Oocytes maintain ROS-free mitochondrial metabolism by suppressing complex I – Nature
Ethical committee permission to work with primordial oocytes from human ovary samples was obtained from the Comité Étic d’Investigació Clínica CEIC-Parc de Salut MAR (Barcelona) and Comité Ético de Investigación Clínica–Hospital Clínic de Barcelona with approval number HCB/2018/0497. Written informed consent was obtained from all participants before their inclusion in the study.
Animals used in this study were housed in the Barcelona Biomedical Research Park, accredited by the International Association for Assessment and Accreditation of Laboratory Animal Care. Animal euthanasia was performed by personnel certified by the competent authority (Generalitat de Catalunya) and conformed to the guidelines from the European Community Directive 2010/63 EU, transposed into Spanish legislation on RD 53/2013 for the experimental use of animals.
Xenopus laevis adult females of between 2 and 4 years old were purchased from Nasco and maintained in water tanks in the following controlled conditions: 18–21 °C, pH 6.8–7.5, O2 4–20 ppm, conductivity 500–1,500 µs, ammonia <0.1 ppm. The C57BL/6J mice used in the experiments were purchased from Charles River Laboratories and maintained in the Animal Facility of the Barcelona Biomedical Research Park under specific-pathogen-free conditions at 22 °C with 40–60% humidity, in a 12 h light/dark cycle, and with access to food and water ad libitum. Female mice of 7 weeks of age were used for extracting muscle tissue.
Oocyte isolation and culture
Human primordial oocytes
Ovaries were provided by the gynaecology service of Hospital Clinic de Barcelona, from women aged 19 to 34 undergoing ovarian surgery and were processed as previously described6. Briefly, ovarian cortex samples were digested in DMEM containing 25 mM HEPES and 2 mg ml−1 collagenase type III (Worthington Biochemical, LS004183) for 2 h at 37 °C with occasional swirling. Individual cells were separated from tissue fragments by sedimentation, and collagenase was neutralized by adding 10% FBS (Thermo, 10270106). Follicles were picked manually under a dissecting microscope. All human oocyte imaging experiments were conducted in DMEM/F12 medium (Thermo, 11330-032) containing 15 mM HEPES and 10% FBS (Thermo, 10270106).
Ovaries were dissected from young adult (aged 3 to 5 years) female X. laevis that had undergone euthanasia by submersion in 15% benzocaine for 15 min. Ovaries were digested using 3 mg ml−1 collagenase IA (Sigma, C9891-1G) in Marc’s modified Ringer’s (MMR) buffer by gentle rocking until dissociated oocytes were visible, for 30 to 45 min. The resulting mix was passed through two sets of filter meshes (Spectra/Mesh, 146424 and 146426). All washes were performed in MMR. For live-imaging experiments with intact granulosa cells, oocytes were transferred to oocyte culture medium (OCM)41 at this stage. For the rest of the experiments, oocytes were stripped of accompanying granulosa cells by treatment with 10 mg ml−1 trypsin in PBS for 1 min, followed by washes in MMR. Removal of granulosa cells was confirmed by Hoechst staining of a small number of oocytes.
HeLa cell culture
HeLa cells were obtained from ATCC (CCL2), authenticated based on morphological inspection and confirmed to be mycoplasma negative. Cells were grown in DMEM (Thermo, 41965039) supplemented with 1 mM sodium pyruvate (Thermo, 11360070) and 10% FBS (Thermo, 102701060).
Human or Xenopus early oocytes were labelled in their respective culture medium (see above). Human oocytes were imaged using a 63× water-immersion objective (NA 1.20, Leica, 506346) with an incubation chamber maintained at 37 °C and 5% CO2. Frog oocytes were imaged using a 40× water-immersion objective (NA 1.10, Leica, 506357) in OCM at room temperature and atmospheric air, unless stated otherwise. All images were acquired using a Leica TCS SP8 microscope with the LAS X software (Leica, v22.214.171.12476). Mean fluorescence intensities in granulosa cells and oocytes were quantified using Fiji software.
