Mouse spermatogenic lineage. When a spermatogonial stem cell (type As spermatogonia) divides, daughter cells can remain type As spermatogonia (self-renewal) or commit to differentiation, giving rise through 12 amplifying divisions (2¹²) to a clone of spermatogenesis that could theoretically contain 4,096 spermatozoa. Once committed to differentiation, an As spermatogonial stem cell gives rise to a pair of undifferentiated spermatogonia (Apr) that are connected by a cytoplasmic bridge. Incomplete cytokinesis is a hallmark of germ cell proliferation. The first four population doubling divisions (2¹–2⁴) generate chains of undifferentiated Apr and Aal spermatogonia; Apr, Aal⁴, Aal⁸, Aal¹⁶, which collectively make up the pool of progenitor spermatogonia. Differentiation becomes evident morphologically when a chain of Aal¹⁶ spermatogonia becomes a chain of type A1¹⁶ spermatogonia. The next five population doubling divisions (2⁵–2⁹) generate types A2, A3, A4, intermediate, and B differentiating spermatogonia. The 10th division (2¹⁰) produces primary spermatocytes (4N) and is followed by two meiotic divisions (2¹¹–2¹²) and spermiogenesis to produce 1N, terminally differentiated spermatozoa. Abbreviations: Aal, A_aligned; Apr, A_paired; As, A_single.

Busulfan-treated (Bu), WT, and cryptorchid (Cr) testis models were evaluated for stem cell activity and gene expression. (A): Macroscopic (top panels; scale bar = 2 mm) and histological (bottom panels; scale bar = 50 μm) examination of Bu (left panels), WT (center panels), and Cr (right panels) testes. Compared to WT testes (92.26 ± 6.33 mg; 35.26 ± 4.1 × 10⁶ cells per testis), Bu and Cr testes were smaller (20.6 ± 0.53 and 23.54 ± 0.62 mg, respectively) and contained fewer cells (1.37 ± 0.17 × 10⁶ and 1.38 ± 0.16 × 10⁶ cells per testis, respectively) because they were nearly devoid of germ cells. Histological sections were stained with hematoxylin and eosin. (B): The spermatogonial stem cell transplantation technique was used to determine the stem cell activity (mean ± SEM) in Bu (0.45 ± 0.13 colonies per 10⁵ cells transplanted), WT (1.55 ± 0.13 colonies per 10⁵ cells transplanted), and Cr (43.30 ± 4.17 colonies per 10⁵ cells transplanted) donor testes. The spermatogonial stem cell transplantation technique has an efficiency of 5%. Therefore, the stem cell concentrations in Bu, WT, and Cr testes were 1 in 11,111 (10⁵ cells transplanted per 0.45 colonies × 5%), 1 in 3,226, and 1 in 115, respectively. Inset, relative stem cell activity in the three testis models is demonstrated by the extent of donor spermatogenesis (blue) produced following transplantation of 500,000 transgenic (lacZ) donor testis cells. Scale bar = 2 mm. (C): Four hypothetical gene expression clusters were predicted on the basis of the morphological and functional (stem cell) analyses in (A) and (B). Gene expression data were organized into clusters predicted to represent somatic cells (WT < Cr, Bu), germ cells (WT > Cr, Bu), stem/ progenitor cluster 1 (Cr > WT, Bu; most specific), and stem/progenitor cluster 2 (Cr > Bu; less restrictive; descriptions given in text). Abbreviation: WT, wild-type.

Functional annotation of candidate gene lists. Stem/progenitor cluster 1 (maroon columns), stem/progenitor cluster 2 (dark blue columns), somatic cell (light blue columns), and germ cell (light gray columns) candidate gene clusters were annotated on the basis of all functional categories in Gene Ontology and compared with the functional distribution of genes on the U74Av2 array (dark gray columns). Results for spermatogenesis, RNA binding, DNA metabolism, and protein biosynthesis functional categories are shown. The percentages of genes in each cluster that fall in the indicated functional categories are indicated on the y-axis. Significant differences (false discovery rate, <0.05) compared with the percentage of functionally annotated genes on the U74Av2 array in each functional category are indicated with an asterisk (*).

Flow cytometric analysis of cryptorchid testis cells for expression of the cell surface marker, CCR2. (A): The α6-integrin⁺, side scatter^lo subpopulation cryptorchid testis cells, which is enriched for SSCs [], was evaluated for CCR2 expression. CCR2⁻ and CCR2⁺ gates were established on the basis of analysis of cryptorchid testis cells stained with an IgG2b isotype control antibody (red line). Distribution of cryptorchid testis cells was 7.26% in fraction R5 (α6-integrin⁺, SSC^lo, CCR2⁻) and 1.37% in fraction R4 (α6-inte-grin⁺, SSC^lo, CCR2⁺). The CCR2⁻ and CCR2⁺ fractions were isolated by fluorescence-activated cell sorting (FACS) and transplanted to the testes of busulfan-treated mice to determine the relative SSC activity. Testes of recipient mice were analyzed 2 months after transplantation for colonies of donor-derived (blue) spermatogenesis (B, C). Colony number was normalized to the number of blue spermatogenic colonies produced per 10⁵ cells transplanted: unsorted cryptorchid testis cells, 20.41 ± 4.83; fraction R5 (CCR2⁻), 67.49 ± 15.85; and fraction R4 (CCR2⁺), 0 ±0 colonies per 10⁵ cells transplanted (mean ± SEM). Representative recipient testes transplanted with CCR2⁻ (B) and CCR2⁺ (C) cells are shown. Scale bars =2 mm (B, C). Abbreviations: APC, allophycocyanin; CCR2, chemokine receptor 2.

The RNA-binding protein Rbmy colocalizes with Plzf in normal adult mouse seminiferous tubules. (A): Rbmy immunoreactivity (red) was restricted to cells on the basement membrane of adult mouse seminiferous tubules. (B): Immunofluorescent staining for Plzf (green), a consensus marker of stem/progenitor spermatogonia that is expressed by rare cells on the basement membrane of mouse seminiferous tubules. (C): Overlay of Plzf and Rbmy fluorescent signals. Inset displays a higher magnification of cells in the dashed white box. (D): 4,6-Di-amidino-2-phenylindole (DAPI) staining of the section depicted in (A–C) demonstrates that all seminiferous tubules contain multiple germ cell layers. (E): Negative control section in which primary antibodies were replaced with an isotype control. (F): DAPI staining of section in (E). Scale bars = 50 μm (F) and 10 μm ([C], inset).