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

Figure 4

(a) Number of reprogrammed PGCs in conditions of normoxia, normoxia and resveratrol (0.5 μM), and hypoxia for 7 days. Resveratrol induces PGC reprogramming. Asterisks show significance at . (b) Electron microscopy photographs showing 0.5 μM resveratrol-induced mitophagy at day 3 (left) and day 6 (right). Autophagic vacuoles are labeled with red stars. M: mitochondria. N: nucleus. (c) Confocal microscopy merge image for immunofluorescence against SSEA1 in red (PGCs) and p62 autophagic vacuoles in green in PGC cytoplasms of cultures subjected to normoxia with 0.5 μM resveratrol for 3 d. Nuclei are seen in blue with DAPI. Scale bars correspond to 25 μm. (d) Embryoid body formation and spontaneous differentiation of reprogrammed cells into cells of the three germ layers by resveratrol (0.5 μM). (e) Immunofluorescence images of ROS levels in PGC (SSEA1+) cultures exposed to normoxia, hypoxia, normoxia and low DCA dose (50 μM), and normoxia and resveratrol (0.5 μM) for 20 h. Scale bars correspond to 20 μm. (f) Number of reprogrammed PGCs in conditions of normoxia, normoxia and ascorbic acid (50 μg/mL), hypoxia, and hypoxia and ascorbic acid (50 μg/mL), for 7 days. Ascorbic acid does not induce reprogramming in normoxia and prevents hypoxia-induced reprogramming. (g) Number of reprogrammed PGCs in conditions of normoxia, normoxia and DCA (50 μM), normoxia and DCA (50 μM), and ascorbic acid (50 μg/mL) for 7 days. Ascorbic acid prevents reprogramming induced by DCA. Asterisks show significance at . (h) Confocal microscopy images for immunofluorescence against SSEA1 and HIF1α in PGC cultures subjected to resveratrol for 48 h. Controls are the same as in Figure 2(b). Scale bars correspond to 25 μm.

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