The retinoblastoma tumor suppressor gene Rb is essential for maintaining the quiescence and for regulating the differentiation of somatic stem cells. Inactivation of Rb in somatic stem cells typically leads to their overexpansion, often followed by increased apoptosis, defective terminal differentiation, and tumor formation. However, Rb's roles in germ-line stem cells have not been explored. We conditionally disrupted the Rb gene in mouse germ cells in vivo and discovered unanticipated consequences for GFRa1-protein-expressing A_single (GFRa1⁺ A_s) spermatogonia, the major source of male germ-line stem cells. Rb-deficient GFRa1⁺ A_s spermatogonia were present at normal density in testes 5 d after birth, but they lacked the capacity for self-renewal, resulting in germ cell depletion by 2 mo of age. Rb deficiency did not affect the proliferative activity of GFRa1⁺ A_sspermatogonia, but their progeny were exclusively transit-amplifying progenitor spermatogonia and did not include GFRa1⁺ A_s spermatogonia. In addition, Rb deficiency caused prolonged proliferation of progenitor spermatogonia, transiently enlarging this population. Despite these defects, Rb deficiency did not block terminal differentiation into functional sperm; offspring were readily obtained from young males whose germ cell pool was not yet depleted. We conclude that Rb is required for self-renewal of germ-line stem cells, but contrary to its critical roles in somatic stem cells, it is dispensable for their proliferative activity and terminal differentiation. Thus, this study identifies an unexpected function for Rb in maintaining the stem cell pool in the male germ line.

In summary, our data demonstrate that Rb plays an unexpected role in maintaining the germ-line stem cell pool in the mouse testis by regulating SSC self-renewal. Although Rb deficiency results in overexpansion of many somatic stem-cell populations, it leads to rapid exhaustion of SSCs. In addition to its role in SSC self-renewal, we also find that Rb plays a separate role, as a cell-cycle regulator, in progenitor spermatogonia. Rb is required for these cells to become quiescent during the mitotically inactive phase (stages III–VIII) in the seminiferous tubules. Despite these defects, however, Rb deficiency does not impair the ability of SSCs to differentiate into mature spermatozoa, which is in contrast to RB’s general roles in fate decision and differentiation of somatic stem cells. Rb’s regulatory roles in spermatogenesis—including its essential function in male germline stem cell renewal—are remarkably different from its known roles in somatic lineages.