Oocytes and associated granulosa cells were incubated in 500 nM MitoTracker Red CM-H2Xros (Thermo, M7513) for 30 min, 5 µM MitoSOX Red (Thermo, M36008) for 10 min, or 5 µM CellROX for 30 min. Cells were then washed and imaged in 35-mm glass-bottom MatTek dishes in culture medium, except for CellROX labelling, for which MMR was used for imaging to satisfy the manufacturer’s instructions.
Mitochondrial membrane potential probes
Oocytes and associated granulosa cells were labelled for 30 min in 500 nM tetramethylrhodamine ethyl ester perchlorate (TMRE) (Thermo, T669), or 45 min in 4 µM JC-1 (Abcam, ab141387). Cells were then washed and imaged in 35-mm glass-bottom MatTek dishes.
Oxygen consumption rate
Oxygen consumption rate (OCR) of Xenopus oocytes was measured using a Seahorse XFe96 Analyser (Agilent) with Seahorse Wave software (Agilent, v2.6). Granulosa-cell-stripped oocytes were placed in XFe96 culture plates immediately after their isolation in Seahorse XF DMEM medium pH 7.4 supplemented with 10 mM glucose, 1 mM pyruvate and 2 mM glutamine (Agilent; 103015-100, 103577-100, 103578-100 and 103579-100). A cartridge was loaded with concentrated inhibitor solution to achieve 5 µM oligomycin, 2 µM carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone or a combination of 0.5 µM rotenone and 0.5 µM antimycin A. Mock medium injections were performed to account for inhibitor-independent decline in OCR. For each sequential injection, at least 4 measurement cycles were acquired consisting of 20 s mix, 90 s wait and 3 min measure, in at least 3 replicates. For basal and maximal respiration rates, assay-independent OCR decline was corrected, and non-mitochondrial respiration (resistant to rotenone–antimycin mix) was subtracted. OCR measurements for growing oocytes (stage III; with a diameter of 450–600 µm (ref. 42)) had to be performed statically because the probe of the analyser compressed and destroyed these large oocytes in long-term measurements. For growing (stage III) oocytes, OCR was acquired during 5 cycles per well, each cycle being 20 s mix, 90 s wait and 3 min measure, in at least 4 replicates. The well size imposed a technical limitation on the maximum number of oocytes per well (100 early and 8 growing oocytes); thus, respiration data were normalized for the total protein amount per sample.
Treatments with OXPHOS inhibitors
At least 50 stage I and stage II, 20 stage III and 10 stage VI oocytes were assayed per condition. Oocytes were placed in 35-mm glass-bottom dishes (MatTek) and incubated for 16 h at 18 °C in OCM with or without the addition of the indicated mitochondrial inhibitors at the following concentrations: 5 µM rotenone (Sigma, R8875), 50 mM malonic acid (Sigma, M1296), 5 µM antimycin A (Abcam, ab141904), 50 mM potassium cyanide (KCN; Merck Millipore, 1049670100), 200 µM N,N′-dicyclohexylcarbodiimide (DCCD) (Sigma, D80002) and 30 µM carbonyl cyanide m-chlorophenyl hydrazone (CCCP) (Abcam, ab141229). Survival was assessed by counting the number of oocytes with intact morphology before and after treatments. Cell death in stage III to VI oocytes was recognized by the development of a mottling pattern in the pigmentation43. Images were taken by a Leica IC90 E stereoscope.
Early (stage I) oocytes were treated with 10 µM menadione (Sigma, M5625) or left untreated, for 2 h in OCM, and washed into fresh OCM. Untreated oocytes were labelled with wheat germ agglutinin 488 (Biotium, 29022-1) to mark their plasma membrane and mixed with menadione-treated oocytes in a glass-bottom MatTek dish 4 h after menadione was removed. The mixed population of oocytes were then labelled with MitoSOX and imaged. At least 50 stage I and II oocytes and at least 10 stage III and VI oocytes were treated with 10 µM menadione (Sigma, M5625) in the presence or in the absence of 10 mM N-acetyl cysteine (NAC) (Sigma, A9165). After 2 h, menadione was removed and N-acetyl cysteine was retained for an overnight incubation. Survival was determined by counting the number of oocytes immediately before menadione treatment (t = 0) and after 16 h in recovery.
