Distinct requirements for energy in mouse primordial germ cells and

their reprogramming to embryonic germ cells

Yohei Hayashi, Kei Otsuka, Masayuki Ebina, Kaori Igarashi, Asuka Takehara, Mitsuyo Matsumoto,

Akio Kanai, Kazuhiko Igarashi, Tomoyoshi Soga, and Yasuhisa Matsui

Supporting Appendix

1 www.pnas.org/cgi/doi/10.1073/pnas.1620915114

SI Materials and Methods

Data reporting. No statistical methods were used to predetermine sample size. The experiments were not randomized. The investigators were not blinded to allocation during experiments and outcome assessment.

Animals. MCH and C57BL/6 mice were purchased from Japan SLC. Oct4-deltaPE-GFP transgenic mice (1) were maintained in a C57BL/6J genetic background. The mice were kept and bred in an environmentally controlled and specific pathogen-free facility, the Animal Unit of the Institute of

Development, Aging and Cancer (Tohoku University), according to the guidelines for experimental animals defined by the facility. Animal protocols were reviewed and approved by the Tohoku

University Animal Studies Committee. Noon on the day of the plug was defined as embryonic day

(E) 0.5. E13.5, E12.5 and E11.5 embryos were obtained from female MCH mice mated with male

Oct4-deltaPE-GFP transgenic mice. Embryos were collected and dissected in Dulbecco’s modified

Eagle medium (DMEM, Gibco 11965-092) containing 10% fetal bovine serum (FBS). The genital ridges of male embryos were dissected.

Flow cytometry. The genital ridges containing PGCs from Oct4-deltaPE-GFP transgenic mice, prepared as described above, were incubated with 1.2 mg/ml collagenase (SIGMA C0130) in PBS containing 10 % FBS for 1 h at 37 oC. To prepare single-cell suspensions for flow cytometry, cells within the samples were dissociated by pipetting, and samples were filtered through a 40 µm pore nylon mesh (BD falcon 352340). A Bio-Rad S3e cell sorter was used to sort and collect viable PGCs with intense Oct4-deltaPE-GFP expression (~ 1 × 105 cells/sorting) and Somas without Oct4- deltaPE-GFP expression. It takes about 30 minutes for a sorting and we have checked the high survival rate (> 93 %) of each cell type immediately after sorting (Fig. S1A). For the metabolomic analysis, sorted cells were immediately treated for metabolite extraction as described below. For the proteomic analyses, sorted cells were washed with PBS, removed supernatant and stored at -80 oC. 2

Cells from ~5 times sorting were suspended to Cell lysis buffer for whole cell extract [20 mM

HEPES (pH = 7.9), 10 % Glycerol, 400 mM KCl, 1 mM EDTA, 1 mM MgCl2, 0.1 % NP-40, 0.5 mM DTT, and 1 × protease inhibitor cocktail (Roche 04 693 132 001)].

ESC culture. Vasa-RFP (VR15) ESCs (2, 3) were cultured in KnockOut DMEM (Gibco 10829-018) supplemented with 15 % FBS, 4 mM L- (Gibco 25030081), 0.01 mM nonessential amino acids (Gibco 11140-050), 0.1 mM b-mercaptoethanol (SIGMA M3148), 1,000 U/ml LIF (ESGRO

Millipore ESG1107) on mouse embryonic fibroblasts inactivated with mitomycin C (SIGMA

M4287). A Bio-Rad S3e cell sorter was used to sort and collect viable VR15 ESCs after 3 days in culture.

Metabolite extraction. The sorted E13.5 male PGCs, Somas and VR15 ESCs (sorted, see “ESC culture”) (~ 1 × 105 cells / sample) were washed twice with 5 % mannitol. Add 1 ml of MeOH containing 2.5 µM each of three IS1s [L-methionine sulfone (Wako 502-76641), 2-(N- morpholino)ethanesulfonic acid (MES, Dojindo 349-01623), D-camphor-10-sulfonic acid (CSA,

Wako 037-01032)]. Leave at rest for 10 min, vortex, and transfer 400 µl to new tube. Add 400 µl of

o CHCl3 and 200 µl of Milli-Q water and mix well. Centrifuge at 10,000 g for 3 min at 4 C, and transfer 400 µl of aqueous layer to an HMT 5 kDa ultrafiltration tube [UltrafreeMC-PLHCC 250 / pk for Metabolome Analysis (UFC3LCCNB-HMT)]. Centrifuge at 9,100 g for 2 h at 20 oC, collect its filtrate and store at -80 oC. Put together the filtrated cell extract from approximately 5 x 105 cells for one specimen of each cell type and dry them using an evacuated centrifuge for 2 h at 40 oC. Add 25

µl of Milli-Q water containing 200 µM each of two IS2s [3-aminopyrrolidine (Aldrich 404624) and trimesate (Wako 206-03641)] for CE-MS analysis. Collected three specimens were then analyzed as three biological replicates.

3

Mass spectrometry for metabolome. The concentrations of all the charged metabolites in samples were measured by capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS, Agilent

Technologies, Santa Clara, CA) using the methods developed by the authors (4-6). Briefly, to analyze cationic compounds, a fused silica capillary (50 µm i.d. × 97 cm) was used with 1 M formic acid as the electrolyte (7). Methanol/water (50% v/v) containing 0.1 µM hexakis(2,2- difluoroethoxy)phosphazene was delivered as the sheath liquid at 10 µl/min. ESI-TOFMS was performed in positive ion mode, and the capillary voltage was set at 4 kV. Automatic recalibration of each acquired spectrum was achieved using the masses of the reference standards ([13C isotopic ion of a protonated methanol dimer (2MeOH+H)]+, m/z 66.0632) and ([hexakis(2,2- difluoroethoxy)phosphazene +H]+, m/z 622.0290). To identify metabolites, relative migration times of all peaks were calculated by normalization to the reference compound 3-aminopyrrolidine. The metabolites were identified by comparing their m/z values and relative migration times to the metabolite standards. Quantification was performed by comparing their peak areas to calibration curves generated using internal standardization techniques with L-methionine sulfone. The other conditions were identical to those described previously (5). To analyze anionic metabolites, a commercially available COSMO(+) (chemically coated with cationic polymer) capillary (50 µm i.d. x 105 cm) (Nacalai Tesque, Kyoto, Japan) was used with a 50 mM ammonium acetate solution (pH

8.5) as the electrolyte. Methanol/5 mM ammonium acetate (50% v/v) containing 0.1 µM hexakis(2,2-difluoroethoxy)phosphazene was delivered as the sheath liquid at 10 µl/min. ESI-

TOFMS was performed in negative ion mode, and the capillary voltage was set at 3.5 kV. For anion analysis, trimesate and CAS were used as the reference and the internal standards, respectively. The other conditions were identical to those described previously (5).

In-Solution Digestion for proteome. Whole cell extracts (5 µg, 3 biological replicates) were diluted over 10-folds with 50 mM NH4HCO3 to final volume of 90 µl. Subsequently, 15 µl of 100 mM DTT

4

(in water) was added followed by incubation for 30 min at 56 oC. Reduced cysteine residues were alkylated by adding 15 µl of 200 mM iodoacetamide (in water) and incubation for 30 min at room temperature in the dark. For in-solution digestion, 1 µg of trypsin (Promega) was added, and samples were incubated overnight at 37 oC. The digest reaction was stopped by adding 3 µl of TFA. Digested peptides were purified with C18 Spin Columns (Thermo Fisher Scientific), dried through vacuum centrifugation and dissolved in 50 µl loading solution [5 % acetonitrile contained 0.5 % TFA].

NanoLC-MS/MS analysis for proteome. Tryptic peptides (10 µl) were loaded on an Easy-nLC

1000 system (Thermo Fisher Scientific) connected with reversed phase C18 columns (Trap column:

Acclaim PepMap 100, 75 µm × 20 mm, Separation column: PepMap RSLC, 75 µm × 250 mm;

Thermo Fisher Scientific). Peptides were eluted with gradient generated by solvent A (0.1 % formic acid in water) and solvent B (0.1 % formic acid in acetonitrile) as followed: 5-23 % B in 180 min at a flow rate of 150 nl/min, 35-90 % B in 5 min at a flow rate of 175 nl/min, maintained at 90 % B in 5 min at a flow rate of 200 nl/min. Peptides were then ionized and analyzed with Orbitrap Elite

(Thermo Fisher Scientific). Full scan MS spectra (from m/z 350 to 2000) were acquired in the

Orbitrap with a resolution 60,000 at m/z 400 with using lock mass option (m/z at 391.284290 and

445.120030), followed by MS/MS fragmentation in the linear ion trap with normalized collision energy of 30 % against 20 most intense ions with +2 or more positive charges. Precursor ions selected for fragmentation once were excluded from selection for 30 s.

Data Processing for proteome. MS/MS data were analyzed with Proteome Discoverer 1.4 (Mascot and Sequest HT) according to manufacturer’s instruction and searched against mouse database for protein identification. For semi-quantification of each protein, the node ‘Precursor Ions

Area Detector’ was used and calculated area values of each protein peak were compared among

E13.5 male PGCs, Somas and ESCs (Dataset S2). Up to two missed cleavages were allowed.

Precursor and fragment mass tolerance were set to 10 ppm and 0.4 Da, respectively. Variable 5

modifications were oxidation of methionine and deamination of asparagine or glutamine, Static modification was carbamidomethylation of cysteine. The obtained sequences were filtered and validated taking into account false discovery rate (FDR) < 5 %.

Analyses of metabolomic and proteomic data. We used MetaboAnalyst 3.0

(http://www.metaboanalyst.ca/MetaboAnalyst/faces/home.xhtml) for statistical analyses of metabolomic and proteomic data (8). In data processing, features with > 50% missing values were removed and remaining missing values were replaced by a half of the minimum positive value in the original data (default configuration). The processed data were normalized using auto scaling method

(mean-centered and divided by the standard deviation of each variable). Statistical differences were calculated using Student’s t-test or one way ANOVA. P < 0.05 was considered as statistically significant differences. The statistically-different features were then classified with K-mean clustering method. Each protein cluster was functionally annotated using the Database for

Annotation, Visualization, and Integrated Discovery (DAVID, https://david.ncifcrf.gov/,

Classification stringency: medium) (9).

Metabolic flux analysis. Seahorse XF24 Analyzer was used to measure consumption rate and extracellular acidification rate of E13.5 male and female PGCs and Somas, E11.5 PGCs, VR15

ESCs and PGCLCs on day4 in culture. The sorted cells were resuspended with DMEM containing 10

o % FBS and 1 mM sodium pyruvate, and incubated for about 1 h at 37 C, 5 % CO2. Centrifuge at

1,000 rpm for 5 min at 4 oC and remove supernatant. Resuspend the cells with XF running medium

[XF Base Medium (Seahorse Bioscience 102353-100), 25 mM glucose, 2 mM L-glutamine and 1 mM sodium pyruvate (Gibco 11360-070)]. Cells were plated in XF24 Cell Culture Microplates

(Seahorse Bioscience 100777-004) at a density of 8 - 24 x 104 cells per well and incubated for more than 30 min at 37 oC. Cells were treated with 0.5 µM oligomycin (an ATP synthase inhibitor,

Alomone labs O-500), 1 µM FCCP (an uncoupler of OXPHOS, SIGMA C2920) and 1 µM rotenone 6

(SIGMA R8875) + 1 µM antimycin (SIGMA A8764) (known ETC inhibitors) and measured following the manufacturer’s instruction (Table S3).

Immunostaining. Immunostaining were performed using genital ridges (E13.5 male, female) from

Oct4-deltaPE-GFP transgenic mice fixed with 2 % paraformaldehyde for 3 h at 4 oC and embedded with Optimum Cutting Temperature (O.C.T.) compound (Sakura Finetek 4583). The embedded samples were sectioned using cryomicrotome CM3050S (Leica) with a section thickness of 10 µm.

The sectioned samples were permeabilized in 1 % Triton X-100 in PBS for 15 min at room temperature, and blocked in 5 % bovine serum albumin (BSA) and 1 % Triton X-100 in PBS for 1 h at room temperature. The sections were then incubated with the primary antibodies diluted by 1 %

BSA and 0.1 % Triton X-100 in PBS overnight at 4 oC, and were incubated with the secondary antibodies in the same buffer with 1 µg/ml DAPI for 2 h at 4 oC. Samples were washed for 5 min × 3 by 0.1 % Triton X-100 in PBS after the primary and the secondary antibody treatments. Samples were mounted with VECTASHIELD (VECTOR H-1000) and observed with confocal laser scan microscope TCS SP8 (Leica). PGCs were detected as Oct4-deltaPE-GFP-positive cells. The Alexa-

568 fluorescence intensities of each single cell were measured from the obtained image using the histogram option of LAS X software (Leica) and average intensities of PGCs and Somas were calculated. The primary antibodies were: NDUFB9 (abcam ab200198, 1:250), ATP5I (mouse

ATP5K, Proteintech 16483-1-AP, 1:50) and GSR (abcam ab16801, 1:1000). Goat anti-rabbit secondary antibody, Alexa Fluor 568 (Thermo Fischer Scientific A11011) was used at 1:500 dilution.

