Journal: Advanced Science
Article Title: AMPK Suppression Due to Obesity Drives Oocyte mtDNA Heteroplasmy via ATF5‐POLG Axis
doi: 10.1002/advs.202307480
Figure Lengend Snippet: AMPK activation improves oocyte maturation and limits mtDNA heteroplasmy in obese females. A) Obese females were fed metformin in drinking water for 4‐weeks (OB+Metf). Immunoblotting of AMPKa, AMPKa phosphorylation at Thr‐172, BMP15, GDF‐9, ATF5, POLG and LonP1 proteins in mature oocytes of control (CON), obese (OB) and OB+Metf females. The β‐tubulin was used as a loading control (n = 5). B) H&E staining in ovary. The primary, secondary and mature oocytes were quantified in each ovary section (n = 5). C) Immunostaining of MitoSpy (green color) and TMRE (red color) in mature oocytes. Fluorescent intensity was qualified by ImageJ and normalized by MitoSpy/TMRE. Five mature oocytes were used in the analyses. Scar bar MitoSpy, 200 µm; bright field, 150 µm; TMRE, 80 µm. D) Number of pups per litter born from female mice. E) Co‐immunoprecipitation of ATF5 in measuring POLG in mature oocytes. ATF5 and POLG were immunoprecipitated followed with SDS‐PAGE separation and measured in immunoblotting. IgG was used as a negative control in immunoprecipitation. F) Percentage of mtDNA heteroplasmy and total mutation sites in mature oocytes from mtDNA‐sequencing (n = 5). Data are presented as mean ± s.e.m. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001; one‐way ANOVA was used in analysis.
Article Snippet: [ , ] Membrane were probed using primary antibodies, including BMP‐15 (#18 982; Proteintech), GDF‐9 (af739; Novus), Caspase‐7 (sc‐56063; Snta‐Cruz), Caspase‐3 (#9662; Cell Signaling), β‐tubulin (#179 513; Abcam), DRP1 (#8570; Cell Signaling), p‐DRP1 (#3455; Cell Signaling), VDAC (#4866; Cell Signaling), TOM20 (sc‐17764; Santa Cruz), ATF5 (ab184923; Abcam), LonP1 (#15 440; Proteintech), POLG (ab128899; Abcam), HSP70 (#4872; Cell Signaling), AMPKa (D63G4; Cell Signaling), p‐AMPKa (#2535; Cell Signaling), GAPDH (#97 166; Cell Signaling).
Techniques: Activation Assay, Western Blot, Staining, Immunostaining, Immunoprecipitation, SDS Page, Negative Control, Mutagenesis, Sequencing