Mitochondrial-enriched fractions were obtained as described previously for gastrocnemius muscle and with minor adaptations for oocyte samples44. Freshly isolated early oocytes from Xenopus were lysed in mitochondria buffer (250 mM sucrose, 3 mM EGTA, 10 mM Tris pH 7.4), and spun at low speed to remove debris. The resulting supernatant was centrifuged at 20,000g for 20 min at 4 °C. Late-stage oocytes were spin-crashed, and yolk-free fraction was combined 1:1 with mitochondria buffer and centrifuged at 20,000g for 20 min at 4 °C to pellet mitochondria. Mitochondrial pellets from early and late-stage oocytes were resuspended in mitochondria buffer and subjected to DNase treatment for 10 min and proteinase K treatment for 20 min. Phenylmethylsulfonyl fluoride was added to stop proteolytic activity and samples were centrifuged again at 20,000g for 20 min at 4 °C. Protein concentration was estimated and aliquots of crude mitochondria were stored at −80 °C until use.
Spectrometric assessment of enzymatic activities of mitochondrial complexes
The specific activities of mitochondrial complex I, complex IV and citrate synthase were determined as described before with minor modifications45. Briefly, mitochondrial extracts were subjected to three freeze–thaw cycles in hypotonic buffer (10 mM Tris-HCl) before activity analysis using an Infinite M200 plate reader (Tecan) with Tecan i-control software (Tecan, v3.23) in black-bottom 96-well plates (Nunc) at 37 °C. For complex I NADH:CoQ activity assessment, reaction solutions (50 mM KP pH 7.5, 3 mg ml−1 BSA, 300 µM KCN and 200 µM NADH) with or without rotenone (10 µM) were distributed into each well first. Mitochondrial extracts were then added and NADH absorbance at 340 nm was measured for 2 min to establish baseline activity. The reaction was then started by the addition of ubiquinone (60 µM). NADH absorbance was recorded for 15 min every 15 s.
For complex IV activity assessment, reaction solutions (50 mM KP pH 7, 60 µM reduced cytochrome c) with or without KCN (600 µM) were distributed into each well first, and absorbance of reduced cytochrome c at 550 nm was recorded for 2 min to establish baseline oxidation. Mitochondrial extracts were then added and absorbance was measured for 15 min every 15 s.
For citrate synthase activity, reaction solution (100 µM Tris pH 8, 0.1% Triton X-100, 100 µM DTNB and 300 µM acetyl CoA) was distributed into each well first. Mitochondrial extracts were then added and absorbance at 410 nm was measured for 2 min to set the baseline; then the reaction was started by addition of the substrate oxaloacetic acid (500 µM). Production of TNB (yellow) was recorded by measuring the absorbance at 410 nm for 15 min every 15 s. Enzymatic assays were plotted with the baseline represented as 1 for simplicity.
Denaturing SDS gel electrophoresis
Oocytes were collected after isolation, frozen in liquid nitrogen and kept at −80 °C until further use. Samples were processed as described previously46. Gastrocnemius total homogenates were obtained as described previously47. HeLa cells were lysed in RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 1% Nonidet P-40, 01% SDS and 1 mM EDTA, supplemented with protease inhibitor cocktail (Complete Roche Mini, 1 tablet per 50 ml)) and spun at 20,000g to eliminate cell debris. Oocyte lysates for determination of the redox state of peroxiredoxin were protected against artefactual oxidation by alkylation as described previously48, but in OCM. Cell lysates or mitochondrial-enriched fractions were resolved by SDS–PAGE using 4–12% NuPAGE Bis-Tris gels.
