Leydig Cells: Testicular Side Population Harbors Transplantable Leydig Stem Cells

The Leydig cell population in the adult testis has a minimal rate of cell turnover (1). Nevertheless, the Leydig cell compartment is fully capable to respond to cell loss or hormonal stimuli to increase cell numbers. Loss of the Leydig cells in the rat testis after administration of the specific Leydig cell toxicant ethane dimethane sulfonate leads to the formation of a completely new population of Leydig cells within a couple of months (2, 3). Furthermore, long-term administration of human chorionic gonadotropin/LH causes substantial increases in Leydig cell numbers (4–7). Most authors agree that peritubular fibroblast-like (also called mesenchymal) cells serve as precursor cells for Leydig cells and that sequential differentiation steps and accompanying proliferation, ultimately lead to the formation of mature Leydig cells (8). In this issue, the above picture has now been significantly supplemented by Lo et al. (9). Genuine stem cells, which just as hemopoietic stem cells have side population (SP) characteristics, are at the start of the Leydig cell lineage. This makes the Leydig cell lineage fully comparable to other renewing cell lineages, like spermatogenesis, because it is composed of stem cells, proliferating and differentiating transit cells in between and functioning cells at the end. Also, transplanted Leydig stem cells can colonize another testis, providing a functional assay for these cells and an important first step is indicated through which these cells can be purified (9). Stem cells of various tissues can be purified by flow cytometry on the basis of their ability to rapidly efflux the fluorescent DNA-binding dye Hoechst 33342 (10). These cells are called SP cells, in the bone marrow comprising 0.05–0.1% of the nucleated cells (11). Transplantation assays indicate that SP cells in the bone marrow to have a long-term reconstituting ability and to be hemopoietic stem cells (10). SP cells are also found in other tissues, e.g. skeletal muscle, brain, mammary gland, and liver, and are suggested to be stem cells there too (12–16). In contrast, SP cells isolated from the mouse epidermis and rat kidney did not possess stem cell properties for epidermal and kidney cell types (17, 18). The enhanced exclusion of the Hoechst dye in SP cells is caused by the expression in these cells of ATP-binding cassette transporter proteins (breast cancer resistance protein 1, multidrug resistance 1). As yet there is no complete picture of the role of these proteins, but they may play a protective role in the sensitivity of SP cells to cytotoxic substrates (19) and to hypoxia (20). In the testis, the presence of SP cells was first described by Kubota et al. (21). Surprisingly, in transplantation assays these cells were unable to colonize the seminiferous epithelium of recipient mice, and it was concluded that testicular SP cells do not represent spermatogonial stem cells. Two other groups did find a positive transplantation assay, indicating the presence of spermatogonial stem cells in their SP cell suspensions (22, 23). As suggested (22, 23), the difference may be caused by the use of different starting materials, Kubota et al. (21) using germ cells from cryptorchid testes, whereas Falciatori et al. (22) and Lassale et al. (23) used young and normal adult mice, respectively. One puzzling aspect was that, although in both studies about 1% of the total cells were SP cells, the enrichment of spermatogonial stem cells, as determined by the transplantation assay, was only about 15-fold. This can now, at least in part, be explained by the presence of contaminating Leydig SP cells (9). The novel findings by Lo et al. (9) open new possibilities to study the formation and maintenance of the Leydig cell population. It will be interesting to see whether the SP cells are identical with the most primitive Leydig cell precursors already described, completing the picture of this cell lineage. Furthermore, an important step has been made in the development of a purification protocol that will allow the biochemical characterization of these cells. Finally, the possibility can be explored to transplant purified Leydig stem cells to an ectopic place in patients in which castration has to be carried out, to maintain testosterone levels. Side population.