High telomerase is a hallmark of undifferentiated spermatogonia and is required for maintenance of male germline stem cells

The germline and the soma face fundamentally different challenges in preserving genetic integrity. Somatic stem cells need to only maintain genome function for the reproductive life of the animal, whereas germline stem cells (GSCs) must ensure that the gametes and resulting embryos can successfully contribute to subsequent generations (Smelick and Ahmed 2005; Cinalli et al. 2008). One aspect of genome integrity that is markedly different between the germline and the soma is the maintenance of telomeres, the nucleoprotein caps that protect chromosome ends (Palm and de Lange 2008). Whereas telomeres shorten with aging in a variety of somatic tissues, telomeres are maintained with advancing age in sperm (de Lange et al. 1990; Allsopp et al. 1992; Zalenskaya and Zalensky 2002; Baird et al. 2003). Challenges in detecting telomerase, the enzyme that synthesizes telomere repeats, have precluded an understanding of the mechanisms governing this dichotomy between germline and somatic telomere dynamics. Telomeres shorten with each cell division due to incomplete replication of the chromosome ends. After an extended lag period during which telomeres continue to shorten, the protective function of telomeres is lost at a subset of chromosome ends, leading to propagation of a DNA damage response that induces senescence or cell death (d'Adda di Fagagna et al. 2003; Takai et al. 2003). Telomerase counters this process by adding telomeric repeats to chromosome ends, a process that is important for long-term cell viability in tissue progenitor cells and cancers (Artandi and DePinho 2010). In vivo, telomere maintenance by telomerase is broadly required for proper homeostasis of proliferative tissues (Lee et al. 1998). In humans, even modest reductions in telomerase levels can result in a collection of somatic tissue failure phenotypes, including aplastic anemia, pulmonary fibrosis, and liver cirrhosis (Batista et al. 2011; Armanios and Blackburn 2012). Complete genetic inactivation of telomerase in model organisms has implicated the male germline as the most telomerase-dependent tissue. Telomere shortening in mice or fish lacking telomerase causes infertility and the eventual loss of male germ cells (Lee et al. 1998; Henriques et al. 2013; Harel et al. 2015). Inactivation of telomerase in worms leads to infertility and a loss of male germ cells as telomeres shorten (Meier et al. 2006). The loss of germ cell cellularity in telomerase-deficient mouse strains is caused by apoptosis triggered by telomere dysfunction (Lee et al. 1998; Hemann et al. 2001). However, the specific cellular compartment adversely affected by telomerase loss is unknown. Previous studies suggested that a telomere surveillance mechanism recognized short telomeres and induced apoptosis at the onset of meiosis (Hemann et al. 2001). The mechanisms underlying the exquisite dependence on telomere maintenance by the germline would be aided by understanding telomerase expression patterns in the male germ cell lineage. Other than mature sperm, every major germ cell population has been previously proposed as the principal cell type expressing telomerase (Zalenskaya and Zalensky 2002). Generation of sperm is fueled by male GSCs, which self-renew and generate differentiated progeny throughout life (Spradling et al. 2011). In mice, male GSCs reside in a population of mitotic cells, termed undifferentiated spermatogonia based on their undifferentiated histological appearance (de Rooij and Russell 2000). These rare cells on the basement membrane of seminiferous tubules are found as single cells, in pairs, and in chains of four to 16 cells (termed A-single, A-paired, and A-aligned, respectively). Incomplete cytokinesis with cell division in this compartment results in elongating cell syncytia. Undifferentiated spermatogonia give rise to differentiated spermatogonia, which are marked by expression of the cell surface receptor cKit (Schrans-Stassen et al. 1999). Differentiated spermatogonia undergo a series of transit-amplifying divisions before entering meiosis. During each cycle of spermatogenesis, the vast majority of spermatogonia migrate luminally to enter meiosis and eventually leave the testis as spermatozoa. Although undifferentiated spermatogonia have not been purified as a population, several lines of evidence indicate that this population harbors the GSCs. Undifferentiated spermatogonia mature according to a cycle that culminates in the production of new (type A1) differentiated spermatogonia. Histological data indicate that only short chain undifferentiated spermatogonia persist into a new cycle, whereas longer chain spermatogonia differentiate, suggesting that GSCs reside in this short chain population (Huckins 1971; Oakberg 1971; de Rooij 1973). Enrichment for certain cell markers such as Thy1, α6-integrin, Oct4, Id4, Pax7, and others, coupled with transplantation, showed that cells within the undifferentiated spermatogonia population possess stem cell activity (Shinohara et al. 1999; Ohbo et al. 2003; Aloisio et al. 2014; Chan et al. 2014). Lineage tracing of cells expressing GDNF family receptor α1 (GFRα1), which marks many of the short chain undifferentiated spermatogonia, yields long-term labeling of the germ cell lineage in mice (Nakagawa et al. 2010; Hara et al. 2014). Together, these findings indicate that GSCs are located within the undifferentiated spermatogonia pool. Telomerase reverse transcriptase (Tert) transcription represents a primary point of control of telomerase levels (Meyerson et al. 1997). To understand where telomerase is expressed within the male germline, we engineered a Tert promoter knock-in mouse strain in which a fluorescent reporter protein is expressed from the endogenous Tert promoter. We used this reporter strain to determine which populations express telomerase within the male germline and exploited this approach to isolate and study phenotypically defined populations of spermatogonia. These advances allow comparisons of telomerase activity between germline progenitor cells and somatic progenitor cells. We leveraged these techniques to determine the underlying reason for germline dependence on telomerase across metazoans, with important implications for understanding germline immortality.