BN-PAGE electrophoresis, and in-gel activity assays
Mitochondrial content in samples of different cell types (different stages of oocytes and muscle tissue) was first assessed by western blotting for their citrate synthase levels (Supplementary Figs. 1b and 2c,d). Next, similar amounts of mitochondrial fractions were solubilized in 1% n-dodecyl-β-d-maltoside (DDM) or digitonin, and were resolved in the native state using NativePAGE 3–12% Bis-Tris (Thermo, BN1001BOX) gradient gels as described before49. The left part of the gel was cut and stained with Coomassie (InstantBlue, Sigma) after BN-PAGE to reveal the native protein molecular weight marker protein (Supplementary Figs. 1a,b and 2a,c,d). Complex I and complex IV activity in-gel assays were performed as described previously24. Briefly, immediately after the run, BN-PAGE gels were incubated in assay solution: for complex I in 2 mM Tris pH 7.4, 0.1 mg ml−1 NADH and 2.5 mg ml−1 nitro blue tetrazolium chloride (NBT) to asses NADH:FMN electron transfer, denoted by the appearance of dark purple colour; and for complex IV in 10 mM phosphate buffer pH 7.4, 1 mg ml−1 cytochrome c and 0.5 mg ml−1 of 3,3′-diaminobenzidine (DAB) in the presence or absence of 0.6 mM KCN to assess the specific cytochrome c oxidation, denoted by the appearance of dark brown colour. The intensities of reduced nitro blue tetrazolium chloride (NBT) were normalized to citrate synthase levels of the same samples, detected by SDS–PAGE followed by immunoblotting. Gels were imaged using an Amersham Imager (GE Healthcare; Supplementary Figs. 1 and 2). Intensity measurements were performed using Fiji software.
Denaturing SDS–PAGE gels were transferred to nitrocellulose membranes through wet transfer using a Mini Trans-Blot Cell (Bio-Rad). Membranes were blocked in Intercept (TBS) Blocking Buffer (LI-COR), and incubated overnight at 4 °C with primary antibodies diluted in Intercept 0.05% Tween-20 as follows: anti-ATP5A1 (Abcam; ab14748; 1:1,000), anti-citrate synthase (Abcam, ab96600; 1:1,000), anti-GAPDH (Thermo, AM4300; 1:5,000), anti-HSPE1 (Thermo, PA5-30428; 1:1,000), anti-NDUFB8 (Abcam, ab110242; 1:1,000), anti-NDUFS1 (Abcam, ab169540; 1:1,000), anti-PRDX3 (Abcam, ab73349; 1:1,000) and anti-SDHB (Abcam, ab14714; 1:1,000). Primary antibodies were washed with TBS-T (0.05% Tween-20) and membranes were incubated in the secondary antibodies anti-mouse IgG DyLight 680 (Thermo, 35518; 1:10,000) or anti-rabbit IgG DyLight 800 4× PEG (Thermo, SA5-35571; 1:10,000). After washing, membranes were imaged by a near-infrared imaging system (Odyssey LI-COR) with Image Studio software (Li-COR, v5.2; Supplementary Figs. 1 and 2). Densitometric analysis of immunoblotting images was performed using Fiji software.
BN-PAGE gels were transferred to polyvinylidene fluoride (PVDF) membranes using a Mini Trans-Blot Cell (Bio-Rad). After wet transfer, polyvinylidene fluoride (PVDF) membranes were destained in methanol, blocked and incubated with antibodies against NDUFS1 (Abcam, ab169540; 1:1,000) and ATP5A1 (Abcam, ab14748; 1:1,000) for complex I and complex V immunodetection, respectively (Supplementary Fig. 2a).
