Telomerase inactivation causes loss of the male germline in worms, fish, and mice, indicating a conserved dependence on telomere maintenance in this cell lineage. Here, using telomerase reverse transcriptase (Tert) reporter
mice, we found that very high telomerase expression is a hallmark of undifferentiated spermatogonia, the mitotic
population where germline stem cells reside. We exploited these high telomerase levels as a basis for purifying
undifferentiated spermatogonia using fluorescence-activated cell sorting. Telomerase levels in undifferentiated
spermatogonia and embryonic stem cells are comparable and much greater than in somatic progenitor compartments. Within the germline, we uncovered an unanticipated gradient of telomerase activity that also enables isolation of more mature populations. Transcriptomic comparisons of TertHigh undifferentiated spermatogonia and
TertLow differentiated spermatogonia by RNA sequencing reveals marked differences in cell cycle and key molecular
features of each compartment. Transplantation studies show that germline stem cell activity is confined to the
TertHigh cKit− population. Telomere shortening in telomerase knockout strains causes depletion of undifferentiated
spermatogonia and eventual loss of all germ cells after undifferentiated spermatogonia drop below a critical
threshold. These data reveal that high telomerase expression is a fundamental characteristic of germline stem cells,
thus explaining the broad dependence on telomerase for germline immortality in metazoans.