Hypoxia inducible factor 2 alpha/insulin-like growth factor receptor signal loop supports the proliferation and Oct-4 maintenance of mouse germline stem cells

Hypoxia inducible factor 2 alpha (HIF-2 alpha) is critical for primordial germ cell (PGC) survival as knockout of HIF-2 alpha (HIF-2 alpha(-/-)) decreases both expression of Oct-4 and PGC number in genital ridge. Hypoxia is known to stabilize HIF-2 alpha protein from proteasomal degradation. However, little is known about the hypoxia-associated endocrinal signaling in HIF-2 alpha expression. The current work demonstrates a role for an endocrine insulin-like growth factor-I receptor (IGF-IR)-PI3K/Akt-mTOR-HIF-2 alpha regulatory loop in the proliferation and Oct-4 maintenance of PGC-like alkaline phosphatase positive mouse germline stem cells (AP(+)GSCs). We found that hypoxia greatly increased the cell proliferation and the levels of nuclear Oct-4/HIF-2 alpha protein of AP(+)GSCs. The hypoxic-AP(+)GSCs presented stronger stemness ability for germ cell differentiation than normoxic, with expressions of c-KIT (differentiation germ cell marker), VASA (differentiation germ cell marker) and SCP3 (meiotic marker) using a renal capsule transplantation assay. Meanwhile, hypoxia significantly increased the expression levels of secreted-IGF-I and IGF-IR. The IGF-I dose dependently increased the HIF-2 alpha expression levels in AP(+)GSCs; and, the inhibition of IGF-IR by RNA interference (shIGF-IR) or LY294002 (PI3K inhibitor)/Rapamycin (mTOR inhibitor) effectively suppressed the IGF-I- and/or hypoxia-induced HIF-2 alpha and Oct-4 expression, suggesting that the IGF-IR and its downstream Akt/mTOR signaling are involved in the IGF-I/hypoxia effects. Additionally, knockdown of HIF-2 alpha dramatically suppressed Oct-4 and IGF-IR protein levels in AP(+)GSC cells. In conclusion, the present study demonstrates a regulatory loop of IGF-IR-PI3K/Akt-mTOR-HIF-2 alpha in proliferation and Oct-4 maintenance of PGC-like AP(+)GSCs under hypoxia. This finding provides insights into the niche endocrinology underlying early germ cell development