RNA from early oocytes and spin-crashed yolk-free late-stage oocyte lysates was extracted using TRI reagent (Sigma, T9424) followed by RNeasy and Oligotex mRNA column (Qiagen) purification, following the manufacturer’s instructions. cDNA was synthesized with a First Strand cDNA synthesis kit (Thermo, K1612). Quantitative real-time PCR was performed using SYBR Green I Master (Roche, 04887352001) in a LightCycler 480 with LightCycler software v1.5.1 (Roche); with the following pairs of primers: ndufs1 forward: 5′-GGTGCGGTATGATGATGTGG-3′, reverse: 5′-ACAGCTTTCACACACTTGGC-3′; ndufs5 forward: 5′-GTCCGAAAGTTGTGCAGTCA-3′, reverse: 5′-CGGATCTGCCCAATTCCATG-3′; ndufv2 forward: 5′-GCATACAATGGAGCAGGTGG-3′, reverse: 5′-CATCCATGCTGTCTCTGTGC-3′; mt-nd3 forward: 5′-ATTTGATCCTCTGGGCTCTG-3′, reverse: 5′-AGCGCAATCTCTAGGTCAAA-3′; mt-nd5 forward: 5′-GGTCATCCACGATCAAATCCA-3′, reverse: 5′-ACCGAAACGATAATAGCTGCC-3′; gapdh forward: 5′-AGTTATCCCTGAGCTGAACG-3′, reverse: 5′-CTGATGCAGTCTTAATGGCG-3′; mt-rnr2 forward: 5′-ACTACCCGAAACTAAGCGAG-3′, reverse: 5′-ATCTTCCCACTCTTTTGCCA-3′. Nuclear-DNA-encoded genes were normalized to gapdh levels and mitochondrial-DNA-encoded genes were normalized to mt-rnr2.
Measurement of FMN and glutathione
Samples were prepared using the automated MicroLab STAR system from Hamilton Company in the presence of recovery standard for quality control by Metabolon. After protein precipitation in methanol, metabolites were extracted and analysed by ultrahigh-performance liquid chromatography with tandem mass spectrometry by negative ionization. Raw data were extracted, peak-identified and processed for quality control using Metabolon’s hardware and software.
Immunostaining paraffin ovary sections
Human and frog ovaries were fixed in 4% PFA in PBS overnight at 4 °C, washed, embedded in paraffin blocks and cut into 5 µM sections. After deparaffinization, antigen retrieval was performed by heating the slides for 15 min in 10 mM sodium citrate at pH 6. Sections were blocked and permeabilized in 3% BSA, 0.05% Tween-20 and 0.05% Triton X-100 for 1 h at room temperature. Sections were incubated overnight at 4 °C in the presence of primary antibodies (1:100): anti-ATP5A1 (Abcam, ab14748) and anti-HSPE1 (Thermo, PA5-30428); then 2 h at room temperature with secondary antibodies (1:500). Antibodies and dyes used were as follows: goat anti-rabbit Alexa488 or Alexa555 (1:500, Thermo, A-11008, A-21428), goat anti-mouse Alexa647 (Thermo, A21236) and Hoechst dye (1:500, Abcam, ab145597). A droplet of mounting medium (Agilent, S302380) was added onto the section before imaging using the LAS X software (Leica, v126.96.36.19976) in a Leica TCS SP8 microscope equipped with 40× (NA 1.30, Leica 506358) and 63× (NA 1.40, Leica 506350) objectives.
Statistics and reproducibility
Sample sizes were chosen based on published studies to ensure reliable statistical testing and to account for variability among outbred populations. Experimental limitations were also taken into account, such as the number of primordial oocytes that could be obtained from human ovaries. All experiments were performed on isolated oocytes or tissues. Sample randomization was performed by two means. First, all outbred frogs used in this study were chosen by blinded animal facility personnel without our knowledge. Second, all isolated oocytes or tissue samples were first grouped together and then randomly distributed to different experimental groups. Blinding during data collection was not required as standard experimental procedures were applied for different groups, such as western blots and immunohistochemistry. Blinding during data analysis was performed in oocyte survival experiments by involving multiple lab members for analysing blinded datasets. Blinding for the analysis of other experiments was not required since the different experimental groups were analysed using the same parameters. All data are expressed as mean ± s.e.m. A simple linear regression was performed to fit a model between the mitochondrial protein abundances of primordial follicle and ovarian somatic cell samples (Fig. 3d,e). Unpaired two-tailed Student’s t-test was used in all other analysis, P values are specified in figure legends, and those <0.05 were considered significant. Multiple t-tests were used in Figs. 1d, 4c and 5b,c and Extended Data Figs. 2c,d, 3k,l and 6b, and were corrected by the Šidák–Bonferroni method using GraphPad Prism. In Xenopus proteomics experiments, q values were calculated as adjusted P values and significance was considered for q value < 0.05 for comparing protein levels. A fold-change heatmap was generated using JMP (version 13.2) software. For Extended Data Fig. 6f, we excised the indicated bands in Extended Data Fig. 6e from one of three gels represented in Fig. 4a; gel-identification MS was performed once.
