Molecular dissection of the male germ cell lineage identifies putative spermatogonial stem cells in rhesus macaques

Abstract

Background: The spermatogonial stem cell (SSC) pool in the testes of non-human primates is poorly defined.

Methods: To begin characterizing SSCs in rhesus macaque testes, we employed fluorescence-activated cell sorting (FACS), a xenotransplant bioassay and immunohistochemical methods and correlated our findings with classical descriptions of germ cell nuclear morphology (i.e. A(dark) and A(pale) spermatogonia).

Results: FACS analysis identified a THY-1+ fraction of rhesus testis cells that was enriched for consensus SSC markers (i.e. PLZF, GFRalpha1) and exhibited enhanced colonizing activity upon transplantation to nude mouse testes. We observed a substantial conservation of spermatogonial markers from mice to monkeys [PLZF, GFRalpha1, Neurogenin 3 (NGN3), cKIT]. Assuming that molecular characteristics correlate with function, the pool of putative SSCs (THY-1+, PLZF+, GFRalpha1+, NGN3+/-, cKIT-) comprises most A(dark) and A(pale) and is considerably larger in primates than in rodents. It is noteworthy that the majority of A(dark) and A(pale) share a common molecular phenotype, considering their distinct functional classifications as reserve and renewing stem cells, respectively. NGN3 is absent from A(dark), but is expressed by some A(pale) and may mark the transition from undifferentiated (cKIT-) to differentiating (cKIT+) spermatogonia. Finally, the pool of transit-amplifying progenitor spermatogonia (PLZF+, GFRalpha1+, NGN3+, cKIT+/-) is smaller in primates than in rodents. CONCLUSIONS These results provide an in-depth analysis of molecular characteristics of primate spermatogonia, including SSCs, and lay a foundation for future studies investigating the kinetics of spermatogonial renewal, clonal expansion and differentiation during primate spermatogenesis.

Figure 1

Rhesus testis cells expressing germ cell and SSC markers are enriched in the THY-1⁺ fraction. (A) Juvenile rhesus testis cells were stained with a THY-1 (CD90) antibody and sorted using FACS into two fractions, THY-1⁺ and THY-1⁻ (polygons). Immunofluorescent staining for VASA (green) was performed in (B) unsorted, (C) FACS-sorted THY-1⁺ and (D) FACS-sorted THY-1⁻ testis cells spotted on glass slides. Cells were counterstained with DAPI (blue). The mean percentage of VASA⁺ cells in each fraction from all replicates is shown in the bottom left of each image. Images are from stained fresh cells. Scale bars: 50 µm. (E) Mean (±SEM) cell sorting statistics from 12 independent FACS experiments (8 animals, using both fresh and cryopreserved cells) are shown for each population. Sort purity is defined as the percentage of sorted cells that fall into the respective sort gate upon re-analysis. The mean (±SEM) percentage of VASA⁺ cells in unsorted and sorted cells is indicated in the last column. (F) Quantitative RT–PCR measured levels of VASA, PLZF and GFRα1 mRNA from unsorted (green bars), THY1⁺ (black bars) and THY-1⁻ (white bars) testis cells. Data are presented as the mean (±SEM) fold-change for each gene relative to the mean levels in the unsorted population (three replicate experiments). Fold-change values were determined by the ΔΔCt method, where levels of each gene were normalized to GAPDH.

Figure 2

THY-1⁺ rhesus testis cells have enhanced xenotransplant colonizing activity. (A–F) The rhesus-to-nude mouse xenotransplantation assay was used to investigate SSCs by detecting donor-derived spermatogonial colonies that arise from transplanted rhesus testis cells (Hermann et al., 2007). White box in (C) is enlarged in (D), and white arrowheads mark characteristic intercellular cytoplasmic bridges in chains of rhesus spermatogonia. Representative colonies from (A–D) THY-1⁺ and (E and F) unsorted donor cells are shown and the source of donor cells is noted. Dashed white lines mark seminiferous tubule margins. Scale bars: 50 µm. (G) Colonization results from the xenotransplant assay are shown for unsorted (green bars) and FACS-sorted [THY-1⁺ (black bars) and THY-1⁻ (white bars)] juvenile rhesus testis cells from four animals in six sorting experiments. Results from each experiment are presented as the mean (±SEM) number of xenotransplant colonies per 10⁵ viable cells transplanted. The number of recipient mouse testes analyzed is shown below each bar.

