Metabolic Reprogramming, Autophagy, and Reactive Oxygen Species Are Necessary for Primordial Germ Cell Reprogramming into Pluripotency

Figure 3

(a) Relative expression of Bnip3 gene carried out by quantitative PCR from PGCs cultured under normoxia or hypoxia. Results are shown normalized with respect to normoxia. (b) Electron microscopy photographs of PGCs in normoxia (top row), hypoxia (second row), and normoxia with 50 μM DCA (bottom). Left column day 3 and right column day 6 cultures. Autophagic vacuoles are labeled with red stars. M: mitochondria. N: nucleus. (c) Confocal microscopy merge images for immunofluorescence against SSEA1 in red (PGCs) and p62 autophagic vacuoles in green in cytoplasms of PGC cultures subjected to normoxia (top row), hypoxia (second row), and normoxia with 50 μM DCA (bottom) for 3 d. Nuclei are seen in blue with DAPI. Scale bars correspond to 25 μm. (d) Quantification of p62-positive autophagic vacuoles in confocal stacks shows a nonstatistically significant increase with respect to normoxia. (e) Inhibition of autophagy with chloroquine (5 μM) for 7 days prevents hypoxia-induced reprogramming. (f) Induction of autophagy with spermidine (1 μM) in normoxia for 7 days does not induce reprogramming. Asterisks show significance at .

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