Conservation of spermatogonial stem cell self-renewal signaling between mouse and rat

Abstract

Self-renewal of spermatogonial stem cells (SSCs) is the foundation for maintenance of spermatogenesis throughout life in males and for continuation of a species. The molecular mechanism underlying stem cell self-renewal is a fundamental question in stem cell biology. Recently, we identified growth factors necessary for self-renewal of mouse SSCs and established a serum-free culture system for their proliferation in vitro. To determine whether the stimulatory signals for SSC replication are conserved among different species, we extended the culture system to rat SSCs. Initially, a method to assess in vitro expansion of SSCs was developed by using flow cytometric analysis, and, subsequently, we found that a combination of glial cell line-derived neurotrophic factor, soluble glial cell line-derived neurotrophic factor-family receptor alpha-1 and basic fibroblast growth factor supports proliferation of rat SSCs. When cultured with the three factors, stem cells proliferated continuously for >7 months, and transplantation of the cultured SSCs to recipient rats generated donor stem cell-derived progeny, demonstrating that the cultured stem cells are normal. The growth factor requirement for replication of rat SSCs is identical to that of mouse; therefore, the signaling factors for SSC self-renewal are conserved in these two species. Because SSCs from many mammals, including human, can replicate in mouse seminiferous tubules after transplantation, the growth factors required for SSC self-renewal may be conserved among many different species. Furthermore, development of a long-term culture system for rat SSCs has established a foundation for germ-line modification of the rat by gene targeting technology.

Fig. 1.

Patterns of FSc and SSc by FCA for fresh MACS EpCAM⁺ and 1-week-cultured MACS EpCAM⁺ cells. (A) Fresh unfractionated rat pup testis cells and MACS EpCAM⁺ cells. The MACS EpCAM⁺ cells are uniform for FSc and SSc. Almost all cells (96.3 ± 0.3%; mean ± SEM, n = 3) in the MACS EpCAM⁺ cell population are in G1, designated the stem cell (containing) gate. Before analysis, cells were stained with PI, and only PI^- cells (live cells) were analyzed. (B) Effect of growth factors on germ cell clump formation. MACS EpCAM⁺ cells (10⁵ cells per well of a 12-well plate) were cultured on STO feeders in mouse serum-free culture medium supplemented with various combinations of growth factors (GDNF, GFRα1, bFGF, and LIF). At 7 days in culture, all cells in the wells were harvested and analyzed by FCA. The cell number in G1, the stem cell gate, was as follows: GDNF = 1.9 ± 0.3 × 10⁴, GDNF + GFRα1 = 6.4 ± 0.9 × 10⁴, GDNF + bFGF = 5.9 ± 0.8 × 10⁴, GDNF + LIF = 2.1 ± 0.3 × 10⁴, GDNF + GFRα1 + bFGF = 10.0 ± 1.4 × 10⁴, and GDNF + GFRα1 + bFGF + LIF = 11.5 ± 2.1 × 10⁴ (mean ± SEM, n = 5). The FSc/SSc patterns for GDNF + GFRα1, GDNF + bFGF, GDNF + LIF, and GDNF + GFRα1 + bFGF are not shown. When 10⁵ cells were placed in wells, the cell number found by FCA in comparable samples in the stem cell gate of fresh MACS EpCAM⁺ cells was 8.2 ± 0.4 × 10⁴ (mean ± SEM, n = 3, 82% of 10⁵ cells). The 18% not in the gate represent cells outside the gate (see A) and PI⁺ cells detected by FCA but not trypan blue staining used to establish the 10⁵ live cells for culture. FCA always identifies more PI⁺ putative dead cells than trypan blue staining. The number of cells in the stem cell gate cultured with four factors is significantly higher than that of GDNF alone or the two-factor combinations (P < 0.01). There is no significant difference between three-factor and four-factor combinations.

Fig. 2.

Expansion of rat SSCs in culture. MACS EpCAM⁺ cells were cultured in rat serum-free medium supplemented with four growth factors (GDNF, GFRα1, bFGF, and LIF) or three growth factors (GDNF, GFRα1, and bFGF) on STO feeders in a 5% oxygen atmosphere. Clump-forming cells were subcultured by pipetting followed by 0.01% trypsin digestion. (A) EpCAM⁺ rat germ cells formed clumps (arrows) after subculturing and continuously proliferated in vitro. (Scale bar: 100 μm.) (B) Macroscopic appearance of recipient testis 2 months after transplantation with 5-month-cultured rat SSCs from MT lacZ rat pup testes. Each blue stained stretch of seminiferous tubule indicates donor cell-derived spermatogenesis from an individual stem cell. (Scale bar: 2 mm.) (C) Fresh EpCAM⁺ cells and cultured cells were transplanted into recipient nude mouse testes. The number of donor-derived spermatogenic colonies per 10⁵ cells originally seeded in culture is shown. Cultured germ cells with four factors were transplanted at 1-month intervals for 5 months. The culture with three factors was transplanted at only 3-5 months. The transplantation assay indicated an increase of rat SSCs in culture with three or four factors for 5 months. Data are shown as means ± SEM for 6-12 recipient testes per time point. Error bars for most points are within the symbol.

Fig. 3.

Rat SSCs express GDNF-receptor molecules and Oct-4 transcriptional factor. (A) Immunocytochemistry for c-Ret receptor tyrosine kinase and NCAM. All clump-forming germ cells express both GDNF receptors. (B) FCA for GFRα1 expression. Cells in the stem cell gate express GFRα1. Closed (red) and open histograms represent GFRα1-stained cells and isotype-stained control cells, respectively. (C) Immunocytochemistry for Oct-4. All germ cell clumps express Oct-4. The Oct-4 immunostaining is localized to the nucleus. (Scale bar: 100 μm.)