Figure 3

All VASA⁺ germ cells are GFRα1⁺ in the juvenile rhesus testis. To characterize the expression of GFRα1 in juvenile rhesus testes, we performed immunohistochemical co-staining for (A and B) GFRα1 and the pan germ cell marker VASA in sections of juvenile rhesus testes. Individual staining profiles are also shown for (C and D) GFRα1 and (E and F) VASA. In all images, DAPI counterstain (blue) identifies all cell nuclei. The white box in the first image (e.g. A) is enlarged in the second image (e.g. B). Markers are noted on each image in the color of the corresponding fluorophore. Scale bars: 50 µm.

Figure 4

GFRα1, PLZF and NGN3 are expressed in spermatogonia of juvenile rhesus testes; cKIT is not expressed. We performed immunohistochemical co-staining for (A–C) GFRα1 and PLZF, (D–F) NGN3 and PLZF and (G–I) cKIT and GFRα1 in sections of juvenile rhesus testes. DAPI counterstain (blue) identifies all cell nuclei. For each marker combination, the area marked by a white box in the first image (e.g. A) is enlarged in the second image (e.g. B). Markers are noted on each image in the color of the corresponding fluorophore. Scale bars: 50 µm. The quantity and relative proportion of cells expressing each pair of markers are indicated in graphs in (C) GFRα1 and PLZF, (F) NGN3 and PLZF or (I) cKIT and GFRα1. Quantification is presented as the mean (±SEM) number of cells per seminiferous cord cross-section exhibiting the staining phenotype noted above the bars. A minimum of 100 seminiferous cord cross-sections per animal were analyzed from three juvenile rhesus macaques.

Figure 5

Differential overlap in the expression between putative markers of stem and/or progenitor spermatogonia (GFRα1, PLZF, NGN3) and cKIT in the adult rhesus testis. We performed immunohistochemical co-staining for (A–C) GFRα1 and PLZF, (D–F) NGN3 and PLZF, (G–I) cKIT and GFRα1, (J–L) cKIT and PLZF, (M–O) cKIT and NGN3 in sections of adult rhesus testes. The quantity and relative proportion of cells expressing each pair of markers from three adults are indicated for (C) GFRα1 and PLZF, (D) NGN3 and PLZF, (I) cKIT and GFRα1, (L) cKIT and PLZF, (O) cKIT and NGN3. Quantification of markers per seminiferous tubule cross-section and figure organization is as shown for Fig. 4.

Figure 6

Correlation between molecular markers (GFRα1, PLZF, NGN3, cKIT) and morphological descriptions of spermatogonia (A_dark, A_pale, B) in the adult rhesus testis. Sections of adult rhesus testes were evaluated by immunohistochemistry for (A–D) GFRα1, (E–H) PLZF, (I–M) NGN3 and (N–R) cKIT. Subsequently, sections were counterstained by the PAS-hematoxylin method to reveal nuclear morphology and identify A_dark and A_pale spermatogonia, as well as differentiating type-B spermatogonia. The first image in each row (A, E, I, N) shows part of one seminiferous tubule cross-section (scale bar: 50 µm). Enlargements are also shown of representative A_dark (B, F, J, O), A_pale (C, G, K, L, P, Q) and B4 spermatogonia (D, H, M, R) (scale bar: 10 µm). (S) For all spermatogonia classified as A_dark or A_pale, the mean labeled percentage (±SEM) for the indicated marker is shown. Quantification was performed on an average of 1088 cells per marker from four adult rhesus macaques.

Figure 7

Summary of marker expression in the adult rhesus and mouse spermatogenic lineages. Colored bars (GFRα1, blue; PLZF, green; NGN3, black; cKIT, red) indicate the extent of marker expression in the adult spermatogenic lineage based on data from Figs 5 and 6 (rhesus, top) or review of the literature (rodents, bottom). Citations for rodent marker expression profiles are indicated within each bar and are as follows: A (Tokuda et al., 2007), B (Schlesser et al., 2008), C (Buaas et al., 2004), D (Greenbaum et al., 2006), E (Yoshida et al., 2004), F (Yoshida et al., 2007b), H (Manova et al., 1990), I (Sorrentino et al., 1991), J (Dym et al., 1995), K (Schrans-Stassen et al., 1999). ‘Stem’, ‘Progenitor’ and ‘Differentiating’ functional descriptors, based on rodent data, are indicated in the middle gray bar and may identify rhesus spermatogonia with corresponding phenotype and function. The transitions from stem to progenitor or progenitor to differentiating are noted by gradient shading between these functional categories. The following abbreviations are used for rodents: A_s, A_single; A_pr, A_paired; A_al, A_aligned.