Culture of PGCs. PGC culture for reprogramming was carried out as described previously (10) with some modifications. The sorted E12.5 PGCs were cultured on a feeder layer of Sl/Sl4-m220 cells

(10) pre-treated with mitomycin C in 4-well tissue culture dishes with EG medium, which was based on the previously reported germline stem cell (GSC) culture medium (11) with modifications

[StemPro34 SFM (Gibco 10640-019) containing StemPro34 Nutrient, 100 µg/ml transferrin (SIGMA 7

T8158), 2 mM L-glutamine, 25 µg/ml insulin (SIGMA I2643), 50 µM b-mercaptoethanol, 20 ng/ml

EGF (SIGMA E4127), 25 ng/ml bFGF (SIGMA F0291), 1,000 U/ml LIF, 100 U/ml penicillin-streptomycin (SIGMA P7359) and 10 % knockout serum replacement (KSR, Gibco

10828-028)]. In some experiments, 1 mM 2-deoxy-D-glucose (2DG, SIGMA D8375) or 0.12 µM rotenone was added. In the experiment for examining glucose-concentration dependency, glucose- free DMEM (Gibco A14430-01) was used instead of StemPro34. After 8-9 days in culture, staining for alkaline phosphatase activity was used to identify EGC colonies, as described previously (12, 13).

The efficiency of EGC formation was determined as a ratio of the number of EGC colonies to every

100 sorted PGCs that were seeded in a culture well. The survival rate of E12.5 PGCs was determined by counting the Oct4-deltaPE-GFP or ALP-positive cells. TdT-mediated dUTP-X nick end labeling

(TUNEL) staining for apoptosis detection in E12.5 PGCs was performed using In Situ Cell Death

Detection Kit, TMR red ver. 11 (Roche 12 156 792 910) according to manufacturer’s instruction after 16 h in culture. Feeder-free EG derivation was performed as reported previously (14). E11.5

PGCs were sorted and cultured for 48 h on 20 µg/ml fibronectin-coated (human plasma fibronectin,

Millipore FC010) 4-well tissue culture dishes in N2B27 medium with 3 µM CHIR99021 (CH, a

GSK3 inhibitor), 1,000 U/ml LIF, 25 nM human bFGF (SIGMA F0291), 2 µM all-trans retinoic acid

(SIGMA R2625), 10 µM forskolin (SIGMA F6886), and 100 ng/ml SCF (R&D 455-MC-010). After

48 h, cultures were transfered to N2B27 with 2i/LIF (3 µM CH, 1 µM PD325901 and 1,000 U/ml

LIF) by daily half-medium changes and GFP-positive colonies were counted on day 7~8.

PGCLC induction. PGCLC induction was performed as described previously (15). In this system, it was used ESCs with the reporter transgenes inserted fluorescent protein in the regulatory regions of PGC-specific genes, Prdm1 (also known as Blimp1) and Developmental Pluripotency

Associated 3 (Dppa3, also known as Stella) (Blimp1-mVenus-Stella-ECFP: BVSC), which were kindly provided from Dr. Mitinori Saitou. 2,000 (for metabolic perturbation) or 20,000 (for flux

8

analyzer) cells of EpiLCs were cultured in a 96U bottom plate (Thermo Fisher Scientific 174925) in

Glasgow’s Modified Eagle’s Medium (GMEM, Gibco 11710-035) supplemented with 15 % KSR,

0.1 mM nonessential , 1 mM sodium pyruvate, 2 mM L-glutamine, 180 µM b- mercaptoethanol with or without cytokines [500 ng/ml BMP4 (R&D 314-BP-050), 500 ng/ml

BMP8a (R&D 1073-BP-010), 100 ng/ml SCF (R&D 455-MC-010), 1,000 U/ml LIF and 50 ng/ml

EGF]. In some experiments, 1 mM 2DG or 0.03 µM rotenone was added. In the experiment for examining glucose-concentration dependency, glucose-free DMEM was used instead of GMEM.

Induced BV-positive PGCLCs were sorted on day 2 or day 4 post induction with a Bio-Rad S3e cell sorter and suspended to buffer RLT containing 1 % b-mercaptoethanol for total RNA extraction.

RNA preparation and reverse real-time PCR. Total RNA samples were purified using RNeasy Micro Kit (QIAGEN 74004) according to the manufacturer’s instruction. RNAs were reverse-transcribed using SuperScript III (Invitrogen 18080044) and random primers (Promega

C118A). Expression levels of genes were quantified using the SYBR Green Master Mix (Applied

Biosystems 4367659) with the primers shown in Table S4. PCR signals were detected using CFX

Connect (Bio-Rad). Arbp was used as an internal control.

Statistical Analysis. All data except for Fig. S10 are expressed as mean ± SEM. Data in Fig. S10 are expressed as box plots with whiskers from minimum to maximum, in which the band inside boxes shows median. The significance of difference was assessed by the unpaired Student’s t test, one-way

ANOVA or Mann–Whitney’s U test. The level of significance was set at P < 0.05.

Data availability. Metabolomic and proteomic data are available as Supporting Information. The microarray data of PGCs analyzed in the previous study (16) was used (GEO accession: GSE40412).

9

A N.S. N.S. N.S. 100

80

60

40

Survivalrate (%) 20

0 PGC Soma VR15 B C D

Soma_3 M1 Soma_2 PGC Soma_1 Normalized ESC PGC_3 M2 signal PGC_1 3 PGC_2 0 PC2(21.8%) ESC_2 -2 Soma ESC_1 M3 ESC_3

PC1 (53.7%) M4 ESC_2 ESC_3 ESC_1 PGC_3 PGC_2 PGC_1 Soma_1 Soma_2 Soma_3

Metabolomic analysis

E F G Soma_2 P1 PGC Soma_1 Soma_3 P2 PGC_2 Normalized signal Soma PGC_1 P3 PGC_3 4 0 PC2(20.8%) ESC_3 P4 -4 ESC ESC_2 ESC_1 P5

PC1 (55.7%) P6 ESC_3 ESC_1 ESC_2 PGC_3 PGC_1 PGC_2 Soma_3 Soma_1 Soma_2

Proteomic analysis

Fig. S1. The profile of metabolomic and proteomic analyses. (A) The survival rates of E13.5 male PGCs, Somas and VR15 ESCs used for metabolomic and proteomic analyses immediately after cell sorting. Values are plotted as mean ± standard error of the mean (SEM) of 3 biological replicates, N.S.: not significant (Student’s t-test). (B and E) Principal component analysis (PCA) of metabolomic (B) and proteomic (E) profiles of E13.5 male PGCs, Somas, and ESCs (3 biological replicates). (C and F) Hierarchical clustering of metabolomic (C) and proteomic (F) profiles of each cell type (clustering distance: Pearson, clustering method: Ward, 3 biological replicates). (D and G) K-mean clustering of statistically different metabolites (D, K = 4) and (G, K = 6) (ANOVA, P < 0.05, 3 biological replicates).

10 A cluster KEGG ID Compound name cluster KEGG ID Compound name C00051 (GSH) C01620 Threonic acid C00127 Oxidized glutathione (GSSG) C00246 Butyric acid C00029 Uridine diphosphate(UDP)-glucose C00077 Ornithine C00063 Cytidine triphosphate (CTP) C00183 L-Valine (Val) C01042 N-Acetyl-L-aspartic acid C05382 Sedoheptulose 7-phosphate (S7P) Cluster C00300 C00064 L-Glutamine (Gln) M1 C00588 Phosphorylcholine C00062 L-Arginine (Arg) PGC- C00346 O-Phosphoethanolamine C00318 L- specific C00114 C00047 L-Lysine (Lys) C00134 Putrescine C00093 Glycerol 3-phosphate (G3P) C01181 gamma-Butyrobetaine Cluster C00002 (ATP) C00044 Guanosine triphosphate (GTP) M3 C00078 L-Tryptophan (Trp) C00791 Creatinine PGC > C00049 L-Aspartic acid (Asp) C02614 Citramalate Soma > C00079 L-Phenylalanine (Phe) C00160 Glycolic acid ESC C00082 L-Tyrosine (Tyr) C00186 L-Lactic acid C00065 L-Serine (Ser) C06104 Adipic acid C00152 L-Asparagine (Asn) - 2-Hydroxyoctanoate C00135 L-Histidine (His) - 2-Hydroxyisobutyrate C00123 L-Leucine (Leu) C00489 Glutaric acid C00092 Glucose 6-phosphate (G6P) C00327 Citrulline C00148 L-Proline (Pro) Cluster C00197 3-Phospho-D-glycerate (3PG) C00085 Fructose 6-phosphate (F6P) M2 C05629 Phenylpropanoate C02571 L- Soma > C01005 O-Phospho-L-serine C00245 PGC > C01089 (R)-3-Hydroxybutyric acid C00073 L-Methionine (Met) ESC C00117 D-Ribose 5-phosphate (R5P) C00043 Uridine diphosphate(UDP)-N-acetylglucosamine Cluster C02129 5-Oxohexanoic acid C00334 Gamma-Aminobutyric acid (GABA) M4 - 4-Oxopentanoate C00103 Glucose 1-phosphate (G1P) ESC- C06337 Terephthalic acid C00354 Fructose 1,6-bisphosphate (F1,6P) specific C02678 Dodecanedioic acid C00111 Dihydroxyacetone phosphate (DHAP) C08277 Sebacic acid -related C01013 Hydroxypropionic acid synthesis-related C05984 2-Hydroxybutyric acid Amino acid-related C02656 Pimelic acid Glutathiones B Cluster P1 (ESC-specific) Cluster P4 (PGC>ESC>Soma)

Glycolysis/ etc. Nucleotide/ATP-binding etc. Nucleotide/ATP-binding etc. DNA repair etc. etc. Purine biosynthesis etc. 0 2 4 6 8 10 12 PPIase cyclophilin-type etc. Cluster P2 (ESC>PGC>Soma) Translation initiation etc.

Nucleotide/ATP-binding etc. 0 2 4 6 8 Carbon metabolism etc. Cluster P5 (PGC-specific) Aminoacyl-tRNA synthetase etc. RNA Helicase etc. etc. Hydlorase/Protease etc. 0 2 4 6 8 10 12 14 16 Oxidoreductase etc. Cluster P3 (PGC>Soma>ESC) /poly(A) binding etc. Glutathione S- etc. NAD etc. Oxidoreductase etc. 0 2 4 6 8 10 12 Fatty acid metabolism etc. Enrichment score biosynthesis etc. Mitochondrion etc.

0 2 4 6 Enrichment score Fig. S2. Metabolomic and proteomic profiling of E13.5 male PGCs, gonadal somatic cells (Somas), and ESCs. (A) A list of the metabolites included in each cluster classified with K-mean clustering (K = 4) (Fig. S1D). The amounts of metabolites per cell (fmol / cell; 3 biological replicates for each cell type) were obtained using capillary electrophoresis–mass spectrometry (CE-MS) and normalized with the volume of cells (nmol / mm3; Table S1 and Dataset S1). Abbreviations of metabolites are shown in this list. (B) Functional annotation of each protein cluster classified with K-mean clustering (K = 6) (Fig. S1G) using the Database for Annotation, Visualization, and Integrated Discovery (9). Experiments consisted of 3 biological replicates for each sample. 11 A Glucose HKs PGLS G6P RU5P G6PDX GPI1 Pentose phosphate pathway RPIA F6P S7P

PFKs FBPs R5P

F1,6P TALDO1 PRPSs

ALDOA PRPP PPAT DHAP GA3P TPI1 GAPDH Nucleotide PGKs synthesis 3PG ATP GTP PGAM1

ENOs CTP UTP PEP PKM

PGC > Soma, P < 0.05 Lactate Pyruvate PGC > Soma, P < 0.05, > 2 fold LDHA PGC < Soma, P < 0.05 PGC < Soma, P < 0.05, > 2 fold B Glycolysis

3PG Ser Gly Amino acids synthesis Pyruvate PDHs GSS PC DLD DLAT Asn Acetyl-CoA Glutathione metabolism GSR Asp CS GSH GSSG

OAA Citrate MDH1 ACO2 MDH2 GCLC Malate Cis-Aconitate GCLM FH1 ACO2

Fumarate TCA cycle Isocitrate IDH1 SDHs IDH3s IDH2 SDHD aKG Glu Succinate GLUD1 SUCLGs Succinyl-CoA OGDH SUCLA2 Mitochondrion OGDHL

