Graduate School of Medicine, Kyoto University, Kyoto, Japan.
² Recognition and Formation, PRESTO, JST, Saitama, Japan.
³ The Sakaguchi Laboratory of Developmental Biology, School of Medicine, Keio University, Tokyo, Japan.

View larger version (125K): [in a new window]   Fig. 1. Appearance of Ngn3-positive spermatogonia and Kit-positive spermatogonia during the first postnatal week. (A) RT-PCR of whole testis mRNA at P1 to P7; amplification for Ngn3, Kit, Ret, and Oct4. No bands are observed in control reactions without reverse transcriptase; no template, amplification product without cDNA. ß-actin was also assayed as a control. (B) ISH of sections from testes at P1 to P7, probed for the same gene set described in A. Green and blue arrowheads indicate the Ngn3 and Kit signals in spermatogonia, respectively; red arrowheads indicate Kit expression in the interstitial cells. Scale bar: 100 µm.

View larger version (74K): [in a new window]   Fig. 2. Distribution of Ngn3-positive spermatogonia and Kit-positive spermatogonia in the P5 testes. (A-D) Double-staining ISH for Ngn3 (purple, A) and Kit (red, B), with overlay images of A and B (C), and of B with DNA staining (blue, D). Purple arrows indicate Ngn3 in spermatogonia; short and long pink and red arrows indicate Kit in spermatogonia and interstitial cells, respectively. Note that the Ngn3 and Kit signals are observed in distinct cells in separate seminiferous tubule cross-sections. Scale bar: 100 µm. (E-G) ISH for Ngn3 and Kit on adjacent sections, under low magnification. The distribution of Ngn3 (E) and Kit (F) germ cell signals are compared and represented in G, where seminiferous tubule cross-sections containing Ngn3 (green), Kit (blue), or both signals (red) are indicated. For statistical evaluation see Table S1 in the supplementary material. Scale bars: 100 µm, in A for A-D, in F for E,F.

View larger version (95K): [in a new window]   Fig. 3. Distribution of the newly-born Ngn3- and Kit-positive spermatogonia, and the seminiferous epithelial cycle prepattern. (A-H) Double-staining ISH of testes sections at the indicated ages with the indicated genes. Overlaid images of Kit or Ngn3 (purple, bright field) and galectin 1 (red fluorescence) are shown in A,C,E,G; galectin 1 signals in the same field are shown in B,D,F,H. Arrows in A and C indicate the Kit- and Ngn3-positive spermatogonia, respectively. E,F and G,H are adjacent sections. Note the variable intensities of galectin 1 signals within the seminiferous tubule cross-sections and their relationship with Kit- or Ngn3-positive spermatogonia (see Results). Asterisks (E-H) represent typical segments with a high galectin 1 mRNA signal; Kit-positive, but not Ngn3-positive, spermatogonia preferentially localize to these segments. Segments marked by hearts show lower galectin 1 signals and exhibit a preference for Ngn3-positive spermatogonia. Scale bars: 100 µm, in D for A-D, in H for E-H. (I) Quantification of Kit- or Ngn3-positive spermatogonia localized in tubule segments categorized by different levels of galectin 1 mRNA (high, medium or low). Tubule cross-sections marked with H, M or L in B and D are examples of segments with high, medium and low levels of galectin 1 mRNA, respectively. Data are represented as `preference' to each category of tubule segment. a and b represent significant deviation with P-values of <0.0001 and <0.05, respectively. See Materials and methods and Tables S2-S7 in the supplementary material. The number of Ngn3-positive spermatogonia at P3 and P4 was too low for statistical analyses. (J) Model for the generation of spermatogonia subpopulations. One area of seminiferous tubules is represented to align tubules with increasing age. The galectin 1 mRNA level is shown by the gradient: black, highest; white, lowest. As galectin 1 expression increases and reduces in cycles (black to gray to white to black, etc.), its expression domain shifts leftward in a wave-like manner. Kit-positive spermatogonia (red ovals) appear specifically at the galectin 1-high segments (red bands). By contrast, Ngn3-positive spermatogonia (green ovals) are generated separately from Kit-positive spermatogonia around the segments of medium level of galectin 1 expression (green bands).

