All aspects of spermatogenesis, including stem cell self-renewal, germline differentiation, and gamete maturation, appear to be affected by aging (Boyle et al. 2007; Cheng et al. 2008; Toledano et al. 2012; Wallenfang et al. 2006). Quantification of GSC number in testes of aged (30–35- and 50-day-old) males revealed a significant decrease in the average number of GSCs over time (Fig. 4.2), which appear to be lost as a consequence of detachment from the hub, followed by differentiation and/or cell death (Boyle et al. 2007; Wallenfang et al. 2006). The remaining GSCs progress through the cell cycle more slowly; therefore, both GSC loss and decreased cell division appear to contribute to the decrease in spermatogenesis observed in older males (Boyle et al. 2007; Wallenfang et al. 2006). Phase contrast analysis of testes from older males also shows increased defects in mitotic amplification divisions, although the mechanisms underlying this aspect of germline aging have not been investigated in depth (Boyle et al. 2007).

Using clonal marking strategies, it was demonstrated that GSCs have an approximate half-life of 14 days (Wallenfang et al. 2006). Therefore, one would predict that, given a starting pool of approximately 10 GSCs, testes should be devoid of germline stem cells by 7 weeks; however, testes of 50-day-old males contained an average of five GSCs/testes (Boyle et al. 2007) (Fig. 4.2). This observation indicated that a mechanism must be in place to maintain a minimum stem cell pool. One intriguing observation that was made by using clonal analysis was that the pool of GSCs appeared to progress toward clonality over time (Wallenfang et al. 2006). Therefore, either a few stem cells are more robust and maintained throughout the lifetime of the fly or, alternatively, dedifferentiating spermatogonia could aid in repopulation of the niche. Interestingly, studies have indicated that spermatogonial dedifferentiation does not decline with age and, therefore, could be a major mechanism supporting GSC maintenance during aging (Wong and Jones 2012).

Some insights were provided by studies examining centrosome positioning in male GSCs (Cheng et al. 2008; Yamashita et al. 2003). As mentioned above, Drosophila male GSCs typically divide in an asymmetric fashion with respect to the hub, with the daughter cell destined to differentiate being displaced away from the hub. During interphase, the mitotic spindle is oriented perpendicularly to the hub to facilitate this asymmetric outcome, and centrosome positioning regulates this stereotypical orientation of the mitotic spindle (Yamashita et al. 2003). The mother centrosome in the GSC is located adjacent to the hub, whereas the daughter centrosome migrates to the opposite side (Yamashita et al. 2007). In young adults, GSCs remain oriented perpendicularly to the hub throughout the cell cycle. However, as flies age, the percentage of GSCs with misoriented centrosomes, in which neither centrosome is located at the GSC-hub interface, increases to about 40 % of total GSCs in 30-day-old flies (Cheng et al. 2008). The age-related accumulation in GSCs bearing misoriented centrosomes was attributed primarily to the dedifferentiation process, as mitotic spindles in mitotically dividing spermatogonia are orientated randomly (Cheng et al. 2008). GSCs with misoriented centrosomes do not enter mitosis, but they can restart the cell division once the centrosomes restore their perpendicular positioning (Cheng et al. 2008).

The extra time required for reorientation of the mitotic spindle could be one contributing factor to the decline in the mitotic index observed in GSCs of aged males (Boyle et al. 2007; Wallenfang et al. 2006). In addition, studies have demonstrated that the level of cell cycle regulatory factors is altered in male germ cells (Boyle et al. 2007; Inaba et al. 2011). For example, the levels of cyclin E, a regulator of progression through the G1 phase of the cell cycle, were increased in GSCs of aged males, consistent with an arrest or extension of G1 (Boyle et al. 2007). In addition, String (Stg), the Drosophila homologue of the dual-specificity phosphatase Cdc25, which promotes cell cycle progression, is highly expressed in both germline and cyst stem cells and is rapidly downregulated in differentiating progeny. However, with increasing age, Stg expression declines specifically in GSCs, but not in the somatic CySCs. Loss and gain of function analysis showed that Stg is required for stem cell maintenance and division, and constitutive expression of Stg in the germline rescued the age-related decrease in GSC division and CySC number (Inaba et al. 2011). Thus, further investigation into the age-related decline in Stg expression might lead to additional insights into mechanisms regulating aging of the male germline.

Interestingly, a recent report noted that one of the core histones, histone H3, is asymmetrically segregated during male GSC divisions (Tran et al. 2012). With every cell cycle, newly synthesized H3 is segregated to the daughter gonialblast, while the original pool of H3, presumably carrying the appropriate posttranslational modifications, is maintained in the GSC. If increasing age were to alter the fidelity of H3 segregation, this could be an epigenetic mechanism leading to changes in gene expression and germline differentiation.