Fig. S3. Integrated analysis of metabolomic and proteomic data between E13.5 male PGCs and Somas. (A and B) An integrated view of glycolysis-related (A) and TCA cycle-related (B) metabolic pathways comparing E13.5 male PGCs with Somas. Ellipses and rectangles show and metabolites, respectively. Orange and red indicate elements that are moderately or strongly (respectively) enriched in PGCs compared with Somas; light blue and blue indicate elements that are moderately or strongly (respectively) depleted in PGCs compared with Somas. Gray indicates elements that were undetected, or those that did not differ significantly between PGCs and Somas. For clarity, these figures exclude several metabolites and enzymes. 12 A B C Glycolysis TCA cycle OXPHOS

G6P F6P Citrate P = 0.21 NAD+ *** *** P = 0.23 P = 0.08 ** 0.6 ** 0.15 1.5 0.4 P = 0.15 3 3

3 0.3 0.4 0.10 1.0 0.2 0.2 0.05 0.5 nmol / mm / nmol

nmol / mm / nmol 0.1 nmol / mm / nmol

0.0 0.00 0.0 0.0 PGC Soma ESC PGC Soma ESC PGC Soma ESC PGC Soma ESC F1,6P DHAP Succinate * * * 2.0 1.0 4.0 P = 0.34 0.8 1.5 3 3 3.0 0.6 N.S. 1.0 2.0 N.S. 0.4 0.5 nmol / mm / nmol 1.0 nmol / mm / nmol 0.2 0.0 0.0 0.0 PGC Soma ESC PGC Soma ESC PGC Soma ESC GA3P 3PG Fumarate * P = 0.10 N.S. N.S. 0.10 0.20 3.0

3 0.15 3 2.0 0.05 0.10 1.0

nmol / mm / nmol 0.05

N.A. N.A. mm / nmol 0.00 0.00 0.0 PGC Soma ESC PGC Soma ESC PGC Soma ESC G3P G1P ** P = 0.06 * 0.3 0.8

3 0.6 0.2 N.S. 0.4 0.1 0.2 nmol / mm / nmol 0.0 0.0 PGC Soma ESC PGC Soma ESC

Fig. S4. Quantitative analyses of metabolites in energy metabolic pathways in E13.5 male PGCs, Somas, and ESCs. (A to C) The concentrations of metabolites involved in glycolysis (A), TCA cycle (B), and OXPHOS (C). Abbreviations are as shown in Fig. S2A. N.A.: not available. Values are plotted as mean ± SEM of 3 biological replicates. N.S.: not significant, *P < 0.05, **P < 0.01, ***P < 0.001 (Student’s t-test).

13 A Pentose phosphate pathway B NTPs

R5P S7P ATP GTP P = 0.16 ** 0.15 0.15 6 * * 8 ** * * 3

0.10 0.10 3 6 4 4 0.05 0.05 2

nmol / mm / nmol 2 nmol / mm / nmol 0.00 N.A. 0.00 0 0 PGC Soma ESC PGC Soma ESC PGC Soma ESC PGC Soma ESC N.S. C Amino acids CTP UTP P = 0.07 P = 0.06 Glu Gln 0.4 * 0.4 *

** 3 10 N.S. 0.3 0.3 6 **

3 8 0.2 0.2 6 4 0.1 0.1 nmol / mm / nmol 4 2 0.0 0.0 nmol / mm / nmol 2 PGC Soma ESC PGC Soma ESC 0 0 D Glutathiones PGC Soma ESC PGC Soma ESC GSSG * GSH Asp Asn *** P = 0.13 * ** 1.5 1.5 1.0 P = 0.11

10 ** 3

3 * 1.0 1.0

0.5 5 0.5 0.5 nmol / mm / nmol nmol / mm / nmol 0 0.0 0.0 0.0 PGC Soma ESC PGC Soma ESC PGC Soma ESC PGC Soma ESC E Ser Gly Epigenetic regulation *** P = 0.08 + 6 * 40 SAM N.S. P = 0.24 N.S. 3 30 0.15 4 3 20 0.10 2

nmol / mm / nmol 10 0.05

0 0 mm / nmol PGC Soma ESC PGC Soma ESC 0.00 PGC Soma ESC

Fig. S5. Quantitative analyses of metabolites in biomaterial synthetic pathways in E13.5 male PGCs, Somas, and ESCs. (A to E) The concentration of metabolites involved in the pentose phosphate pathway (A), nucleotide synthesis (B), amino acid synthesis (C), glutathione metabolism (D), and epigenetic regulation (E). Abbreviations are as shown in Fig. S2A. N.A.: not available. Values are plotted as mean ± SEM of 3 biological replicates. N.S.: not significant, *P < 0.05, **P < 0.01, ***P < 0.001 (Student’s t-test).

14 A E13.5 male genital ridge 1.5 P = 6.395E-13

DAPI GFP 1.0

0.5 Relative mean value mean Relative ATP5K Merge 0.0 PGC Soma n = 109 n = 66 B E13.5 male genital ridge

1.5 P = 4.581E-21

DAPI GFP 1.0

0.5 Relative mean value mean Relative GSR Merge 0.0 PGC Soma n = 60 n = 60 C E13.5 female genital ridge 1.5 P = 3.962E-17

DAPI GFP 1.0

0.5 Relative mean value mean Relative GSR Merge 0.0 PGC Soma n = 60 n = 60

Fig. S6. The expression of OXPHOS- and glutathione-related enzymes in E13.5 genital ridges, related to Fig. 3. (A) ATP5K immunostaining of E13.5 male genital ridges (left panel). The relative intensities of ATP5K fluorescence (right panel, PGC: n = 109, Soma: n = 66). (B and C) GSR immunostaining of E13.5 male (B) and female (C) genital ridges (left panel). The relative intensities of GSR fluorescence (right panel, n = 60). PGCs were detected as Oct4-deltaPE-GFP-positive cells. Experiments consisted of 2 biological replicates for each sample. Scale bar: 25 µm. Values are plotted as mean SEM (Student’s t-test).

15 A N.S. B C ** * 3.00 * 1.20 2.50 1.00 1.00)

= 2.00 0.80 mM 0.60 0 mM 2.5 mM 5.0 mM 1.50 0.40 (control = 1.00) = (control 1.00 Relative efficiency Relative 0.20 efficiency Relative 0.50 (Glucose 10 10 (Glucose 0.00 0.00 0 2.5 5.0 7.5 10 7.5 mM 10 mM Glucose conc. (mM) 2DG

D E F N.S. 1.40 1.0 * 1.20 ** * 40 0.8 35 1.00 30 0.80 PGC 25 0.6 * 0.60 DMSO 20 Soma 15 0.40 0.4

10 efficiency Relative 0.20 VR15 5 1.00) = (DMSO~72hr TUNEL positive (%) positive TUNEL 0.00 0.2 0 Survival rate (DMSO = 1.0) = (DMSO Survivalrate 0.0 DMSO Rotenone Rotenone day1 day2 day3

G N.S. *** 1.20 *** 1.00 GFP 0.80 1.00) 0.60

0.40 (Control = (Control

Relative efficiency efficiency Relative PH 0.20

0.00

Fig. S7. Effects of metabolic perturbations on E12.5 PGC reprogramming, related to Fig. 4. (A) Relative efficiency of EGC colony formation with 2DG up to or from 72 h after the initiation of culture. (B) Representative images of ALP staining of EGC colonies with glucose at 0 to 10 mM (as indicated). The 6th photo is an enlargement of the box indicated in the 5-mM plate image. Black and red arrows indicate EGC colony and non-specific staining, respectively. Scale bar: 500 µm. (C) Glucose concentration dependency of EGC formation rate (data from experiment portrayed in panel B). (D) TUNEL positive rate of E12.5 PGCs in culture with DMSO or rotenone. (E) Relative efficiency of EGC colony formation with DMSO or rotenone up to or from 72 h after the initiation of culture (left panel). Representative images of ALP staining of EGC colonies grown with DMSO or rotenone from 72 h after the initiation of culture (right panel, scale bar = 200 µm). (F) Temporal survival rate of E12.5 PGCs, Somas and VR15 ESCs with rotenone on day 1 ~ day 3 in culture. (G) Relative efficiency of EGC colony formation from E11.5 PGCs in the feeder-free culture (left panel). Representative images of Oct4-deltaPE-GFP fluorescence of EGC colonies (right panel, scale bar = 250 µm). Values are plotted as mean SEM of 3 biological replicates, N.S.: not significant, *P < 0.05, **P < 0.01, ***P < 0.001 (Student’s t-test). 16 Cyto+ A Day4 Cyto- - 2DG

0.58 % 15.13 % 18.53 % Cellcount

Venus intensity B Day4 C Day4 *** Cyto+ ** Glucose 25 0 1.25 2.5 5.0 25 1.60 P = 0.05 (mM) 1.40 1.20

Venus 1.00) 1.00

+= 0.80 PGCLC 0.60 0.40 day4 day4 0.20 (G25,cyto PH 0.00 cyto- 0 1.25 2.5 5 25

cyto+, Glucose(mM) D Day4 Cyto- Cyto+ Glucose 25 0 1.25 2.5 (mM)

0.13 % 14.86 % 9.97 % 12.72 % Cellcount

E Day2 Cyto+ Cyto+ Glucose 5.0 25 DMSO (mM) Rotenone

10.74 % 10.83 % 23.60 % 3.92 % Cellcount

Venus intensity

Fig. S8. Effects of metabolic perturbations on PGCLC induction, related to Fig. 5. (A, D, and E) Representative histograms of flow cytometry showing the rate of Blimp1-mVenus (BV) -positive cells. PGCLC induction with or without a glycolysis inhibitor, 2DG (A), PGCLC induction at various glucose concentrations (D), or PGCLC induction with or without rotenone (E). The BV-positive zone is highlighted in green. (B) Glucose concentration dependence of PGCLC induction. (C) Relative proportion of PGCLCs after 4 days of induction (data from experiment portrayed in panel B). For relevant panels: Venus: Blimp1-mVenus. PH: phase difference. Scale bar: 250 µm. Values are plotted as mean SEM of 3-4 biological replicates, **P < 0.01, ***P < 0.001 (Student’s t-test).

17 A Glucose depletion Aggregate day2

Blimp1 Stella Nanos3 Fgf5 Hoxb1 * 400 4 P = 0.080 40 P = 0.072 1.2 300 * 1.0 250 300 3 30

= 1) = 0.8 200 200 2 20 0.6 150

EpiLC 0.4 100 ( 100 1 10 0.2 P = 0.060 50 Relativeexpression 0 0 0 0.0 0 G0+ G0+ G0+ G0+ G0+ G25+ G25+ EpiLC G25+ G25+ EpiLC G25+ EpiLC EpiLC EpiLC B Glucose depletion BV-positive cells day4 Blimp1 Stella Nanos3 Fgf5 Hoxb1 * ** P = 0.086 250 80 80 2.0 15 ** 200 60 60 1.5 10 = 1) = 150 40 40 1.0 100 5 EpiLC 20 20 ( 50 0.5 N.S.

Relativeexpression 0 0 0 0.0 0 G0+ G0+ G0+ G0+ G0+ G25+ G25+ G25+ G25+ G25+ EpiLC EpiLC EpiLC EpiLC EpiLC

C Addition of 2DG Aggregate day2 Blimp1 Stella Nanos3 Fgf5 Hoxb1

250 * 5 N.S. 1.2 600 ** * 30 200 4 25 1.0 0.8 400 150 20 = 1) = 3 15 0.6 100 2 200 10 0.4 *** EpiLC ( 50 1 5 0.2 0.0 0 Relativeexpression 0 0 0 2DG 2DG 2DG cyto+ cyto+ 2DG cyto+ 2DG EpiLC EpiLC cyto+ EpiLC cyto+ EpiLC EpiLC

D Addition of 2DG BV-positive cells day4 Blimp1 Stella Nanos3 Fgf5 Hoxb1 N.S. 400 1.5 20 50 ** 80 *** * 300 40 60 15 1.0

= 1) = 30 200 40 10 20 0.5

EpiLC 20 5 ( 100 10 N.S.

