Testosterone regulation of the spermatogonial stem cell niche in mice

Testosterone, a steroid hormone, is not only important for testes development but also regulates sperm production. Sperm differentiate from spermatogonial stem cells (SSCs) a cell population that continually differentiates into sperm and must proliferate to replenish the germ cells in the testes. The regulation of spermatogonial stem cell proliferation or differentiation is an important aspect of male fertility. This purpose of this project was to determine how testosterone regulates SSCs homeostasis. To determine how testosterone regulates SSC, a protocol to immunize mice against GnRH was used to stop pituitary gland secretion of LH and FSH to establish a depleted hormone environment in mice. Subsequent experiments determined the testosterone regulated cell mechanisms regulating SSC homeostasis. We established that immunization of mice against GnRH suppressed SSC activity such that these cells did not have stem cell activity. However, treating immunized mice with testosterone propionate (TP) stimulated progressive SSC activity over 48 hr. The data show that testosterone functionally enhances SSC proliferation in immunized mice. Glial cell-derived neurotrophic factor (GDNF) is critical for the establishment and maintenance of SSCs. Therefore, it was hypothesized that testosterone regulates GDNF. Real-time RT-PCR showed that gdnf expression was induced in GnRH immunized mice testis after TP injection. In addition, the expression of gdnf and leukemia inhibitory factor (lif), another factor that regulates SSC activity, increased in the testes of flutamide-treated mice when the flutamide block of androgen receptor was lost. To determine the specific testicular cell responding to testosterone to support SSCs, Sertoli and peritubular myoid cells were treated with testosterone. Testosterone inhibited gdnf expression in the Sertoli cells but significantly increased gdnf mRNA and protein expression in peritubular myoid cells. In contrast, pregnenolone, which is produced by Sertoli cells, suppresses gdnf expression in the PM cells. Based on these data, the SSC niche includes FSH regulation of GDNF expression by Sertoli cells while pregnenolone and testosterone regulate peritubular myoid cell expression of GDNF. Together, these factors regulate SSC proliferation and self-renewal in the testis. This regulatory loop provides a physiological and cellular model of SSC homeostasis in mammals