Deleterious mtDNA mutations are common in mature oocytes

Abstract

Heritable mitochondrial DNA (mtDNA) mutations are common, yet only a few recurring pathogenic mtDNA variants account for the majority of known familial cases in humans. Purifying selection in the female germline is thought to be responsible for the elimination of most harmful mtDNA mutations during oogenesis. Here we show that deleterious mtDNA mutations are abundant in ovulated mature mouse oocytes and preimplantation embryos recovered from PolG mutator females but not in their live offspring. This implies that purifying selection acts not in the maternal germline per se, but during post-implantation development. We further show that oocyte mtDNA mutations can be captured and stably maintained in embryonic stem cells and then reintroduced into chimeras, thereby allowing examination of the effects of specific mutations on fetal and postnatal development.

Keywords: mitochondria; mtDNA; oocyte.

Figure 1

Spectrum of mtDNA mutations in somatic cells of PolG^mut/wt females and their germline transmission. (A) Schematic illustration of experimental design. To investigate the timing when purifying selection for mtDNA mutations operates, somatic mutations in the SF of PolG^mut/wt mothers (Mother) and germline transmitted variants in MII oocytes (MII), PolG^wt/wt blastocysts, PolG^wt/wt viable fetuses at E9.5, and PolG^wt/wt live offspring SF (Pup) were examined by mtDNA whole genome sequencing (MiSeq). (B) Quantification of mtDNA mutations in PolG^mut/wt female SFs (Mother, n = 20) and their MII oocytes (MII, n = 81), and PolG^wt/wt pup SFs (Pup, n = 20). (C) Quantification of heteroplasmy levels of mtDNA variants in PolG^mut/wt female SFs (n = 20) and their MII oocytes (n = 81) and PolG^wt/wt pup SFs (n = 20). (D) Quantification of mtDNA mutations in PolG^wt/wt blastocysts at E3.5 (n = 20) and PolG^wt/wt viable fetuses at E9.5 (n = 20). (E) Effect of heteroplasmy levels on germline transmission of mtDNA mutations in PolG^mut/wt females SFs (n = 20) and their MII oocytes (n = 81) and PolG^wt/wt pup SFs (n = 20). Purple, pink, and blue colors represent mtDNA variants with high (≥50%), moderate (10–49%), and low (<10–2%) heteroplasmy levels, respectively. In B–D, error bars are mean ± SEM; ns denotes P ≥ 0.05.

Figure 2

Gene-specific negative selection of mtDNA mutations. (A) Quantification of non-synonymous mtDNA mutations in genes encoding OXPHOS complexes I, III, IV, and V in PolG^mut/wt female SFs (Mother, n = 20) and their MII oocytes (MII, n = 81) and PolG^wt/wt pup SFs (Pup, n = 20). (B) Germline transmission of non-synonymous mtDNA mutations in mt-Nd4l, mt-Nd5, mt-CoxI, mt-CoxII, mt-CoxIII, and mt-Atp6 measured in PolG^mut/wt female SFs (Mother, n = 20) and their MII oocytes (MII, n = 81) and PolG^wt/wt pup SFs (Pup, n = 20). (C) Quantification of non-synonymous protein-coding mutations with different heteroplasmy levels in PolG^mut/wt female SFs (Mother, n = 20) and their MII oocytes (MII, n = 81) and PolG^wt/wt pup SFs (Pup, n = 20). Heteroplasmy levels were grouped into high (≥50%), moderate (10-49%), and low (≤10%) groups. In A–C, error bars are mean ± SEM; ns denotes P ≥ 0.05.

Figure 3

Fetal lethality of COXIII-81 chimeras. (A) Heteroplasmy levels (%) and locations of non-synonymous protein-coding mutations in COXIII-81, ND6-100, ND2-50, and ATP8-62 ESC lines. ND indicates that mtDNA mutations were not detected in Control-1 ESC line. (B) Proportion of GFP-positive ESCs in COXIII-81 and chimeric blastocysts (E3.5). ns denotes P ≥ 0.05. (C) Detection of COXIII-81 ESC contribution by GFP in chimeric blastocysts. The upper panel is a bright field image and the lower panel is a fluorescent image. Scale bar = 100 μm. (D) Implantation rate of COXIII-81 and Control-1 chimeric embryos. The implantation rate was calculated as the number of implantation site/number of embryos transferred. ns denotes P ≥ 0.05. (E) Percent of viable E9.5 fetuses in COXIII-81 and Control-1 chimeras. The viable fetus rate was estimated as the number of viable fetuses/number of implantation sites. (F) Morphology of degenerated E9.5 fetus (Fetus #1) from COXIII-81 chimeras. The upper panel shows a bright field image and the lower panel shows a fluorescent image. Scale bar = 1 mm. (G) Heteroplasmy levels of 9138 G > A mutation in mt-CoxIII in degenerated E9.5 fetuses and in live chimeric pups. (H) Heteroplasmy level of 9278 A > T mutation in mt-CoxIII in degenerated E9.5 fetuses and in live chimeric pups.

Figure 4

Phenotypes of chimeras carrying deleterious mtDNA mutations. (A) Survival rate of chimeras with COXIII-81 (n = 12), ND6-100 (n = 35), ND2-50 (n = 34), ATP8-62 (n = 25), and control (n = 17) ESC lines at 21 days (dotted line). Premature death was observed in ATP8-62 chimeras, resulting in a significantly lower survival rate compared to controls. (B) Two hour blood glucose levels (mg/dl) in high contribution chimeras with COXIII-81 (n = 1), ND6-100 (n = 3), ND2-50 (n = 2), ATP8-62 (n = 2), and Control-1(n = 3) ESCs after 1 mg/g glucose injection. For each animal, two measurements were performed. Each dot represents biological replicates, ns denotes P ≥ 0.05. Reduced glucose tolerances were observed in ND6-100, ND2-50, and ATP8-62 chimeras compared to Control-1 animals. (C) Optokinetic response spatial frequency (c/d) of pigmented eye chimeras from COXIII-81 (n = 2), ND6-100 (n = 4), ND2-50 (n = 4), ATP8-62 (n = 1), and Control-1(n = 4) ESCs. Measurement was performed on both eyes of animals. Each dot represents biological replicates, ns denotes P ≥ 0.05. Optic neuropathy was observed as low optokinetic response in COXIII-81, ND6-100, and ND2-50 chimeras compared to Control-1 animals. (D–G) Mean cardiac ultrasound values documenting reduced ejection fraction (D) and increased left ventricular (LV) volume (E) in ATP8-62 chimeras (n = 6) compared with age- and sex-matched control PolG^wt/wt (n = 6). In tandem, right ventricles (RV) were significantly enlarged in ATP8-62 chimeric hearts (F–G). (H) Echocardiography of ATP8-62 chimeras showing biventricular chamber dilation and reduced pump function in hearts as dilated cardiomyopathy. LV/RV, left/right ventricle. Vertical scale bars = 2 mm.