Interestingly our transplantation study revealed

Interestingly, our transplantation study revealed that Bbc3 depletion could not protect SSCs, suggesting that the Bindarit that survived irradiation were spermatogonia progenitors. Bbc3-depleted cells retained their proliferative activity but lost their recolonizing ability. This result was unexpected because several studies showed the involvement of Bbc3 in the Trp53-induced depletion of adult stem cells, including spermatogonia (Qiu et al., 2008; Liu et al., 2010; Yu et al., 2010). An additional study reported that Bbc3 is upregulated in ITGA6+ side population (SP) phenotype spermatogonia, which are thought to be SSC enriched (Coureuil et al., 2010). The viability of Bbc3 KO SP cells was decreased by 1.6-fold, compared with a 2.7-fold reduction in the WT SP cells. However, because the presence of SSCs was not assessed in that study, it was unclear whether apoptosis occurred in SSC or progenitor cells. Our results suggest that Bbc3 is upregulated in KIT+ cells and that radiation-induced apoptosis of progenitor cells, rather than SSCs, may be Bbc3 dependent. Given the beneficial roles of Cdkn1a and Bbc3 in other self-renewing tissues, SSCs appear to have radiation-response features distinct from these stem cell types despite their common dependence on Trp53. Another possible regulator of Trp53-mediated apoptosis is the TNFSF10-TNFRSF10B pathway. In stressed conditions, germ cell apoptosis depends on both extrinsic and intrinsic pathways. Radiation-induced apoptosis caused by DNA damage generally requires the intrinsic, rather than the extrinsic, pathway (Forand and Bernardino-Sgherri, 2009). One study showed that spermatogonia upregulated Tnfrsf10b after irradiation, whereas Tnfsf10 deficiency could not inhibit radiation-induced apoptosis as efficiently as Bbc3 deficiency (Coureuil et al., 2010). Consistent with this, the TNFSF10-TNFRSF10B pathway did not protect against apoptosis in gonocytes, which are spermatogonia precursors (Forand and Bernardino-Sgherri, 2009). Nevertheless, radiation-induced apoptosis in GSCs was rescued by depleting Tnfrsf10b, highlighting the importance of the extrinsic pathway in SSC radioprotection. Because TNFSF10 expression is upregulated in both germ cells and somatic cells, we speculate that increased TNFRSF10B expression in SSCs makes them more sensitive to apoptosis induced by upregulated TNFSF10 expression. Taken together, our results suggest that quality control of the male germline against genotoxic damage is unique in that differentiation induces a switch in the cell death machinery. Although the induction of Tnfrsf10b is Trp53 dependent, up to now, its mechanism of induction has been elusive. Our study reveals that Trp53inp1 induces Tnfrsf10b. Despite its ubiquitous expression, suppression of Trp53inp1 could rescue GSCs, but not mGSCs or MEFs, suggesting that it may confer a germ cell-specific response. Trp53inp1 is induced by ROS (Cano et al., 2009) and was previously isolated as a Trp53-inducible protein that participates in Trp53-dependent apoptosis by regulating Trp53 function (Okamura et al., 2001). More recent studies showed that it also regulates ROS and autophagy (Cano et al., 2009; Sancho et al., 2012; Seillier et al., 2012). TRP53INP1 binds to HIPK2, PRKCD, and TRP53 to mediate the phosphorylation of TRP53 at Ser46. Colocalization of these proteins in promyelocytic leukemia nuclear bodies facilitates the protein interactions. This increases TRP53 stability and transcriptional activity, leading to transcriptional activation of Trp53 target genes such as TP53AIP1, cell growth arrest, and apoptosis upon DNA damage stress (Tomasini et al., 2003; Yoshida et al., 2006). Trp53inp1 is a major mediator of the antioxidant function of Trp53 (Cano et al., 2009). Although Trp53inp1 KO mice do not exhibit an overt phenotype, they are susceptible to induction of colorectal tumorigenesis and acute colitis, which is thought to be due to increased ROS production (Gommeaux et al., 2007). In a previous study (Morimoto et al., 2013), we showed that although moderate ROS levels were required to stimulate self-renewal, increasing ROS levels by H2O2 supplementation did not increase SSC activity, and high concentrations of H2O2 killed the SSCs. In this context, the degree of ROS generation after irradiation was apparently toxic. Our results suggest that Trp53inp1 also induces Tnfrsf10b expression after irradiation. Because Tnfrsf10b is expressed more strongly in GSCs, such cells are probably more sensitive to apoptosis stimuli, which may explain the germ cell-specific rescue of radiation-induced apoptosis. In addition, the relatively lower ATM levels in GSCs also may have contributed to this phenomenon. Like Trp53 KO mice, Trp53inp1 KO mice have no apparent fertility phenotype. However, given Trp53inp1’s diverse functions and functional redundancy with Trp53inp2 (Nowak et al., 2009), future studies are needed to delineate the physiological roles of these genes in germ cell biology.