Relativeexpression 0 0 0 0 0.0 2DG 2DG 2DG 2DG cyto+ cyto+ cyto+ 2DG cyto+ EpiLC EpiLC EpiLC cyto+ EpiLC EpiLC

E Addition of rotenone Aggregate day2

Blimp1 Stella Nanos3 Fgf5 Hoxb1 ** 100 1.2 10 * 1.4 350 *** 1.2 300 80 1.0 N.S. 8 1.0 250

= 1) = 0.8 60 6 0.8 200 0.6 40 4 0.6 150

EpiLC 0.4 0.4 ( 100 20 0.2 2 0.2 N.S. 50 Relativeexpression 0 0.0 0 0.0 0 EpiLC EpiLC EpiLC EpiLC EpiLC DMSO DMSO DMSO DMSO DMSO Rotenone Rotenone Rotenone Rotenone Rotenone Fig. S9. The expression changes of early PGC markers (Blimp1, Stella, Nanos3), an epiblast marker (Fgf5), and a mesoderm marker (Hoxb1) in the cell aggregates on day2 or in the purified BV-positive PGCLCs on day4. PGCLC induction in the depletion of glucose on day 2 (A) and day 4 (B), in the presence of 2DG on day 2(C) and day 4 (D), or in the presence of rotenone on day 2 (E) were shown. Values are plotted as mean SEM of 3 biological replicates. N.S.: not significant, *P < 0.05, **P < 0.01, ***P < 0.001 (Student’s t- test). 18 A Glycolysis B OXPHOS

* *** *** N.S. *** N.S. NormalizedValueIntensity

E9.5 E11.5 E13.5 E13.5 E9.5 E11.5 E13.5 E13.5 male female male female

PGC PGC

C NTPs Amino acids Glutathiones

Male PGCs

PSCs PGCLCs E11.5 PGCs (~E9.5 PGCs)

Female PGCs

Glycolysis

OXPHOS (male)

OXPHOS (female)

Fig. S10. (A and B) Comparative expression (transcript levels) of genes encoding energy metabolism- related enzymes in PGCs. Expression comparison of glycolysis (A, KEGG pathway: mmu00010) and OXPHOS (B, KEGG pathway: mmu00190) genes among E9.5, E11.5, and E13.5 PGCs (GEO accession: GSE40412) (16). Values are presented as box plots with whiskers from minimum to maximum. The band inside the boxes indicates the median value. N.S.: not significant, *P < 0.05, ***P < 0.001 (Mann–Whitney U test). (C) Schematic diagram summarizing metabolic changes observed in the process of cell conversion between PSCs and PGCs.

19 Table S1. Measurement of cell volumes of E13.5 male PGCs, Somas and ESCs.

Diameter ( m) Diameter ( m) E13.5 mPGCs E13.5 mSomas Major Minor Ave. (Max:Min) Major Minor Ave. (Max:Min) 1 9.122 8.724 8.923 1 8.368 8.039 8.204 2 11.139 10.718 10.929 2 9.319 8.416 8.868 3 10.484 8.654 9.569 3 8.905 7.755 8.330 4 10.657 10.039 10.348 4 9.038 8.422 8.730 5 10.07 9.659 9.865 5 8.619 8.36 8.490 6 10.118 9.424 9.771 6 8.665 7.85 8.258 7 11.244 10.4 10.822 7 10.866 9.335 10.101 8 9.992 9.394 9.693 8 9.02 7.389 8.205 9 10.429 9.192 9.811 9 7.478 7.075 7.277 10 9.274 8.651 8.963 10 8.996 8.032 8.514 11 12.045 11.275 11.660 11 8.263 7.641 7.952 12 10.352 9.526 9.939 12 10.332 9.368 9.850 13 9.05 8.684 8.867 13 10.32 9.608 9.964 Average diameter (m) 9.935 14 9.813 9.354 9.584

Standard deviation 0.830 Average diameter ( m) 8.737 Radius (m) 4.968 Standard deviation 0.839

Volume of a sphere (m3) 513.491 Radius (m) 4.369

Volume of a sphere (m3) 349.252

Diameter (m)

VR15 ESCs Major Minor Ave. (Max:Min)

1 11.897 10.264 11.081

2 11.477 10.358 10.918

3 13.281 12.374 12.828

4 11.992 11.183 11.588

5 10.746 9.895 10.321

6 12.815 12.197 12.506

7 12.818 12.441 12.630

8 12.72 12.552 12.636

9 12.407 11.94 12.174

10 11.903 10.887 11.395

11 12.28 11.206 11.743

12 12.088 11.325 11.707

13 11.275 10.926 11.101

14 12.528 11.524 12.026

15 12.38 10.998 11.689

16 12.798 12.131 12.465

17 11.432 11.333 11.383

Average diameter (m) 11.384

Standard deviation 0.799

Radius (m) 5.888

3 Volume of a sphere ( m ) 854.968

20

Table S2. Proteomic comparison of OXPHOS-related enzymes among E13.5 male PGCs, Somas, and ESCs.

PGC vs Soma PGC vs ESC

Gene Name Uniplot Fold change P value Fold change P value Symbol Atp5a1 ATP synthase, H+ transporting, mitochondrial F1 complex, alpha subunit 1 AccessionQ03265 1.603 0.244 1.130 0.507 Atp5b ATP synthase, H+ transporting mitochondrial F1 complex, beta subunit P56480 1.956 0.050 1.569 0.088 Atp5c1 ATP synthase, H+ transporting, mitochondrial F1 complex, gamma polypeptide 1 Q91VR2 1.683 0.173 1.040 0.763 Atp5f1 ATP synthase, H+ transporting, mitochondrial F0 complex, subunit B1 Q9CQQ7 1.659 0.176 0.463 0.019 Atp5j ATP synthase, H+ transporting, mitochondrial F0 complex, subunit F P97450 1.934 0.141 0.852 0.550 Atp5k ATP synthase, H+ transporting, mitochondrial F1F0 complex, subunit E Q06185 4.193 NA 19.328 0.286 Atp6v1a ATPase, H+ transporting, lysosomal V1 subunit A P50516 1.626 0.374 1.531 0.387 Atp6v1b2 ATPase, H+ transporting, lysosomal V1 subunit B2 P62814 1.514 0.238 0.910 0.498 Atp6v0d1 ATPase, H+ transporting, lysosomal V0 subunit D1 P51863 0.844 0.821 0.661 0.469 Atp6v1e1 ATPase, H+ transporting, lysosomal V1 subunit E1 P50518 NA NA 0.705 0.081 Cox17 assembly protein 17 P56394 2.348 NA 0.353 0.017 Cox4i1 cytochrome c oxidase subunit IV isoform 1 P19783 2.496 0.077 1.925 0.111 Cox5a cytochrome c oxidase subunit Va P12787 2.609 0.119 1.727 0.244 Cox5b cytochrome c oxidase subunit Vb P19536 1.055 0.925 0.955 0.874 Cox6c cytochrome c oxidase subunit VIc Q9CPQ1 4.015 0.156 2.869 NA Cox7a2 cytochrome c oxidase subunit VIIa 2 P48771 3.557 0.077 3.235 0.045 ATP8 ATP synthase F0 subunit 8 P03930 1.853 0.164 1.509 0.136 COX2 cytochrome c oxidase subunit II P00405 1.539 0.511 1.669 0.456 ND3 NADH dehydrogenase subunit 3 P03899 1.989 0.072 NA NA Ndufa2 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 2 Q9CQ75 1.997 0.140 2.051 0.045 Ndufa4 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 4 Q62425 1.785 0.117 1.345 0.198 Ndufs4 NADH dehydrogenase (ubiquinone) Fe-S protein 4 Q9CXZ1 2.195 0.042 0.468 0.239 Ndufv1 NADH dehydrogenase (ubiquinone) flavoprotein 1 Q91YT0 1.865 0.082 1.202 0.193 Cox7a2l cytochrome c oxidase subunit VIIa polypeptide 2-like Q61387 1.984 0.279 1.464 0.256 Uqcrq ubiquinol-cytochrome c reductase, complex III subunit VII Q9CQ69 1.597 0.381 1.518 0.396 Uqcrc1 ubiquinol-cytochrome c reductase core protein 1 Q9CZ13 2.579 0.044 2.115 0.041 Atp5l ATP synthase, H+ transporting, mitochondrial F0 complex, subunit G Q9CPQ8 1.363 0.412 1.172 0.250 Atp5o ATP synthase, H+ transporting, mitochondrial F1 complex, O subunit Q9DB20 1.756 0.151 1.378 0.182 Atp5j2 ATP synthase, H+ transporting, mitochondrial F0 complex, subunit F2 P56135 1.794 0.152 1.354 0.241 Atp5d ATP synthase, H+ transporting, mitochondrial F1 complex, delta subunit Q9D3D9 2.049 0.201 1.631 0.193 Ndufb5 NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 5 Q9CQH3 0.483 NA 0.542 NA Sdhc succinate dehydrogenase complex, subunit C, integral membrane protein Q9CZB0 0.783 NA 1.221 0.465 Ndufa9 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 9 Q9DC69 1.319 0.496 1.118 0.396 Atp6v1f ATPase, H+ transporting, lysosomal V1 subunit F Q9D1K2 1.817 0.090 0.887 0.435 Uqcr10 ubiquinol-cytochrome c reductase, complex III subunit X Q8R1I1 2.291 0.046 2.146 0.016 Ndufb9 NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 9 Q9CQJ8 3.241 0.035 2.086 0.159 Atp6v1g1 ATPase, H+ transporting, lysosomal V1 subunit G1 Q9CR51 1.860 0.229 0.487 0.027 Atp6v1c1 ATPase, H+ transporting, lysosomal V1 subunit C1 Q9Z1G3 NA NA 1.546 0.291 Ndufa12 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 12 Q7TMF3 2.042 0.050 1.564 0.069 Ndufa7 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 7 (B14.5a) Q9Z1P6 0.933 0.886 4.066 NA Cyc1 cytochrome c-1 Q9D0M3 2.463 0.023 2.522 0.004 Uqcrh ubiquinol-cytochrome c reductase hinge protein P99028 2.461 NA NA NA Uqcrfs1 ubiquinol-cytochrome c reductase, Rieske - polypeptide 1 Q9CR68 1.638 0.466 0.900 NA Ndufb7 NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 7 Q9CR61 1.435 0.397 2.143 NA Sdha succinate dehydrogenase complex, subunit A, flavoprotein (Fp) Q8K2B3 1.478 0.415 1.150 0.555 Uqcrc2 ubiquinol cytochrome c reductase core protein 2 Q9DB77 2.224 0.074 1.475 0.098 Atp5e ATP synthase, H+ transporting, mitochondrial F1 complex, epsilon subunit P56382 1.286 0.766 2.742 0.391 Ndufa6 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 6 (B14) Q9CQZ5 2.424 0.014 2.305 NA Ndufa13 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 13 Q9ERS2 3.367 0.295 3.329 0.292 Ndufb8 NADH dehydrogenase (ubiquinone) 1 beta subcomplex 8 Q9D6J5 1.634 0.182 1.725 0.015 Ndufa10 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex 10 Q99LC3 1.202 0.707 0.804 0.651 Uqcrb ubiquinol-cytochrome c reductase binding protein Q9D855 2.809 0.045 2.158 0.039 Sdhb succinate dehydrogenase complex, subunit B, iron sulfur (Ip) Q9CQA3 1.469 0.376 1.581 0.073 Ppa1 pyrophosphatase (inorganic) 1 Q9D819 3.766 0.003 1.459 0.031 Ndufc2 NADH dehydrogenase (ubiquinone) 1, subcomplex unknown, 2 Q9CQ54 2.248 0.049 2.581 0.026 Ndufa5 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 5 Q9CPP6 0.933 NA 0.579 NA Ndufb10 NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 10 Q9DCS9 5.920 NA NA NA Ndufs3 NADH dehydrogenase (ubiquinone) Fe-S protein 3 Q9DCT2 2.255 0.046 1.865 0.032 Ndufa8 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 8 Q9DCJ5 1.737 0.179 1.257 0.264 Ndufa11 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex 11 Q9D8B4 NA NA 1.561 NA Ndufab1 NADH dehydrogenase (ubiquinone) 1, alpha/beta subcomplex, 1 Q9CR21 1.522 0.318 2.517 0.028 Atp5h ATP synthase, H+ transporting, mitochondrial F0 complex, subunit D Q9DCX2 1.815 0.080 1.100 0.544 Ndufv2 NADH dehydrogenase (ubiquinone) flavoprotein 2 Q9D6J6 1.749 NA 2.387 NA Ppa2 pyrophosphatase (inorganic) 2 Q91VM9 1.932 0.154 0.865 0.684 Ndufv3 NADH dehydrogenase (ubiquinone) flavoprotein 3 Q8BK30 1.721 NA 1.810 NA Atp6v1h ATPase, H+ transporting, lysosomal V1 subunit H Q8BVE3 21.846 NA 0.259 0.122 Cox6b1 cytochrome c oxidase, subunit VIb polypeptide 1 P56391 2.354 0.005 1.397 0.053 Ndufs2 NADH dehydrogenase (ubiquinone) Fe-S protein 2 Q91WD5 2.310 0.290 0.231 NA Ndufs1 NADH dehydrogenase (ubiquinone) Fe-S protein 1 Q91VD9 2.867 0.026 2.132 0.024 Ndufb6 NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 6 Q3UIU2 0.972 0.878 1.385 NA Ndufs6 NADH dehydrogenase (ubiquinone) Fe-S protein 6 P52503 0.704 0.466 0.716 0.403 PGC > Soma or ESC, P < 0.05 Comparison of enzymes involved in oxidative phosphorylation (KEGG pathway, mmu00190). PGC > Soma or ESC, P < 0.05, > 2 fold Statistical analysis was performed by Student’s t-test. PGC < Soma or ESC, P < 0.05

