pkc inhibitor br Experimental Procedures br Acknowledgments

Experimental Procedures
Acknowledgments
Introduction Spermatogonial stem pkc inhibitor (SSCs) give rise to the spermatogenesis that endures throughout the life of male animals (de Rooij and Russell, 2000; Meistrich and van Beek, 1993). SSCs reside in a special microenvironment termed a niche, which is located in the basal lamina facing the interstitium of the testis (Chiarini-Garcia et al., 2001; Yoshida et al., 2007). SSCs self-renew in this germline niche, giving rise to progenitor cells while remaining undifferentiated. Although the behavior of SSCs in the niche has been well-described in Drosophila (Li and Xie, 2005), little is known about SSCs of the mammalian testis, partly because the testis contains relatively few SSCs (0.02%–0.03%) (de Rooij and Russell, 2000; Meistrich and van Beek, 1993) and partly because SSCs are difficult to distinguish from committed progenitor cells via morphological analyses. Previous studies showed that SSCs produce either two stem cells after a self-renewal division or two differentiating cells after a differentiating division (de Rooij and Russell, 2000). These two types of division occur at the same frequency to maintain the SSC population at a constant level. Detailed analysis of SSCs is challenging because no SSC-specific markers are yet known. Additionally, in principle, SSCs must be defined by the ability to undergo self-renewal division, which is not easy to assess. Such problems have made it difficult to analyze SSCs and the interactions thereof within the niche described above. In 2000, glial cell line-derived neurotrophic factor (GDNF) was shown to be involved in SSC self-renewal (Meng et al., 2000). GDNF belongs to the transforming growth factor β superfamily molecules and binds to glycosylphosphatidylinositol (GPI)-anchored GFRA1, triggering signaling via the transmembrane receptor tyrosine kinase RET, which does not directly bind to GDNF (Sariola and Saarma, 2003). In transgenic mice overexpressing Gdnf, undifferentiated spermatogonia accumulate in seminiferous tubules and cease differentiation. In contrast, mice heterozygous for Gdnf gradually lose spermatogenesis and become infertile as spermatogonia are lost (Meng et al., 2000). Knockout (KO) animals with defects in Ret or Gfra1 also exhibit similar phenotypes (Jain et al., 2004; Jijiwa et al., 2008; Naughton et al., 2006). Such results suggest that SSCs undergo self-renewal when GDNF level is high, but differentiate when the GDNF concentration is low. This feature has been exploited to develop a long-term culture system for SSCs; SSC numbers increase exponentially over a 2-year period (Kanatsu-Shinohara et al., 2003). Cultured SSCs, termed germline stem (GS) cells can be subjected to gene targeting and reinitiate spermatogenesis upon transplantation into seminiferous tubules (Kanatsu-Shinohara et al., 2006). These results suggested that GDNF is a bona fide self-renewal factor for SSCs. As GDNF plays a critical role in determining the fate of SSCs, controls on the GDNF receptor components have been investigated extensively. However, the question of whether SSCs express such receptor components remains controversial (Buageaw et al., 2005; Ebata et al., 2005; Grisanti et al., 2009). Some authors have claimed that SSCs express GFRA1, whereas others have raised the possibility that the situation is otherwise. A transplantation assay showed that GFRA1 was transiently expressed in SSCs of immature pup testes, but not in neonate or adult SSCs (Ebata et al., 2005). Another group found that 10% of Asingle (As) spermatogonia did not express GFRA1 and that 5% of Apaired (Apr) spermatogonia asymmetrically expressed GFRA1 (Grisanti et al., 2009). Cells positive in terms of GFRA1 expression (selected using magnetic beads) were not clonogenic, whereas cells lacking GFRA1 produced colonies after transplantation. Thus, although a positive influence of GDNF in the context of SSC self-renewal has been suggested, it remains unknown why a significant proportion of As spermatogonia lack SSC activity and whether such spermatogonia express the GDNF receptor.