AB198. Nodal regulates the differentiation of iPS cells to male germ cells via Smad2/3, Oct4 and Foxh1 activation

OBJECTIVE: The differentiation of male germ cells from iPS cells provides an ideal model for unveiling molecular mechanisms of spermatogenesis. Nodal could promote proliferation of mouse spermatogonial stem/progenitor cells via Smad2/3 and oct-4 activation. The objective of this study was to determine the role of Nodal signaling in the differentiation of iPS cells to male germ cells. DESIGN: Comparative and controlled study. MATERIALS AND METHODS: In this study, embryoid body (EB) formation and the exposure of Nodal induction were applied to induce the male germ cells from mouse iPS in vitro Germ cell-specific genes and proteins were assessed using real-time PCR, immunoblotting and flow cytometry. The moleculars of Nodal signaling pathway were detected by immunoblotting. RESULTS: We found that Nodal and its receptors Alk4, ActR-IIB except Alk7 were expressed in the mouse iPS cells, whereas both Nodal and its receptors were detected in the EBs. Nodal could promote the propagation of iPS cells and Nodal RNAi disrupted the proliferation of iPS cells. The results of real-time PCR and western blots showed that Nodal could up-regulate the expression of germ-cell marker genes and proteins in iPS-derived EBs. Moreover, the level of Smad2/3 phosphorylation, Oct4 and Foxh1 transcription, and cyclin D1 and E were increased with graded Nodal signaling. CONCLUSIONS: Collectively, the above results suggest that Nodal promotes the generation of male germ cells from iPS cells via the activation of Smad2/3 and Oct4 and Foxh1 transcription. This study offers novel insights into molecular mechanisms of male germ cell development. SUPPORT: This work was supported by National Science Foundation of China (31201109) and China National Key Project (2010CB945200).