PGC < Soma or ESC, P < 0.05, > 2 fold

21

Table S3. OCR and ECAR of PGCs, PGCLCs, Somas, and ESCs (related to Fig. 2)

OCR (pmol/min/1000cells) E13.5 E11.5 - male female experiment # PGC Soma PGC Soma PGC Soma PGCLC ESC 1 1.277 ------0.281 2 1.460 0.130 - - - - - 0.246 3 1.042 0.216 0.654 0.195 - - - - 4 1.096 - 0.911 - - - - - 5 1.367 0.276 0.702 0.396 - - - 0.514 6 1.303 0.358 0.678 0.202 - - - 0.426 7 - - - - 1.175 0.567 0.485 - 8 - - - - 1.220 0.248 0.612 0.298 9 - - - - 1.103 0.188 0.532 - Average (pmol/min/1000cells) 1.257 0.245 0.736 0.264 1.166 0.334 0.543 0.353 Standard error of mean 0.065 0.048 0.059 0.066 0.034 0.118 0.037 0.050 Count 6 4 4 3 3 3 3 5 p value (vs E13.5 male PGC) 3.50.E-06 0.0005 3.06.E-05 0.3829 0.0001 0.0002 2.20.E-06 p value (vs E13.5 male Soma) 0.0007 0.8163 2.83.E-05 0.4670 0.0058 0.1717 p value (vs E13.5 female 0.0032 0.0023 0.0208 0.0533 0.0016 pPGC) value (vs E13.5 female 0.0003 0.6309 0.0210 0.3242 pSoma) value (vs E11.5 PGC) 0.0025 0.0002 2.81.E-05 p value (vs E11.5 Soma) 0.1652 0.8694 p value (vs PGCLC) 0.0392

ECAR (mPH/min/1000cells) E13.5 E11.5 - male female

experiment #(correspond to PGC Soma PGC Soma PGC Soma PGCLC ESC OCR#)

3 0.0248 0.0104 0.0261 0.0075 - - - - 4 0.0228 - 0.0333 - - - - - 5 0.0321 0.0225 0.0178 0.0278 - - - 0.1600 6 0.0668 0.0425 0.0493 0.0167 - - - 0.1031 7 - - - - 0.1552 0.0667 0.1084 - 8 - - - - 0.1488 0.0367 0.1183 0.1167 9 - - - - 0.1754 0.0875 0.4007 - Average (mPH/min/1000cells) 0.0366 0.0251 0.0316 0.0173 0.1598 0.0636 0.2091 0.1266 Standard error of mean 0.0103 0.0094 0.0067 0.0059 0.0080 0.0148 0.0958 0.0172 Count 4 3 4 3 3 3 3 3 p value (vs E13.5 male PGC) 0.4619 0.6974 0.1998 0.0003 0.1799 0.0867 0.0049 p value (vs E13.5 male Soma) 0.5848 0.5176 0.0004 0.0924 0.1286 0.0065 p value (vs E13.5 female 0.1849 0.0001 0.0812 0.0788 0.0022 pPGC) value (vs E13.5 female 0.0001 0.0434 0.1164 0.0038 pSoma) value (vs E11.5 PGC) 0.0046 0.6351 0.1542 p value (vs E11.5 Soma) 0.2077 0.0496 p value (vs PGCLC) 0.4442

22

Table S4. Primers used in this study. real-time quantitative PCR

Target Forward Reverse

Blimp1 TTCTCTTGGAAAAACGTGTGGG GGAGCCGGAGCTAGACTTG

Stella CCAAGAGAAGGGTCCGCACTTT GCAGAGACATCTGAATGGCTCAC

Nanos3 GGGCTACACTTCTGTCTACTGC TTCCTGCCACTTTTGGAACCT

Fgf5 TGTGTCTCAGGGGATTGTAGG AGCTGTTTTCTTGGAATCTCTCC

Hoxb1 GAATCGCCTTGCTCGTCAGAAC CGTGGTGAAGTTTGTGCGGAGA

Arbp AGATTCGGGATATGCTGTTGGC TCGGGTCCTAGACCAGTGTTC

23

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25

Dataset S1. Metabolite concentrations of E13.5 male PGCs, Somas, and ESCs. E13.5 male E13.5 male E13.5 male E13.5 male E13.5 male E13.5 male VR15 ES cells 1 VR15 ES cells 2 VR15 ES cells 3 KEGG ID Compound Name PGCs 1 PGCs 2 PGCs 3 Somas 1 Somas 2 Somas 3 KEGG ID(others) (nmol/mm^3) (nmol/mm^3) (nmol/mm^3) (nmol/mm^3) (nmol/mm^3) (nmol/mm^3) (nmol/mm^3) (nmol/mm^3) (nmol/mm^3) C00103 G1P NA 0.067 0.071 NA 0.062 0.049 0.260 0.180 0.150 C00663 C00092 G6P 0.530 0.520 0.510 0.400 0.380 0.300 0.070 0.084 0.062 C00668 C00085 F6P 0.120 0.140 0.130 0.090 0.101 0.094 0.030 0.031 0.023 C05345 C00354 F1,6P 0.220 0.250 0.140 0.310 0.300 0.118 2.050 1.500 0.980 C05378 C00111 DHAP 0.390 0.390 0.250 0.260 0.420 0.249 1.060 0.790 0.600 C00093 Glycerophosphate 0.850 0.650 0.630 0.410 0.490 0.340 0.170 0.200 0.130 C03189 C00661 G3P NA NA NA NA NA NA 0.086 0.083 0.077 C00118 C01159 2,3-DPG NA NA NA 0.070 0.080 NA NA NA NA C03339 C00197 3PG 0.140 0.090 0.120 0.170 0.180 0.132 0.070 0.066 0.079 C00597 C00631 2PG NA NA NA NA NA NA NA NA NA C00074 PEP 0.050 0.040 0.060 0.080 0.060 0.049 0.050 0.049 0.049 C00022 Pyruvate NA NA NA NA NA NA NA NA NA C00186 Lactate 148.920 128.750 150.000 140.880 210.390 209.000 83.650 97.000 81.000 C00256 C00352 6-phosphate NA NA NA NA NA NA NA NA NA C00024 Acetyl CoA NA 0.020 NA NA NA NA NA NA NA C00158 Citrate 1.760 0.650 0.610 0.610 0.480 0.250 0.190 0.710 0.300 C00417 cis-Aconitate NA NA 0.040 NA NA NA 0.016 NA NA C00311 Isocitrate NA 0.056 0.048 NA NA 0.043 0.021 NA NA C00026 2-Oxoglutarate NA NA NA NA NA NA NA NA NA C02630 2-Hydroxyglutarate 0.160 0.220 0.210 0.180 0.270 0.410 0.080 0.150 0.105 C01087 C03196 C00091 Succinyl CoA NA NA NA NA NA NA NA NA NA C00042 Succinate 1.390 2.150 2.100 1.460 2.850 3.300 0.950 1.140 0.950 C00122 Fumarate 1.020 2.320 0.176 1.010 2.960 0.284 0.160 NA 0.140 C00711 Malate 0.670 1.040 0.970 0.870 1.370 1.350 0.840 1.080 0.640 C00149 C00497 C00345 6-Phosphogluconate NA NA NA NA NA NA NA NA NA C00199 Ru5P NA NA NA NA NA NA NA 0.061 NA C01101 C00117 R5P 0.100 0.101 0.130 0.120 0.148 0.128 NA NA NA C03736 C05382 S7P 0.100 0.100 0.128 0.070 0.070 0.070 0.031 0.063 0.042 C00048 Glyoxylate NA 1.048 NA NA NA NA 0.845 NA NA C00163 Propionate 3.400 3.075 1.380 2.730 4.473 2.790 1.105 1.600 0.970 C00160 Glycolate 6.260 7.540 8.600 6.760 12.260 14.800 3.860 5.400 4.500 C01771 Crotonate NA NA NA NA NA NA NA NA NA C00246 Butyrate 1.410 1.710 1.290 1.180 1.680 1.140 0.710 0.780 0.510 C01013 3-Hydroxypropionate 8.580 7.230 7.200 10.990 11.322 11.300 3.890 6.800 5.000 C11145 Methanesulfonate NA NA 0.480 NA NA 0.500 NA 0.280 NA C08279 Tiglate 0.190 0.312 NA 0.190 0.238 0.235 0.120 NA NA C00109 2-Oxobutyrate NA NA NA NA NA NA NA NA NA C00803 Pentanoate 0.400 0.510 0.182 0.360 0.710 0.214 0.107 0.180 0.066 C08262 3-Methylbutanoate 0.240 0.520 0.290 0.330 0.610 0.300 0.150 0.160 0.130 C00383 Malonate 0.590 0.420 0.530 NA 0.727 0.810 0.300 0.480 0.330 - 2-Hydroxyisobutyrate 0.190 0.163 0.180 0.180 0.250 0.300 0.090 0.130 0.103 C05984 2-Hydroxybutyrate 0.150 0.166 0.139 0.180 0.258 0.230 0.072 0.108 0.082 C01089 3-Hydroxybutyrate 0.740 0.522 0.500 0.740 0.602 0.720 0.273 0.270 0.260 C03197 C03173 (Methylthio)acetate NA NA NA NA NA NA NA NA NA C00258 Glycerate 1.770 1.104 2.500 2.420 2.414 5.500 1.045 1.600 1.400 C05942 Pyrrole-2-carboxylate NA NA NA NA NA NA NA NA NA C02704 Methyl sulfate NA NA 0.071 0.030 NA NA NA NA NA C01546 2-Furoate 0.180 0.450 0.210 0.290 0.610 0.450 0.059 0.200 0.100 C07091 3-Acetylacrylate NA 4.262 0.000 NA 8.821 0.000 4.125 0.000 0.000 C00141 2-Oxoisopentanoate NA NA NA NA NA NA NA NA NA - 4-Oxopentanoate 2.080 2.130 1.620 2.160 2.860 2.280 0.950 0.940 0.780 C01585 Hexanoate 0.610 2.030 0.530 1.550 3.160 0.640 0.210 0.780 0.340 - 2-Hydroxypentanoate 3.270 NA 2.500 3.390 NA 3.200 NA 1.300 1.080 C00180 Benzoate 3.250 2.074 NA 3.190 1.221 NA NA NA NA C00253 Nicotinate NA NA NA NA NA NA NA NA NA C03782 1-Aminoethylphosphonate NA NA NA NA NA NA NA NA NA C03557 2-Aminoethylphosphonate NA NA NA NA NA NA NA NA NA C05123 Isethionate NA NA NA NA NA NA NA NA NA C00813 NA NA NA NA NA NA NA NA NA C01879 5-Oxoproline 1.860 1.550 2.500 1.550 2.950 2.770 1.090 1.130 1.300 C00490 Itaconate 0.050 0.050 0.046 0.070 0.070 0.070 0.030 0.026 0.079 C02226 Citraconate NA 0.120 0.086 NA 0.190 0.161 0.060 0.075 0.073 C00233 4-Methyl-2-oxopentanoate NA NA NA NA NA NA NA NA NA - 4-Oxohexanoate NA 0.200 0.114 NA 0.224 0.186 0.068 0.069 0.060 C02129 4-Acetylbutyrate 1.200 1.190 0.920 1.390 1.440 1.180 0.570 0.430 0.370 C17714 Heptanoate 0.250 0.630 0.098 0.570 0.790 0.118 0.040 0.290 0.086 C01073 N-Acetyl-beta-alanine NA NA NA NA NA NA NA NA NA C00489 Glutarate 0.380 0.390 0.400 0.350 0.670 0.760 0.200 0.260 0.230 C02642 3-Ureidopropionate NA NA NA NA NA NA NA NA NA C03264 2-Hydroxy-4-methylpentanoate NA 0.050 0.044 NA 0.080 0.062 0.013 0.037 0.018 C06103 6-Hydroxyhexanoate NA 0.109 0.116 NA NA 0.165 0.075 0.076 0.042 C01620 Threonate 0.970 1.090 0.670 0.940 0.870 0.600 0.153 0.200 0.190 C00805 o-Hydroxybenzoate NA 0.034 NA NA 0.066 NA NA NA NA C00156 p-Hydroxybenzoate NA NA NA NA NA NA NA NA NA C00587 m-Hydroxybenzoate NA NA NA NA NA NA NA NA NA C05682 Phosphonoacetate NA NA NA NA NA NA NA NA NA C00169 Carbamoylphosphate NA NA NA NA NA NA NA NA NA C00346 Ethanolamine phosphate 26.680 25.370 22.000 9.060 9.770 6.800 0.590 0.360 0.370 C02595 2-Thiopheneacetate NA NA NA NA NA NA NA NA NA C06423 Octanoate 0.550 1.890 0.210 1.420 2.510 0.264 0.110 0.710 0.160 C06104 Adipate 0.690 0.810 0.760 0.770 1.180 1.190 0.380 0.490 0.350 C03618 threo-beta-methylaspartate NA NA NA NA NA NA NA NA NA C01180 4-Methylthio-2-oxobutyrate NA NA NA NA NA NA NA NA NA C02614 Citramalate 0.130 0.150 0.200 0.160 0.250 0.320 0.100 0.120 0.093 C00815 C02612 C00423 trans-Cinnamate NA NA NA NA NA NA NA NA NA C00898 Tartrate NA 0.040 0.048 NA NA NA NA NA NA C02107 C02137 Benzoylformate NA NA NA NA NA NA NA NA NA

