FUNCTIONAL DECIPHERING OF THE CORE SIGNALING MECHANISM FOR SPERMATOGONIAL STEM CELL SELF-RENEWAL AND SURVIVAL

Abstract

The biological fate decisions of self-renewal and differentiation by spermatogonial stem cells (SSCs) provide the foundation for continual spermatogenesis in the mammalian testis. Since SSCs are rare in the testis, it is extremely difficult to study the mechanisms that regulate their biological functions. The SSC self-renewal fate decision has been shown to be dependent on glial cell line-derived neurotrophic factor (GDNF) influence. However, the intracellular mechanisms utilized by GDNF in SSCs are unknown. Recently we identified GDNF-regulated genes in SSCs, which likely encode for molecules that regulate self-renewal. From these data significant up-regulated expression of the transcription factors Bcl6b, Erm, and Lhx1 was identified and Bcl6b was subsequently shown to be important for SSC self-renewal in vitro and in vivo. The objective of this study was to begin deciphering the intracellular signaling mechanisms utilized by GDNF to regulate SSC self-renewal and survival. We utilized a mouse GDNF-dependent culture system that maintains an undifferentiated germ cell population enriched for self-renewing SSCs. Continuous in vitro culture and transplantation analysis are critical elements to define the presence of SSCs in any population of cells subject to examination for factors regulating SSC function. Using siRNA to reduce expression in cultured SSCs and transplantation to quantify stem cell numbers, Bcl6b, Erm, and Lhx1 were all shown to be important for SSC self-renewal in vitro. Next, GDNF was shown to activate AKT in SSCs and this activation could be blocked by inhibition of both PI3K and Src Family Kinases (SFKs). Culture of SSCs with inhibitors to AKT (AKT inhibitor IV), PI3K (LY294002), or SFKs (SU6656) followed by transplantation analysis showed significant impairment of SSC maintenance in vitro compared to control cultures. Apoptosis analysis revealed a significant increase in the percentage of apoptotic cells when both AKT and PI3K inhibitors, but not SFK inhibitor, was added to culture media indicating that multiple signaling pathways are responsible for SSC selfrenewal and survival. Up-regulation of Bcl6b, Erm, and Lhx1 gene expression by GDNF in self-renewing SSCs could be blocked by inhibition of AKT. However, expression of both Plzf and Oct4, which are expressed in SSCs but not GDNF-regulated, were also significantly reduced when AKT was inhibited. GDNF was further shown to phosphorylate SFKs in cultured SSCs and inhibition of SFK function could specifically block GDNF-regulated expression of Bcl6b, Erm, and Lhx1 without effecting either Plzf or Oct4 expression. These data demonstrate that GDNFactivated AKT signaling is essential for SSC survival, but not a specific pathway used for GDNF-regulated gene expression. Importantly, these data also demonstrate that SFK stimulation is an essential intracellular signaling mechanism utilized by GDNF to specifically regulate SSC selfrenewal. (platform)