AMPK Suppression Due to Obesity Drives Oocyte mtDNA Heteroplasmy via ATF5‐POLG Axis

Abstract Due to the exclusive maternal transmission, oocyte mitochondrial dysfunction reduces fertility rates, affects embryonic development, and programs offspring to metabolic diseases. However, mitochondrial DNA (mtDNA) are vulnerable to mutations during oocyte maturation, leading to mitochondrial nucleotide variations (mtSNVs) within a single oocyte, referring to mtDNA heteroplasmy. Obesity (OB) accounts for more than 40% of women at the reproductive age in the USA, but little is known about impacts of OB on mtSNVs in mature oocytes. It is found that OB reduces mtDNA content and increases mtSNVs in mature oocytes, which impairs mitochondrial energetic functions and oocyte quality. In mature oocytes, OB suppresses AMPK activity, aligned with an increased binding affinity of the ATF5‐POLG protein complex to mutated mtDNA D‐loop and protein‐coding regions. Similarly, AMPK knockout increases the binding affinity of ATF5‐POLG proteins to mutated mtDNA, leading to the replication of heteroplasmic mtDNA and impairing oocyte quality. Consistently, AMPK activation blocks the detrimental impacts of OB by preventing ATF5‐POLG protein recruitment, improving oocyte maturation and mitochondrial energetics. Overall, the data uncover key features of AMPK activation in suppressing mtSNVs, and improving mitochondrial biogenesis and oocyte maturation in obese females.

Clpp, Dnaja3, Hspd1, Chop and Hspe1 (n = 6).mRNA was normalized to 18s rRNA.D) Immunoblotting of PLOG in mature oocytes.GAPDH was used as a loading control (n = 5).E) The percentage of mtDNA heteroplasmy and total mutation sites in ATF5immunoprecipitation (n = 5).F, G) Circular plot of the mitochondrial genome shows the genome annotation on the outer circle.Five green inner circles and five red outside cycles show the genomic locations and the relative frequencies of mtDNA mutations from POLG immunoprecipitants in mature oocytes of CON (green inner cycle) and OB females (red outside cycle) (F).Relative fold changes of mtDNA heteroplasmy between CON and OB mature oocytes were mapped (G).The heteroplasmic change was OB compared with controls.Regions corresponding to the different mtDNA genes (green, protein coding genes; pink, rRNA; red, tRNA; blue, non-coding regions) (n = 5).Data are presented as mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001; unpaired two-tail Student's t test was used in analyses.

Figure S3 .
Figure S3.Excessive UPR mt in mature oocytes of obese females.A) Immunostaining of mitospy (green color) and HSP70 (red color) in mature oocytes collected in both control (CON) and obese (OB) females.Blue is DAPI to indicate nucleus.Scale bar 20 μm.B) Immunoblotting of HSP70 in mature oocytes.GAPDH was used as a loading control (n = 5).C) The mRNA expression of mitochondrial unfolded protein response indicators, including

Figure S4 .
Figure S4.ATF5 activation impairs oocyte maturation and mtDNA heteroplasmy.A) Fluoresecent intensity in mature oocytes of control (CON) and ATF5-overexpressed (ATF5-OE) females was quantified by image J, and further normalized by MitoSpy/TMRE.B) Immunoblotting of BMP15, GDF-9, LonP1 and POLG in mature oocytes.β-tubulin was used as a loading control.C) Co-immunoprecipitation of ATF5 in measuring POLG in mature oocytes.ATF5 and POLG were immunoprecipitated followed with SDS-PAGE separation

Figure S5 .
Figure S5.AMPK inactivation impairs mitochondrial energetics and oocyte maturation.A) Relative folds of protein content in mature oocytes, including LonP1, AMPKa and phosphorylated AMPKa Thr172 (n = 5).B) Immunoblotting of AMPKa1 in mature oocytes in wild type (WT) and Prkaa1-knockout (AMPKa1-KO) mice.GAPDH was used as a loading control.C) H&E staining of ovaries in WT and AMPKa1-KO mice.D) Immunostaining of MitoSpy (green color) and TMRE (red color) in mature oocytes, and fluorescent intensity was qualified by ImageJ and normalized by MitoSpy/TMRE.At least 10 mature oocytes were

Figure S6 .
Figure S6.AMPK activation improves oocyte maturation and limiting ATF5-POLG binding in obese females.A) Oocytes were isolated in control (CON) and obese females (OB), and treated AMPKa agonists metformin in vitro.Immunoblotting of AMPKa, AMPKa phosphorylation at Thr-172, LonP1, ATF5 and POLG proteins in mature oocytes.GAPDH was used as loading controls (n = 4).B) ADP/ATP ratio in mature oocytes.C) Coimmunoprecipitation 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 (n = 4).D, E) Oxygen

Figure S7 .
Figure S7.AMPK activation improves oocyte quality and reduces mtDNA heteroplasmy in obese females.A) The mature oocytes were collected in the fallopian tubes in control (CON), obese (OB) and obese females fed metformin (OB + Metf).Stars indicate the abnormal oocytes.B) Fluorescent intensity of MitoSpy and TMRE in mature oocytes.Intensity was quantified by Image-J.C) The number of mtDNA mutations in D-loop and protein coding genes in mature oocytes collected in CON, OB and OB+Metf females.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 data analysis.

Figure S8 .
Figure S8.Uncropped immunoblotting figures shown in the study.