26 E13.5 male E13.5 male E13.5 male E13.5 male E13.5 male E13.5 male VR15 ES cells 1 VR15 ES cells 2 VR15 ES cells 3 KEGG ID Compound Name PGCs 1 PGCs 2 PGCs 3 Somas 1 Somas 2 Somas 3 KEGG ID(others) (nmol/mm^3) (nmol/mm^3) (nmol/mm^3) (nmol/mm^3) (nmol/mm^3) (nmol/mm^3) (nmol/mm^3) (nmol/mm^3) (nmol/mm^3) C05629 3-Phenylpropionate 1.790 1.780 1.260 2.000 2.790 1.320 0.850 0.840 0.670 C05852 2-Hydroxyphenylacetate NA NA NA NA NA NA NA NA NA C00642 p-Hydroxyphenylacetate NA NA NA NA NA NA NA NA NA C00606 Cysteine sulfinate NA NA NA NA NA NA NA NA NA C00628 2,5-Dihydroxybenzoate NA NA NA NA NA NA NA NA NA C00295 Orotate NA NA NA NA NA NA NA NA NA C03030 2,4-Dihydroxypyrimidine-5-carboxylate NA NA NA NA NA NA NA NA NA C00337 Dihydroorotate NA NA NA NA NA NA NA NA NA - 2-Oxooctanoate NA NA NA NA NA NA NA NA NA C01601 Pelargonate 1.920 5.040 0.710 3.970 6.470 1.050 0.500 1.600 0.590 C00322 2-Oxoadipate NA NA NA NA NA NA NA NA NA C02656 Pimelate 0.140 0.130 0.115 0.140 0.210 0.188 0.060 0.088 0.065 - 2-Hydroxyoctanoate 0.020 0.020 0.022 0.020 0.035 0.034 NA 0.014 0.009 C01044 N-Formylaspartate NA NA NA NA NA NA NA NA NA C03761 3-Hydroxy-3-methylglutarate NA 0.031 NA NA 0.039 NA 0.018 NA NA C00166 Phenylpyruvate NA NA NA NA NA NA NA NA NA C00811 p-Coumarate NA NA NA NA NA NA NA NA NA C01772 o-Coumarate NA NA NA NA NA NA NA NA NA C06337 Terephthalate 0.140 0.120 0.113 0.120 0.220 0.177 0.090 0.065 0.061 C01606 Phthalate 0.530 0.170 0.173 0.360 0.240 0.216 0.070 0.109 0.064 C05607 3-Phenyllactate NA NA NA NA NA NA NA NA NA C01198 3-(2-Hydroxyphenyl)propionate NA NA NA NA NA NA NA NA NA C01744 3-(4-Hydroxyphenyl)propionate NA NA NA NA NA NA NA NA NA C03722 2,3-Pyridinedicarboxylate NA NA NA NA NA NA NA NA NA C00366 Urate NA 0.320 NA NA NA NA NA 0.120 NA C11527 4-Hydroxymandelate NA NA NA NA NA NA NA NA NA C03198 C05343 C00506 Cysteate NA NA NA NA NA NA NA NA NA C05672 2-Amino-3-phosphonopropionate NA NA NA NA NA NA NA NA NA C01571 Decanoate 0.500 0.630 0.200 0.800 0.880 0.330 0.140 0.320 0.130 C06325 2-Quinolinecarboxylate NA NA NA NA 0.102 NA NA NA NA C02710 N-Acetylleucine NA 0.120 NA NA 0.080 NA 0.022 NA NA C02734 Phenyl phosphate NA NA NA NA NA NA NA NA NA C02341 trans-Aconitate NA NA NA NA NA NA NA NA NA C00493 Shikimate NA NA NA NA NA NA NA NA NA C01042 N-Acetylaspartate 0.440 0.380 0.380 0.060 0.060 0.072 0.030 0.036 0.029 C00438 Carbamoylaspartate NA NA NA NA NA NA NA NA NA C00499 Allantoate NA NA NA NA NA NA NA NA NA C02504 2-Isopropylmalate NA NA NA NA NA NA NA NA NA C03145 N-Formylmethionine NA NA NA NA NA NA NA NA NA C01586 Hippurate NA NA NA NA NA NA NA NA NA C05582 Homovanillate NA NA NA NA NA NA NA NA NA C00847 4-Pyridoxate NA NA NA NA NA NA NA NA NA C02703 Serine O-sulfate NA NA NA NA NA NA NA NA NA C01005 O-Phosphoserine 0.150 0.130 0.113 0.200 0.190 0.200 NA NA NA C17715 Undecanoate NA 0.120 0.047 NA 0.120 0.074 0.028 0.069 0.033 C08261 Azelate 1.510 1.400 0.870 1.030 1.760 1.560 0.710 0.760 0.710 C02774 10-Hydroxydecanoate NA 0.030 0.028 NA 0.050 0.057 0.012 0.017 0.014 C00624 N-Acetylglutamate NA NA NA NA NA NA NA NA NA C05829 N-Carbamylglutamate NA NA NA NA NA NA NA NA NA C02712 N-Acetylmethionine NA NA NA NA NA NA NA NA NA C00296 Quinate NA NA NA NA NA NA NA NA NA C05598 Phenaceturate NA NA NA NA NA NA NA NA NA C00191 Glucuronate NA NA NA NA NA NA NA NA NA C16245 C01494 trans-4-Hydroxy-3-methoxycinnamate NA 0.055 NA NA NA NA NA NA 0.180 C07588 o-Hydroxyhippurate NA NA NA NA NA NA NA NA NA C00257 Gluconate NA NA NA NA NA NA NA NA NA C10833 Syringate NA NA NA NA NA NA NA NA NA C05584 4-Hydroxy-3-methoxymandelate NA NA NA NA NA NA NA NA NA C02679 Dodecanoate 3.530 3.520 1.620 4.570 5.740 2.360 1.370 1.200 1.040 C05824 Cysteine S-sulfate NA NA NA NA NA NA NA NA NA C08277 Sebacate 0.130 0.110 0.076 0.110 0.190 0.139 0.060 0.052 0.051 C11284 3-Indolebutyrate NA NA NA NA NA NA NA NA NA C02470 Xanthurenate NA NA NA NA NA NA NA NA NA C03519 N-Acetylphenylalanine NA NA NA NA NA NA NA NA NA C09816 Benzylsuccinate NA NA NA NA NA NA NA NA NA C00818 Saccharate NA NA NA NA NA NA NA NA NA C00879 Mucate 0.080 0.140 0.040 0.170 0.110 NA NA NA NA - 3-Indoxyl sulfate NA NA NA NA NA NA NA NA NA C00672 2-Deoxyribose 1-phosphate NA NA NA NA NA NA NA NA NA C00864 Pantothenate 0.070 0.050 0.062 0.050 NA NA 0.030 0.029 NA C00482 Sinapate NA NA NA NA NA NA NA NA NA C00931 Porphobilinogen NA NA NA NA NA NA NA NA NA C02678 Dodecanedioate 0.100 0.090 0.065 0.080 0.130 0.097 0.040 0.043 0.037 C06369 2-Deoxyglucose 6-phosphate NA NA NA NA NA NA NA NA NA C00120 Biotin NA NA NA NA NA NA NA NA NA C01096 Sorbitol 6-phosphate NA NA NA NA NA NA NA NA NA C04037 Galacturonate 1-phosphate NA NA NA NA NA NA NA NA NA C02713 N-Acetylmuramate NA NA NA NA NA NA NA NA NA C04501 N-Acetylglucosamine 1-phosphate NA 0.080 0.048 NA 0.060 0.048 NA NA NA C04256 C00357 N-Acetylglucosamine 6-phosphate 0.110 NA NA 0.070 NA NA NA NA NA C02354 2',3'-cCMP NA NA NA NA NA NA NA NA NA C00239 dCMP NA NA NA NA NA NA NA NA NA C00365 dUMP NA NA NA NA NA NA NA NA NA C00270 N-Acetylneuraminate NA NA NA NA NA NA NA NA NA C01182 Ribulose 1,5-diphosphate NA NA NA NA NA NA NA NA NA C01151 C00968 3',5'-Cyclic dAMP NA NA NA NA NA NA NA NA NA C00364 dTMP NA NA NA NA NA NA NA NA NA C00055 CMP NA NA NA NA NA NA NA NA NA C00105 UMP NA NA NA NA NA NA NA NA NA