For isobaric-tag-based quantification for Xenopus, mitochondrial extracts from early (stage I) oocytes, late (stage VI) oocytes, gastrocnemius muscle, heart, liver and white adipose tissues were processed in two parallel experiments: stage I, stage VI and muscle in triplicates; and stage I, heart, liver and white adipose tissue in duplicates. Samples were quantified and 100 μg of each sample was processed with slight modifications from ref. 46. In brief, methanol-precipitated proteins were dissolved in 6 M guanidine hydrochloride (GuaCl). Samples were then digested with LysC (20 ng µl−1) in 2 M GuaCl overnight at room temperature. The next morning, samples were further diluted to 0.5 M GuaCl and digested with trypsin (10 ng µl−1) and further LysC (20 ng µl−1) for 8 h at 37 °C. Later, samples were speed-vacuumed, and the resulting pellet was resuspended in 200 mM EPPS pH 8.0. Ten-microlitre volumes of tandem mass tag (TMT) stock solutions (20 µg µl−1 in acetonitrile) were added to 50 μl of samples, and samples were incubated 3 h at room temperature. The TMT reaction was quenched with a 0.5% final concentration of hydroxylamine. The samples were combined in one tube, acidified by 10% phosphoric acid, and subjected to a MacroSpin C18 solid-phase extraction (The Nest Group) to desalt and isolate peptides. TMT mixes were fractionated using basic pH reversed-phase fractionation in an Agilent 1200 system. Fractions were desalted with a MicroSpin C18 column (The Nest Group) and dried by vacuum centrifugation50.
For label-free proteomics for human oocytes, human primordial follicles and ovarian somatic cells were collected from two individuals who underwent ovarian surgery. Samples were dissolved in 6 M GuaCl pH 8.5, diluted to 2 M GuaCl and digested with LysC (10 ng µl−1) overnight. Samples were further diluted down to 0.5 M GuaCl and digested with LysC (10 ng µl−1) and trypsin (5 ng µl−1) for 8 h at 37 °C. Samples were acidified by 5% formic acid and desalted with home-made C18 columns.
For detection of complex I and II subunits from BN-PAGE gels, gel bands were destained, reduced with dithiothreitol, alkylated with iodoacetamide and dehydrated with acetonitrile for trypsin digestion. After digestion, peptide mix was acidified with formic acid before analysis through liquid chromatography with MS/MS.
Chromatographic and MS analysis
TMT and label-free samples were analysed using a Orbitrap Eclipse mass spectrometer (Thermo) coupled to an EASY-nLC 1200 (Thermo). Peptides were separated on a 50-cm C18 column (Thermo) with a gradient from 4% to 32% acetonitrile in 90 min. Data acquisition for TMT samples was performed using a Real Time Search MS3 method51. The scan sequence began with an MS1 spectrum in the Orbitrap. In each cycle of data-dependent acquisition analysis, following each survey scan, the most intense ions were selected for fragmentation. Fragment ion spectra were produced through collision-induced dissociation at a normalized collision energy of 35% and they were acquired in the ion trap mass analyser. MS2 spectra were searched in real time with data acquisition using the PHROG database52 with added mitochondrially encoded proteins. Identified MS2 spectra triggered the submission of MS3 spectra that were collected using the multinotch MS3-based TMT method53.
Label-free samples were acquired in data-dependent acquisition mode and full MS scans were acquired in the Orbitrap. In each cycle of data-dependent acquisition analysis, the most intense ions were selected for fragmentation. Fragment ion spectra were produced through high-energy collision dissociation at a normalized collision energy of 28%, and they were acquired in the ion trap mass analyser.
