We further explored the ploidy levels of human SSCs

We further explored the ploidy levels of human SSCs with RA and SCF treatment by detecting DNA content. Notably, the percentage of haploid cantharidin was significantly increased in human SSCs by RA and SCF treatment, although spontaneous differentiation of SSCs into haploid cells was observed in control samples without RA or SCF induction. As such, RA and SCF promote the differentiation of human SSCs into haploid spermatids. The SCF/KIT signaling pathway has been proven to be essential for human ESCs to differentiate into human germ cells (West et al., 2010). Additionally, RA has a crucial role in pushing complete meiosis and generating haploid cells from mouse ESCs and human iPSCs (Eguizabal et al., 2011; Nayernia et al., 2006; Riboldi et al., 2012). Taken together, these studies from our peers and us suggest that RA and SCF are effective to coax the second meiosis of germ cells into haploid spermatids. It remains unknown whether round spermatids derived from mouse spermatogonia have fertilization ability (Feng et al., 2002). Of unusual significance, haploid spermatids generated from human SSCs of cryptorchid patients had the potential to fertilize oocytes to form embryos that were capable of developing into eight-cell stages.
Experimental Procedures
Acknowledgments
Introduction The testis is a tissue that is highly sensitive to DNA damage by ionizing radiation. Compared with somatic cells in the testis, spermatogenic cells are easily damaged by radiation insults: irradiated animals undergo germ cell loss and become sterile (Meistrich, 1982; Creemers et al., 2002). However, depending on the radiation dose, spermatogenesis can regenerate to regain fertility (Meistrich et al., 1978). Spermatogonial stem cells (SSCs) are important for regeneration. Although there are only 2–3 × 104 SSCs in the testis (Meistrich and van Beek, 1993; de Rooij and Russell, 2000), their robust regenerative activity supports spermatogenesis throughout adult life. It is generally believed that germ cells have a lower mutation rate than somatic cells (Provost et al., 1993; Walter et al., 1998; Hill et al., 2004). Moreover, the survival of spermatogonia after radiation damage varies depending on their stage of differentiation. Differentiating spermatogonia (including A1–A4, intermediate, and B spermatogonia) are the most sensitive, whereas undifferentiated spermatogonia (including SSCs) can survive moderate radiation doses (Erickson, 1976; Dym and Clermont, 1970). A relatively higher apoptosis rate of progenitors has also been reported in other self-renewing tissues (Etienne et al., 2012; Qiu et al., 2008), but the mechanism for this remains unclear. Double-strand breaks (DSBs), which are created by radiation and are the most hazardous type of DNA damage, are generally repaired by nonhomologous end joining (NHEJ) and homologous recombination (HR) (Branzei and Foiani, 2008). Whereas NHEJ is error prone and functions throughout the cell cycle, HR is error free and occurs in S and G2 phases when sister chromatids are available as templates. The cellular response is initiated by ataxia telangiectasia-mutated (ATM) and DNA protein kinase, which associates with DSBs and phosphorylates histone H2AX. Phosphorylated H2AX (γH2AX) recruits damage repair proteins such as MDC1. Additional factors, such as 53BP1, then bind and initiate DNA repair (Eliezer et al., 2009). However, several studies have suggested that a unique DNA repair mechanism operates in SSCs. γH2AX is not detected in undifferentiated spermatogonia, possibly including SSCs, whereas differentiated spermatogonia exhibit distinct foci formation (Rübe et al., 2011). It was shown that these cells also do not express MDC1 after irradiation, although nuclear 53BP1 foci were detected (Ahmed et al., 2007; Rübe et al., 2011). More surprisingly, several groups suggested that the tumor suppressor Trp53, a key molecule in the response to DNA damage, does not play a role in the radiation-induced apoptosis of SSCs (Hendry et al., 1996; Beumer et al., 1998; Hasegawa et al., 1998). In Trp53 knockout (KO) mice, negligible spermatogonia apoptosis was observed after doses of up to 5 Gy, whereas the number of spermatogonia was reduced by 60% within 1 day in wild-type (WT) mice (Beumer et al., 1998). In addition, TRP53 was not detected in undifferentiated spermatogonia in either nonirradiated or irradiated conditions. Therefore, the reduction in the number of spermatogonia resulted from Trp53-dependent apoptosis in differentiating spermatogonia (Beumer et al., 1998; Hasegawa et al., 1998).