27 E13.5 male E13.5 male E13.5 male E13.5 male E13.5 male E13.5 male VR15 ES cells 1 VR15 ES cells 2 VR15 ES cells 3 KEGG ID Compound Name PGCs 1 PGCs 2 PGCs 3 Somas 1 Somas 2 Somas 3 KEGG ID(others) (nmol/mm^3) (nmol/mm^3) (nmol/mm^3) (nmol/mm^3) (nmol/mm^3) (nmol/mm^3) (nmol/mm^3) (nmol/mm^3) (nmol/mm^3) C00575 cAMP NA NA NA NA NA NA NA NA NA C01204 Phytate NA NA NA NA NA NA NA NA NA C00943 cIMP NA NA NA NA NA NA NA NA NA C00360 dAMP NA NA NA NA NA NA NA NA NA C00942 cGMP NA NA NA NA NA NA NA NA NA C01367 3'-AMP 0.140 NA NA 0.100 NA NA NA NA NA C00020 AMP NA 0.100 0.092 NA 0.090 0.085 0.040 0.031 0.039 C00130 IMP NA NA NA NA NA NA 0.030 0.060 NA C00584 Prostaglandin E2 NA 2.081 NA NA NA NA 0.893 NA NA C00639 Prostaglandin F2alpha NA NA NA NA NA NA NA NA NA C00144 GMP NA NA NA NA NA NA NA NA NA C00655 XMP NA NA NA NA NA NA NA NA NA C01103 Orotidine 5'-monophosphate NA NA NA NA NA NA NA NA NA C02273 Digalacturonate NA NA NA NA NA NA NA NA NA C00005 NADPH 0.140 0.040 NA NA NA NA NA NA NA C00010 CoA NA NA NA NA NA NA NA NA NA C00705 dCDP NA NA NA NA NA NA NA NA NA C01346 dUDP NA NA NA NA NA NA NA NA NA C00119 PRPP NA NA NA NA NA NA NA NA NA C00363 dTDP NA NA NA NA NA NA NA NA NA C00112 CDP NA NA NA NA NA NA NA NA NA C00015 UDP NA 0.047 0.040 NA NA NA 0.022 NA NA C00695 Cholate NA NA NA NA NA NA NA NA NA C00206 dADP NA NA NA NA NA NA NA NA NA C01260 P1, P4-Di(adenosine-5') tetraphosphate NA NA NA NA NA NA NA NA NA C00630 Isobutyryl CoA NA NA NA NA NA NA NA NA NA C00689 Trehalose 6-phosphate 0.170 0.050 0.200 NA NA NA NA NA NA C00083 Malonyl CoA NA NA NA NA NA NA NA NA NA C00224 Adenosine 5'-phosphosulfate NA NA NA NA NA NA NA NA NA C00054 Adenosine 3',5'-diphosphate 0.450 NA NA 0.250 NA NA NA NA NA C00008 ADP NA 0.480 0.410 NA 0.380 0.320 0.110 0.120 0.106 C00104 IDP NA NA NA NA NA NA NA NA NA C00504 NA NA NA NA NA NA NA NA NA C00035 GDP NA 0.150 0.156 NA 0.080 NA 0.047 NA NA C03794 Adenylosuccinate NA NA NA NA NA NA NA NA NA C01921 Glycocholate NA NA NA NA NA NA NA NA NA C00458 dCTP NA NA NA NA NA NA NA NA NA C00460 dUTP NA NA NA NA NA NA NA NA NA C00459 dTTP NA NA NA NA NA NA NA NA NA C00063 CTP 0.370 0.230 0.330 0.140 0.130 0.106 0.180 0.240 0.140 C00075 UTP 0.380 0.230 0.380 0.230 0.160 0.165 0.290 0.350 0.210 C00131 dATP NA NA NA NA NA NA NA NA NA C01345 dITP NA NA NA NA NA NA NA NA NA C00002 ATP 6.300 4.250 5.500 3.190 2.920 2.470 0.890 1.600 1.050 C00286 dGTP NA NA NA NA NA NA NA NA NA C00081 ITP NA NA NA NA NA NA NA NA NA C05122 Taurocholate NA NA NA NA NA NA NA NA NA C00044 GTP 3.910 2.240 4.400 1.930 1.240 1.490 0.640 1.300 0.920 C00700 XTP NA NA NA NA NA NA NA NA NA C00307 CDP-choline 0.240 NA NA NA NA NA NA NA NA C00301 ADP-ribose NA NA NA NA NA NA NA NA NA C01882 C00842 TDP-glucose NA NA NA NA NA NA NA NA NA C00029 UDP-glucose 0.170 0.130 0.107 0.090 0.070 0.065 0.090 0.085 0.079 C00167 UDP-glucuronate NA NA NA 0.120 0.120 NA 0.070 0.073 0.074 C00498 ADP-glucose NA NA NA NA NA NA NA NA NA C00096 GDP-mannose NA NA NA NA NA NA NA NA NA C00043 UDP-N-acetylglucosamine 0.490 0.380 0.430 0.300 0.270 0.231 0.560 0.670 0.490 C00128 CMP-N-acetylneuraminate NA 0.030 NA NA NA NA NA NA NA C00003 NAD+ 0.390 0.200 0.270 NA 0.170 0.118 0.160 0.160 0.140 C00857 Deamido-NAD+ NA NA NA NA NA NA NA NA NA C00004 NADH NA NA NA NA NA NA NA NA NA C00006 NADP+ NA NA NA NA NA NA NA NA NA C00016 FAD NA NA NA NA NA NA NA NA NA C00086 Urea 67.260 40.180 88.000 62.310 63.990 111.000 24.000 52.000 47.000 C00481 Pyrazole NA NA NA NA NA NA NA NA NA C05670 3-Aminopropionitrile NA NA NA NA NA NA NA NA NA C02294 Methylguanidine NA NA NA NA NA NA NA NA NA C02787 Isobutylamine NA NA NA NA NA NA NA NA NA C00986 1,3-Diaminopropane NA NA NA NA NA NA NA NA NA C00037 Gly 15.760 10.790 38.000 9.100 8.700 12.000 1.500 2.700 2.400 C01104 Trimethylamine N-oxide 0.130 NA NA 0.150 NA NA NA NA NA C05771 Isopropanolamine NA 0.073 0.280 NA 0.112 0.410 0.044 0.112 0.111 C03194 C07044 Hydroxyurea NA NA NA NA NA NA NA NA NA C01008 Trimethylsulfonium NA NA NA NA NA NA NA NA NA C01678 Cysteamine NA NA NA NA NA NA NA NA NA C02640 Isoamylamine 0.020 NA NA 0.010 NA NA NA NA NA C00134 Putrescine(1,4-Butanediamine) 0.160 0.151 0.143 0.100 NA 0.086 NA 0.035 0.027 C00099 beta-Ala 0.150 0.102 0.270 0.140 NA 0.184 0.130 0.150 0.090 C00041 Ala 0.100 5.300 6.400 0.060 4.110 3.300 0.890 0.950 0.680 C00133 C00213 Sarcosine 6.050 NA 0.660 4.260 NA 0.340 NA NA NA C06057 3-Aminopropane-1,2-diol NA NA NA NA NA NA NA NA NA C00292 Aniline NA NA NA NA NA NA NA NA NA C05562 4-Hydroxymethylimidazole NA NA NA NA NA NA NA NA NA C11118 1-Methyl-2-pyrrolidinone NA NA NA NA NA NA NA NA NA C00571 Cyclohexylamine 0.190 0.300 0.440 0.210 0.370 0.600 0.170 0.290 0.200 C01234 1-Aminocyclopropane-1-carboxylate NA NA NA NA NA NA NA NA NA C00576 Betaine aldehyde 2.240 NA NA 1.450 NA NA NA NA NA C00750 Spermine NA NA NA NA NA NA NA NA NA C08306 Hexylamine NA NA NA NA NA NA NA NA NA

28 E13.5 male E13.5 male E13.5 male E13.5 male E13.5 male E13.5 male VR15 ES cells 1 VR15 ES cells 2 VR15 ES cells 3 KEGG ID Compound Name PGCs 1 PGCs 2 PGCs 3 Somas 1 Somas 2 Somas 3 KEGG ID(others) (nmol/mm^3) (nmol/mm^3) (nmol/mm^3) (nmol/mm^3) (nmol/mm^3) (nmol/mm^3) (nmol/mm^3) (nmol/mm^3) (nmol/mm^3) C01672 Cadaverine NA NA NA NA NA NA NA NA NA C00334 GABA 0.060 0.082 0.097 0.050 0.102 0.067 0.160 0.200 0.130 C05145 3-Aminoisobutyrate 0.080 0.073 0.052 0.100 NA NA NA NA NA C02356 2AB NA 0.113 0.143 NA NA NA NA NA NA C02261 C02721 N-Methylalanine 0.090 NA NA 0.060 NA NA NA NA NA C01026 N,N-Dimethylglycine 0.050 NA 0.450 0.020 NA 0.204 NA NA NA C00114 Choline 0.830 0.740 0.860 0.400 0.430 0.320 0.360 0.250 0.210 C06393 2,3-Diaminopropionate NA NA NA NA NA NA NA NA NA C03401 C00065 Ser 5.070 3.970 5.200 3.140 3.740 2.370 1.050 0.990 0.930 C00740 C00716 C06772 Diethanolamine 10.510 11.920 17.500 8.860 10.840 39.000 5.500 13.000 7.900 C02299 N-Methylaniline NA NA NA NA NA NA NA NA NA C05341 beta-Ala-Lys NA NA NA NA NA NA NA NA NA C00519 Hypotaurine 0.400 0.423 NA 0.390 0.733 NA 0.530 0.530 0.380 C01987 2-Aminophenol NA NA NA NA NA NA NA NA NA C00380 Cytosine 0.020 NA NA NA NA NA NA NA NA C00388 Histamine NA NA NA NA NA NA NA NA NA C00106 Uracil NA NA NA NA NA NA NA NA NA C00386 Carnosine NA NA NA NA NA NA NA NA NA C00791 Creatinine 0.180 0.110 0.220 0.120 NA 0.087 NA 0.032 0.028 C08311 Muscimol NA NA NA NA NA NA NA NA NA C02512 beta-Cyanoalanine NA NA NA NA NA NA NA NA NA C00429 Dihydrouracil NA NA NA NA NA NA NA NA NA C00148 Pro 2.230 1.930 2.200 1.550 1.430 1.040 0.310 0.260 0.230 C00763 C16435 C00581 Guanidinoacetate 0.330 NA 0.390 0.030 NA 0.071 NA NA NA C00431 5-Aminovalerate NA NA NA NA NA NA NA NA NA C00183 Val 1.040 1.190 1.670 0.280 1.118 0.820 0.360 0.280 0.170 C06417 C16436 C00719 Betaine 1.410 0.853 1.640 0.920 NA NA NA NA NA C02009 0.020 NA NA 0.020 NA NA NA NA NA C03283 2,4-Diaminobutyrate NA NA NA NA NA NA NA NA NA C03678 4-Amino-3-hydroxybutyrate NA NA NA NA NA NA NA NA NA C00263 Homoserine 2.070 NA NA 2.680 NA NA NA NA NA C02115 Alpha-Methylserine NA NA NA NA NA NA NA NA NA C00188 Thr 0.030 2.110 2.100 0.030 2.750 2.050 0.560 0.420 0.310 C00820 C15587 Purine NA NA NA NA NA NA NA NA NA C01262 Anserine NA NA NA NA NA NA NA NA NA C00884 Homocarnosine NA NA NA NA NA NA NA NA NA C01784 Benzamidine NA NA NA NA NA NA NA NA NA C00097 Cys NA NA NA NA NA NA NA NA NA C00793 C00736 C09815 Benzamide NA NA NA NA NA NA NA NA NA C05332 Phenethylamine NA NA NA NA NA NA NA NA NA C02455 alpha-Methylbenzylamine NA NA NA NA NA NA NA NA NA C11003 2,4-Dimethylaniline NA NA NA NA NA NA NA NA NA C02421 Isonicotinamide 0.220 NA NA 0.260 NA NA NA NA NA C00153 Nicotinamide NA 0.244 0.146 NA 0.362 0.155 0.089 0.069 0.077 C01950 Picolinamide NA NA NA NA NA NA NA NA NA C02567 N1-Acetylspermine NA NA NA NA NA NA NA NA NA C02635 3-Chloroalanine NA NA NA NA NA NA NA NA NA C00245 Taurine 1.520 1.720 1.520 1.150 1.552 0.710 NA NA NA C02376 5-Methylcytosine NA NA NA NA NA NA NA NA NA C05127 1-Methylhistamine NA NA NA NA NA NA NA NA NA C02835 Imidazole-4-acetate NA NA NA NA NA NA NA NA NA C00178 Thymine NA NA NA NA NA NA NA NA NA C08737 Melamine 0.330 0.178 0.106 0.300 0.312 0.131 0.130 0.130 0.054 C03969 1-Amino-1-cyclopentanecarboxylate 0.030 NA NA 0.030 NA NA NA NA NA C00408 Pipecolate NA NA NA 0.010 NA NA NA NA NA C07151 Metformin NA NA NA NA NA NA NA NA NA C01740 Octylamine NA NA NA NA NA NA NA NA NA C02714 N-Acetylputrescine 0.030 NA NA 0.010 NA NA NA NA NA C00179 NA NA NA NA NA NA NA NA NA C01015 Hydroxyproline NA 0.430 0.470 NA 0.293 0.220 NA NA NA C00430 5-Aminolevulinate 0.490 NA NA 0.250 NA NA NA NA NA C00300 Creatine 9.110 6.070 7.100 1.170 1.150 1.060 0.360 0.410 0.290 C02378 6-Aminohexanoate 1.090 NA NA 0.750 NA NA NA NA NA C02486 beta-Leucine NA NA NA NA NA NA NA NA NA C00407 Ile NA 1.010 1.220 NA 0.870 0.530 0.200 0.170 0.170 C06418 C16434 C00123 Leu 1.130 1.000 1.520 0.740 0.890 0.580 0.200 0.160 0.170 C01570 C16439 C03375 Bis(3-aminopropyl)amine NA NA NA NA NA NA NA NA NA C02037 Gly-Gly NA NA 0.122 NA NA NA NA NA NA C00152 Asn 0.830 0.850 1.000 0.500 0.581 0.370 0.160 0.113 0.073 C01905 C16438 C00077 Ornithine 0.360 0.210 0.520 0.390 0.420 0.263 0.114 0.082 0.109 C00515 C01602 C00049 Asp 6.000 4.620 6.700 4.240 3.630 2.900 0.840 1.000 0.870 C00402 C16433 C10901 2-Aminobenzimidazole NA NA NA NA NA NA NA NA NA C00155 Homocysteine NA NA NA NA NA NA NA NA NA C05330 C00147 Adenine 0.030 NA NA 0.040 NA NA NA NA NA C00262 Hypoxanthine NA NA NA NA NA NA NA NA NA C02918 1-Methylnicotinamide NA NA NA NA NA NA NA NA NA C00568 p-Aminobenzoate NA NA NA NA NA NA NA NA NA C01004 Trigonelline NA NA NA NA NA NA NA NA NA C00108 Anthranilate NA NA NA NA NA NA NA NA NA C02735 Phenylethanolamine NA NA NA NA NA NA NA NA NA C00483 Tyramine NA NA NA NA NA NA NA NA NA C00785 Urocanate 0.110 0.158 0.220 0.090 0.305 0.165 0.092 0.070 0.107 C01020 6-Hydroxynicotinate NA NA NA NA NA NA NA NA NA C00783 Tropinone NA NA 0.029 NA NA NA NA NA NA C00860 Histidinol NA NA NA NA NA NA NA NA NA C00729 Tropine 0.010 NA NA NA NA NA NA NA NA C06231 Ectoine NA NA NA NA NA NA NA NA NA C04294 4-Methyl-5-thiazoleethanol 0.020 NA NA 0.020 NA NA NA NA NA C10172 Proline betaine 0.030 NA NA 0.010 NA NA NA NA NA