Gel bands were analysed using a LTQ-Orbitrap Velos Pro mass spectrometer (Thermo) coupled to an EASY-nLC 1000 (Thermo). Peptides were separated on a 25-cm C18 column (Nikkyo Technos) with a gradient from 7% to 35% acetonitrile in 60 min. The acquisition was performed in data-dependent acquisition mode and full MS scans were acquired in the Orbitrap. In each cycle, the top 20 most intense ions were selected for fragmentation. Fragment ion spectra were produced through collision-induced dissociation at a normalized collision energy of 35%, and they were acquired in the ion trap mass analyser.
Digested bovine serum albumin was analysed between each sample and QCloud (ref. 54) was used to control instrument performance.
Acquired spectra were analysed using the Proteome Discoverer software suite (v2.3, Thermo) and the Mascot search engine (v2.6, Matrix Science55). Label-free data were searched against the SwissProt Human database. Data from the gel bands were searched against a custom PHROG database52 that includes 13 further entries that correspond to mitochondrially encoded proteins for the Xenopus samples and the SwissProt mouse database for the mouse samples. TMT data were searched against the same custom ‘PHROG’ database. False discovery rate in peptide identification was set to a maximum of 5%. Peptide quantification data for the gel bands and the label-free experiments were retrieved from the ‘Precursor ion area detector’ node. The obtained values were used to calculate an estimation of protein amount with the top3 area, which is the average peak area of the three most abundant peptides for a given protein. For the TMT data, peptides were quantified using the reporter ion intensities in MS3. Reporter ion intensities were adjusted to correct for the isotopic impurities of the different TMT reagents according to the manufacturer’s specifications. For final analysis, values were transferred to Excel. For all experiments, identified proteins were selected as mitochondrial if they were found in MitoCarta 3.0 (ref. 56). MS3 spectra with abundance less than 100 or proteins with fewer than 2 unique peptides were excluded from the analysis. Each TMT channel was normalized to total mitochondrial protein abundance. A total of 926 mitochondrial proteins were identified (and 807 quantified) in 3 biological replicates from wild-type outbred animals, representing 80% of known mitochondrial proteins (Supplementary Table 1 and Extended Data Fig. 3b). Although the mitochondrial proteome in diverse cell types could be quite different57, we found comparable levels of mitochondrial housekeeping proteins (such as the import complexes TIMMs and TOMMs) across different maturity stages (Extended Data Fig. 3c and Supplementary Table 1), enabling us to compare and contrast changes in other pathways.
For human somatic cell samples, we analysed three dilutions: the 1× reference had a similar level of protein loading to that of the primordial follicle sample (0.55 µg total protein); a twofold dilution (0.25 µg total protein); and a fivefold dilution (0.1 µg total protein). In scatter plots (Fig. 3d,e), we estimated differences in mitochondrial complex I protein abundance using the twofold somatic cell dilution, a conservative approach that compared primordial follicle samples (0.55 µg total protein) to somatic cells half their loading concentration (0.25 µg total protein), nevertheless observing similar levels of the mitochondrial import machinery subunits TOMMs and TIMMs. The fivefold-dilution somatic cell sample was useful for establishing detection limits; indeed, many complex I subunits absent in oocytes were detected with high confidence even at this dilution. In the heatmap (Extended Data Fig. 5), we considered normalizing our data using the mitochondrial loading controls citrate synthase and COX4I1 to estimate differences in protein abundance. The abundance of COX4I1 fell within the linear range of our proteomic methodology (R2 = 0.99), in contrast to that for citrate synthase (R2 = 0.89) whose higher abundance led to measurement saturation at higher concentrations. Therefore, COX4I1 was chosen to normalize protein abundances in the heatmap representation. We identified 454 mitochondrial proteins (Supplementary Table 3; 298 and 397 proteins were quantified for early oocyte and somatic cell samples, respectively), representing 40% of all known mitochondrial proteins. Here too, levels of the mitochondrial import proteins TIMMs and TOMMs were similar between oocytes and ovarian somatic cells (Fig. 3d,e), demonstrating an equivalent mitochondrial abundance that facilitated comparison of protein levels between different cell types.
Further information on research design is available in the Nature Research Reporting Summary linked to this paper.
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