View larger version (90K): [in a new window]   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 CreERTM, resulting in the labeling of cells with ß-gal expression. (B) The experiment schedule. (C,D) ISH on adjacent sections from a P8 Ngn3/CreERTM transgenic mouse testis detecting Ngn3 and CreERTM expression and indicating their overlapped expression (arrowheads). (E,F) Whole-mount X-Gal staining of a seminiferous tubule of a P8 Ngn3/CreERTM; 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/Wv 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.

View larger version (123K): [in a new window]   Fig. 5. Chase of cells of Ngn3+ and Ngn3- lineages during the maturation of testes using Ngn3/Cre;CAG-CAT-Z double-transgenic mice. (A) Experimental design. In Ngn3-positive cells of Ngn3/Cre;CAG-CAT-Z mice, the reporter gene is irreversibly recombined between loxP sites by Cre recombinase driven by the Ngn3 regulatory sequence, thereby labeling their progenies with ß-gal (encoded by lacZ), while CAT expression is lost (Ngn3+ lineage). The reporter gene remains intact in cells that have never expressed Ngn3; these cells express CAT, but not lacZ (Ngn3- lineage). (B-D) Serial sections of P3 testes probed for Ngn3, Cre and lacZ, representing the overlapping expression of these genes in a single seminiferous tubule (arrowheads). (E,F) A pair of adjacent sections of a P5 testis, which were hybridized for Kit and lacZ expression. The Kit-positive spermatogonia apparently outnumber the lacZ-positive ones. (G-I) P21 testis probed for CAT. H shows a higher magnification of a part of G; I shows a PI-stained fluorescence image of H. CAT signals are preferentially detected at the center of the seminiferous tubules (arrowheads). Arrows indicate CAT signals in the interstitial cells. (J-L) P28 testis probed for CAT and lacZ. (K) Higher magnification of a part of J; (L) the same location but in the next section. CAT-positive and lacZ-negative (Ngn3- lineage) spermatogenic cells are preferentially found in the innermost layer (arrowheads). Seminiferous tubules marked 1-3 are examples of the different degrees of maturation (see Results). (M,N) P56 testis probed for CAT and lacZ. All germ cells are CAT negative and lacZ positive. Arrows indicate somatic cells positive for CAT (Sertoli and interstitial cells). Scale bars: 100 µm.

View larger version (22K): [in a new window]   Fig. 6. Contribution of cells of the Ngn3+ and Ngn3- lineages to functional spermatozoa. (A) Experimental design. Ngn3/Cre;CAG-CAT-Z double-transgenic male mice were mated with non-transgenic females, and the offspring genotyped to determine whether the contributed spermatozoa carried an intact (CAG-CAT-Z) or recombined (CAG-Z) form of the reporter gene. (B) Frequency of offspring with the intact (gray) and recombined (white) forms of the reporter gene, classified according to the paternal age at fertilization.

View larger version (30K): [in a new window]   Fig. 7. Model for the two lineages in the mouse spermatogenesis. In the first postnatal week, gonocytes directly give rise to Kit-positive differentiating spermatogonia and Ngn3-positive undifferentiated spermatogonia in parallel. This process is closely related to the presumptive seminiferous epithelial cycle pre-pattern, which initiates before birth. Kit-positive spermatogonia are specifically generated in the galectin 1-high segments (pink arrow 1). These cells do not pass through a Ngn3-positive, undifferentiated spermatogonia stage and differentiate in the first round of spermatogenesis, resulting in the formation of fertile spermatozoa (Ngn3- lineage). By contrast, Ngn3-positive undifferentiated spermatogonia are generated preferably at galectin 1-medium segments (green arrow). They subsequently act as a self-renewing stem cell population, while also providing cells that transform into differentiating spermatogonia. Thus, these cells support steady-state spermatogenesis following the first round of spermatogenesis (Ngn3+ lineage). The transformation of undifferentiated spermatogonia into differentiating spermatogonia is tightly related to the seminiferous epithelial cycle, and Kit-positive differentiating spermatogonia are established in stages of high galectin 1 expression (stage IX-X), indicated by pink arrow 2. See Results for more details. PGC, primordial germ cells.