|
Fig. 1: Early oocytes have undetectable levels of ROS. a, Live-cell imaging of human and Xenopus early oocytes, both with attached granulosa cells. The ROS level was measured using MitoTracker Red CM-H2XRos (H2X), a reduced mitochondrial dye that does not fluoresce until it is oxidized by ROS. The boxed area is magnified in the top right image. Xenopus granulosa cells were imaged at the basal plane of the oocyte. DIC, differential interference contrast. Scale bars, 15 µm (human oocytes), 50 µm (Xenopus oocytes), 3 µm (human granulosa cells) and 10 µm (Xenopus granulosa cells). b,c, Quantification of the mean fluorescence intensity (MFI) of H2X in the oocyte and in the population of granulosa cells surrounding the equatorial plane of the oocyte for human (b) and Xenopus (c) oocytes. The data represent the mean ± s.e.m. of three biological replicates, shown in different colours. **P = 0.0001 and ***P = 4.13 × 10−11 using a two-sided Student’s t-test. d, Overnight survival of oocytes at the indicated stages of oogenesis after treatment with menadione, N-acetyl cysteine (NAC) or the combination of both (see Extended Data Fig. 1j for experimental design). At least ten oocytes were incubated per condition. The data represent the mean ± s.e.m. across four biological replicates. *P = 1.94 × 10−9, **P = 3.77 × 10−18 and ***P = 2.37 × 10−19 compared with the untreated condition using a two-sided Student’s t-test with Šidák–Bonferroni-adjusted P values for multiple comparisons.
|
|
Fig. 2: OXPHOS is low, but essential, in early oocytes. a,b, Live-cell imaging of human and Xenopus early oocytes with attached granulosa cells labelled with tetramethylrhodamine ethyl ester perchlorate (TMRE) to detect mitochondrial membrane potential (ΔΨm; a) and JC-1, a membrane potential sensitive binary dye (b). Green JC-1 fluorescence is a sign of low membrane potential; red fluorescence indicates JC-1 aggregation inside mitochondria, and thus high membrane potential. The insets in the Xenopus images show granulosa cells imaged in the basal plane of the oocyte. DIC, differential interference contrast. Scale bars, 10 µm (human oocytes) and 50 µm (Xenopus oocytes). Representative images are shown (see Extended Data Fig. 2 for quantification of independent experiments). c, The basal oxygen consumption rate in early (stage I) and growing (stage III) Xenopus oocytes, normalized for total protein per sample (n = 17 for stage I and n = 43 for stage III). The data represent mean ± s.e.m. ***P = 2.98 × 10−8 using a two-sided Student’s t-test. d, Overnight survival of early (stage I) and late (stage VI) oocytes after treatments with mitochondrial poisons: complex I (CI) to V (CV) inhibitors and an ionophore (5 µM rotenone, 50 mM malonic acid, 5 µM antimycin A, 50 mM KCN, 200 µM N,N′-dicyclohexylcarbodiimide (DCCD) or 30 µM carbonyl cyanide m-chlorophenyl hydrazone (CCCP), respectively). At least 50 early and 10 late-stage oocytes were incubated per condition. ΔΨm, mitochondrial membrane potential. The data represent the mean ± s.e.m. across three biological replicates.
|
|
Fig. 3: The mitochondrial proteomes of Xenopus and human oocytes. a, A volcano plot showing P values versus fold changes of mitochondrial proteins between early (stage I) and late (stage VI) oocytes. The subunits of the mitochondrial OXPHOS machinery are indicated in colour, according to the key in the plot. Other mitochondrial proteins significantly changing (q value < 0.05, >1.5-fold change) are depicted in black. n = 3 outbred animals, P values were calculated using two-sided Student’s t-test, and q values were obtained by multiple-comparison adjustment. b, The early Xenopus oocyte proteome ranked by protein abundance. The inset shows data for the top 5% most abundant proteins, corresponding to the grey area of the graph. UPRmt proteins are indicated in red. The data are the mean ± s.e.m. from n = 3 outbred animals. c, The human primordial follicle proteome ranked by protein abundance. The inset shows data for the top 5% most abundant proteins, corresponding to the grey area of the graph. Oocytes were collected from ovaries of two patients and pooled together. UPRmt proteins are indicated in red. d,e, Scatter plots comparing mitochondrial (d) and OXPHOS (e) protein abundance in human primordial follicles and ovarian somatic cells. The dashed line represents the identity line x = y and the solid line shows the linear regression estimate relating protein abundance between mitochondrial proteomes of primordial follicles and ovarian somatic cells. IM, import machinery.
|
|
Fig. 4: Complex I is not assembled in early oocytes. a, Mitochondrial fractions solubilized in n-dodecyl-β-D-maltoside (DDM) were resolved by BN-PAGE and complex I activity was assayed by reduction of nitro blue tetrazolium chloride (NBT) in the presence of NADH. n ≥ 3 (see Extended Data Fig. 6b for quantifications). b, Spectrophotometric analysis of complex I (green, rotenone-specific activity) and complex IV (orange, KCN-specific activity) activities in mitochondrial extracts from early (stage I) and late (stage VI) oocytes and muscle. cyt c, cytochrome c; abs, absorbance; a.u., arbitrary units. The data represent the mean ± s.e.m.; n = 3 biological replicates. c, Flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) levels in early (stage I) and late (stage VI) Xenopus oocytes. The data represent the mean ± s.e.m.; n = 6. ***P = 6.92 × 10−9 and **P = 3.57 × 10−5 using two-sided Student’s t-test with Šidák–Bonferroni-adjusted P values for multiple comparisons.
|
|
Fig. 5: Complex I and ROS levels correlate throughout oogenesis. a, Mitochondrial fractions from early (stage I), maturing (stage II and stage III) and late-stage (stage VI) Xenopus oocytes and muscle solubilized in n-dodecyl-β-D-maltoside (DDM) were resolved by BN-PAGE and complex I activity was assayed. One representative gel from three independent experiments is shown. CS, citrate synthase. b, Overnight survival of early (stage I), maturing (stage II and III) and late-stage (stage VI) Xenopus oocytes after treatment with the complex I inhibitor rotenone (5 µM). At least 10 oocytes were incubated per condition. The data represent the mean ± s.e.m.; n = 3 biological replicates. *P = 0.0028 and **P = 0.0002 using two-sided Student’s t-test with Šidák–Bonferroni-adjusted P values for multiple comparisons. c, Prdx3 dimer/monomer ratio assessed in oocytes in the indicated stages of oogenesis. The data represent the mean ± s.e.m; n = 4 biological replicates. NS, not significant (P = 0.1128), *P = 0.0376 and **P = 0.0164 using two-sided Student’s t-test with Šidák–Bonferroni-adjusted P values for multiple comparisons.
|
|
Supplementary Figure 1. Caption in second photo.
|
|
Supplementary Figure 1. Caption in photo.
|
|
Supplementary Figure 2. Caption in second photo.
|
|
Supplementary Figure 2. Caption in photo.
|
