Traditionally the effects of radiation on

Traditionally, the effects of radiation on spermatogonia have been evaluated by morphological analyses. It is considered that the percentage of seminiferous tubule cross-sections that show type A spermatogonia 10 days after irradiation is representative of SSC survival (van der Meer et al., 1992). Among the several different types of undifferentiated spermatogonia, Asingle (As) spermatogonia are thought to have SSC potential (de Rooij and Russell, 2000). However, it is difficult to distinguish SSCs from committed progenitors morphologically. This is particularly true after genotoxic stress, which may influence the Bindarit and morphology. Moreover, because germ cells contact Sertoli cells directly within seminiferous tubules, the potential effects of paracrine interactions with neighboring peritubular and interstitial cells must be considered. Radiation-induced damage to Sertoli cells causes hormonal imbalance and dysregulated cytokine secretion (Guitton et al., 1999; Legué et al., 2001), which impairs the analysis of SSCs after irradiation. In this study, we used two techniques to investigate the mechanism of radiation-induced apoptosis of SSCs. First, we used germ cell transplantation. Transplantation of testis cells into the seminiferous tubules of infertile testes results in the regeneration of spermatogenesis from donor SSCs (Brinster and Zimmermann, 1994). This allows the functional identification of SSCs. Second, we used an SSC culture technique (Kanatsu-Shinohara et al., 2003). SSCs undergo self-renewal division and proliferate as grape-like clusters of spermatogonia when the media are supplemented with fibroblast growth factor 2 (FGF2) and glial cell line-derived neurotrophic factor (GDNF). These cultured spermatogonia, designated germline stem cells (GSCs), are enriched for SSCs. Assuming 10% colonization efficiency (Nagano et al., 1999), 1%–2% of GSCs can colonize seminiferous tubules (Kanatsu-Shinohara et al., 2005). GSCs proliferate logarithmically and recolonize seminiferous tubules, resulting in spermatogenesis and normal offspring. Because it is possible to manipulate GSCs genetically, the combination of transplantation and culture techniques for SSCs provides a powerful approach for investigating the effect of candidate genes involved in radiation response. Using these strategies, we assessed the molecular mechanism underlying the DNA damage response in SSCs.
Results
Discussion In contrast to previous studies, we have shown increased survival of Trp53 KO SSCs after irradiation. At least two factors must be considered to explain this discrepancy. First is the method used to identify SSCs. Whereas we identified SSCs using a functional transplantation assay, previous studies identified them by observing in situ colony regeneration. It is challenging to distinguish whether poor colony development is due to defects in the SSCs or their microenvironment. Second is the abnormal cell division of Trp53 KO spermatogonia. A previous study showed that loss of Trp53 caused a 40%–50% increase in the total number of type A spermatogonia in nonirradiated testes and also stimulated a 7-fold increase in the number of giant spermatogonia after irradiation (Beumer et al., 1998). Giant spermatogonia were mostly single cells, suggesting their As spermatogonia origin. The fate of giant spermatogonia is unknown, but it is possible that they represent SSCs that cannot differentiate normally. Because TRP53 expression is highest in meiotic cells (Hendry et al., 1996), it is also possible that Trp53 KO cells experience difficulties in undergoing meiotic differentiation. Although we did not analyze the effects of Trp53 signaling on spermatocyte or spermatid development, this may result in smaller colony formation, which could be underrepresented by a histological detection method. To analyze the molecules downstream of Trp53, we first focused on the role of CDKN1A and the BH3-only protein family. Several previous studies suggested that CDKN1A-mediated cell-cycle arrest and Bbc3-induced apoptosis contribute to radiation damage in self-renewing tissues (Yu et al., 2003; Leibowitz et al., 2011). For example, DNA damage in hematopoietic and mammary stem cells activated CDKN1A and induced symmetrical self-renewal divisions by suppressing Trp53 activation, thus inhibiting apoptosis (Insinga et al., 2013). Moreover, Bbc3 deficiency protected both intestinal crypt cells and hematopoietic stem cells from radiation damage (Yu et al., 2010; Qiu et al., 2008). In our study, Cdkn1a KO GSCs were comparable to WT GSCs, whereas Bbc3 depletion enhanced GSC survival. Cell rescue by Bbc3 depletion could also be applicable to MEFs and mGSCs, suggesting that this effect of Bbc3 is not specific to germ cells.