29 E13.5 male E13.5 male E13.5 male E13.5 male E13.5 male E13.5 male VR15 ES cells 1 VR15 ES cells 2 VR15 ES cells 3 KEGG ID Compound Name PGCs 1 PGCs 2 PGCs 3 Somas 1 Somas 2 Somas 3 KEGG ID(others) (nmol/mm^3) (nmol/mm^3) (nmol/mm^3) (nmol/mm^3) (nmol/mm^3) (nmol/mm^3) (nmol/mm^3) (nmol/mm^3) (nmol/mm^3) C03413 N1,N12-Diacetylspermine NA NA NA NA NA NA NA NA NA C01035 gamma-Guanidinobutyrate 0.040 NA 0.051 0.030 0.066 0.083 0.022 0.035 NA C01996 NA NA 0.026 NA NA NA 0.008 NA NA C01181 gamma-Butyrobetaine 0.030 0.033 0.043 0.020 NA NA NA 0.009 NA C01930 Lysinamide NA NA NA NA NA NA NA NA NA C00315 Spermidine NA NA NA NA NA NA NA NA NA C00064 Gln 4.320 3.660 4.900 2.020 2.160 1.660 1.200 0.780 0.300 C00819 C00303 C03114 5,6-Dimethylbenzimidazole NA NA NA NA NA NA NA NA NA C00047 Lys 1.560 1.120 2.100 1.020 1.030 0.740 0.430 0.450 0.390 C00739 C16440 C07166 Trientine NA NA NA NA NA NA NA NA NA C00025 Glu NA 8.310 6.400 NA 8.590 5.600 3.300 3.200 2.000 C00217 C00302 C00979 O-Acetylserine 7.530 NA NA 7.630 NA NA NA NA NA C06524 Nornicotine NA NA NA NA NA NA NA NA NA C00073 Met 0.270 0.320 0.300 0.170 0.270 0.139 0.047 0.042 0.033 C00913 3-Methyladenine NA NA NA NA NA NA NA NA NA C08434 6-Methylaminopurine NA NA NA NA NA NA NA NA NA C00242 Guanine NA NA NA NA NA NA NA NA NA C06818 NA NA NA NA NA NA NA NA NA C00385 Xanthine NA NA NA NA NA NA NA NA NA C02518 4-Aminosalicylate NA NA NA NA NA NA NA NA NA C00632 3-Hydroxyanthranilate NA NA NA NA NA NA NA NA NA C03758 Dopamine NA NA NA NA NA NA NA NA NA C05135 4-(beta-Acetylaminoethyl)imidazole 0.010 NA NA 0.010 NA NA NA NA NA C10865 Pseudopelletierine NA NA NA NA NA NA NA NA NA C00135 His 0.380 0.350 0.480 0.250 0.310 0.229 0.092 0.081 0.066 C06419 C00768 C05568 beta-Imidazolelactate NA NA NA NA NA NA NA NA NA C10600 Ibotenate NA NA NA NA NA NA NA NA NA C01551 Allantoin NA 3.550 NA NA 13.750 NA 5.000 NA NA C02350 C02348 C00637 Indole-3-acetaldehyde 3.110 4.368 2.600 2.520 4.882 NA 2.600 NA NA C08269 Betonicine NA NA NA NA NA NA NA NA NA - N-Acetylvaline NA NA NA NA NA NA NA NA NA C00993 Ala-Ala NA NA NA NA NA NA NA NA NA C00398 Tryptamine NA NA NA NA NA NA NA NA NA C00956 alpha-Aminoadipate NA NA NA NA NA NA NA NA NA C01046 N-Methylglutamate 0.020 NA NA 0.070 NA NA NA NA NA C00955 Indole-3-ethanol NA 0.371 0.450 NA 0.487 0.510 0.190 0.240 0.200 C00318 Carnitine 0.510 0.430 0.720 0.280 0.210 0.158 NA 0.017 0.024 C00487 C15025 C02627 2-Deoxystreptamine NA NA NA NA NA NA NA NA NA C16741 5-Hydroxylysine NA NA NA NA NA NA NA NA NA C00745 0.040 0.081 0.051 0.040 0.127 0.062 0.034 0.029 0.024 C16150 C16386 C00715 Pterin NA NA NA NA NA NA NA NA NA C03172 S-Methylmethionine NA NA NA NA NA NA NA NA NA C02989 Methionine sulfoxide NA NA NA NA NA NA NA NA NA C02242 7-Methylguanine NA NA NA NA NA NA NA NA NA C02230 3-Methylguanine NA NA NA NA NA NA NA NA NA C00079 Phe 0.620 0.510 0.660 0.390 0.510 0.257 0.113 0.077 0.077 C01959 Taurocyamine NA NA NA NA NA NA NA NA NA C00250 Pyridoxal NA NA NA NA NA NA NA NA NA C05587 3-Methoxytyramine NA NA NA NA NA NA NA NA NA C04548 Synephrine NA NA NA NA NA NA NA NA NA C01869 C00534 Pyridoxamine NA NA NA NA NA NA NA NA NA C00314 Pyridoxine NA NA NA NA NA NA NA NA NA C00547 Noradrenaline NA NA NA NA NA NA NA NA NA C01152 3-Methylhistidine NA NA NA NA NA NA NA NA NA C11016 Diphenylamine NA NA NA NA NA NA NA NA NA C02693 Indole-3-acetamide NA NA NA NA NA NA NA NA NA C00437 N-Acetylornithine NA NA NA NA NA NA NA NA NA C01047 N-gamma-Ethylglutamine NA NA NA NA NA NA NA NA NA C00062 Arg 0.550 0.470 0.830 0.270 0.320 0.255 0.210 0.190 0.180 C00792 C08304 Gramine NA NA NA NA NA NA NA NA NA C06213 N-omega-Methyltryptamine NA NA NA NA NA NA NA NA NA C03139 Guanidinosuccinate NA NA NA NA NA NA NA NA NA C00954 Indole-3-acetate NA NA 0.120 NA NA NA NA NA NA C10898 2-Guanidinobenzimidazole NA NA NA NA NA NA NA NA NA C00327 Citrulline 0.140 0.108 0.151 0.120 0.190 0.107 0.059 0.081 0.052 C00308 Canavanine NA NA NA NA NA NA NA NA NA C00780 Serotonin NA NA NA NA NA NA NA NA NA C08265 Alliin NA NA NA NA NA NA NA NA NA C01419 Cys-Gly NA NA NA NA NA NA NA NA NA - Xanthopterin NA NA NA NA NA NA NA NA NA C03570 Mannosamine NA NA NA NA NA NA NA NA NA C00329 Glucosamine NA NA NA NA NA NA NA NA NA - n-Butyl a-Picolinate NA NA NA NA NA NA NA NA NA C03510 Methionine sulfoximine NA NA NA NA NA NA NA NA NA C03829 C00082 Tyr 0.760 0.660 0.830 0.480 0.550 0.390 0.160 0.150 0.130 C06420 C01536 C09209 Harman NA NA NA NA NA NA NA NA NA C00588 Phosphorylcholine 17.610 12.780 12.600 5.630 4.900 3.800 3.300 3.800 2.700 C00788 Epinephrine NA NA NA NA NA NA NA NA NA C04259 N-alpha,N-alpha-Dimethylhistidine NA NA NA NA NA NA NA NA NA C01029 N8-Acetylspermidine 0.010 NA NA NA NA NA NA NA NA C00612 N1-Acetylspermidine NA NA NA NA NA NA NA NA NA C02155 Gly-Leu NA NA NA NA NA NA NA NA NA C02727 N-epsilon-Acetyllysine NA NA NA NA NA NA NA NA NA C03793 N6,N6,N6-Trimethyllysine 0.010 NA NA 0.010 NA NA NA NA NA C02256 Castanospermine NA NA NA NA NA NA NA NA NA C05659 5-Methoxytryptamine NA NA NA NA NA NA NA NA NA C05635 5-Hydroxyindoleacetate NA NA NA NA NA NA NA NA NA C00021 SAH NA NA NA NA NA NA NA NA NA C03752 Glucosaminate NA NA NA NA NA NA NA NA NA

30 E13.5 male E13.5 male E13.5 male E13.5 male E13.5 male E13.5 male VR15 ES cells 1 VR15 ES cells 2 VR15 ES cells 3 KEGG ID Compound Name PGCs 1 PGCs 2 PGCs 3 Somas 1 Somas 2 Somas 3 KEGG ID(others) (nmol/mm^3) (nmol/mm^3) (nmol/mm^3) (nmol/mm^3) (nmol/mm^3) (nmol/mm^3) (nmol/mm^3) (nmol/mm^3) (nmol/mm^3) C00355 DOPA NA NA NA NA NA NA NA NA NA C02997 N-Acetylhistidine NA NA NA NA NA NA NA NA NA C00019 SAM+ 0.110 0.097 0.141 0.100 0.122 0.116 0.079 0.099 0.066 C01404 Arginine ethyl ester NA NA NA NA NA NA NA NA NA C03626 ADMA NA NA NA NA NA NA NA NA NA - SDMA NA NA NA NA NA NA NA NA NA C02571 o-Acetylcarnitine 0.320 0.290 0.280 0.180 0.270 0.177 NA 0.013 0.010 C00078 Trp 0.150 0.189 0.210 0.110 0.188 NA NA NA NA C00525 C00806 C05660 5-Methoxyindoleacetate 3.880 NA NA 4.690 NA NA NA NA NA C00328 Kynurenine NA NA NA NA NA NA NA NA NA C01718 - Cysteine-glutathione disulphide -Divalent NA NA NA NA NA NA NA NA NA C01909 Desthiobiotin 0.080 NA NA 0.080 NA NA NA NA NA C01118 o-Succinylhomoserine NA NA NA NA NA NA NA NA NA C00371 trans-Zeatin NA NA NA NA NA NA NA NA NA C00643 5-Hydroxytryptophan NA NA NA NA NA NA NA NA NA C01239 N-Acetylglucosylamine NA NA NA NA NA NA NA NA NA C00140 N-Acetylglucosamine NA NA NA NA NA NA NA NA NA C02291 Cystathionine NA NA NA NA NA NA NA NA NA C00542 C02794 3-Hydroxykynurenine NA NA NA NA NA NA NA NA NA C03227 C00881 2'-Deoxycytidine NA NA NA NA NA NA NA NA NA - N1,N8-Diacetylspermidine 0.010 NA NA NA NA NA NA NA NA - gamma-Glu-2AB NA NA NA NA NA NA NA NA NA C01598 NA NA NA NA NA NA NA NA NA C00835 Sepiapterin NA NA NA NA NA NA NA NA NA C02953 7,8-Dihydrobiopterin NA NA NA NA NA NA NA NA NA C00491 Cystine NA NA NA NA NA NA NA NA NA C01420 C03592 5-Methyl-2'-deoxycytidine NA NA NA NA NA NA NA NA NA C00214 Thymidine 15.780 15.670 11.700 12.130 25.780 15.400 9.000 6.900 6.300 C00475 Cytidine NA NA NA NA NA NA NA NA NA C00299 Uridine NA NA NA NA NA NA NA NA NA C04137 Octopine NA NA NA NA NA NA NA NA NA C00647 Pyridoxamine 5'-phosphate NA NA NA NA NA NA NA NA NA C00669 gamma-Glu-cys NA NA NA NA NA NA NA NA NA C06470 Muramate NA NA NA NA NA NA NA NA NA C05198 5'-Deoxyadenosine NA NA NA NA NA NA NA NA NA C01736 Purine riboside NA NA NA NA NA NA NA NA NA C05512 2'-Deoxyinosine NA NA NA NA NA NA NA NA NA C04874 7,8-Dihydroneopterin NA NA NA NA NA NA NA NA NA C00670 Glycerophosphorylcholine 0.250 NA 0.169 NA NA NA 0.200 0.230 0.130 C00378 Thiamine 0.020 NA NA 0.010 NA NA NA NA NA C03795 N6-Methyl-2'-deoxyadenosine NA NA NA NA NA NA NA NA NA C00212 Adenosine NA NA NA NA NA NA NA NA NA C00330 2'-Deoxyguanosine NA NA NA NA NA NA NA NA NA C01817 Homocystine NA NA NA NA NA NA NA NA NA C00294 Inosine NA NA NA NA NA NA NA NA NA C01425 Glu-Glu NA NA NA NA NA NA NA NA NA C00449 Saccharopine NA NA NA NA NA NA NA NA NA C02494 1-Methyladenosine NA NA NA NA NA NA NA NA NA C00387 Guanosine NA NA NA NA NA NA NA NA NA C01762 Xanthosine NA NA NA NA NA NA NA NA NA - Ophthalmate 0.030 NA NA NA NA NA NA NA NA C03406 Argininosuccinate NA NA NA NA NA NA NA NA NA C00170 5-Methylthioadenosine NA NA NA NA NA NA NA NA NA C00127 Glutathione(ox) 1.610 1.090 0.790 0.780 0.730 0.530 0.240 0.350 0.250 C01670 N-alpha-Benzenolarginine ethylester NA NA NA NA NA NA NA NA NA C02515 3-Iodotyrosine NA NA NA NA NA NA NA NA NA C00051 Glutathione(red) 0.380 1.250 0.920 0.150 NA 0.080 0.180 0.055 0.043 - Phe-Phe NA NA NA NA NA NA NA NA NA C04677 AICAR NA NA NA NA NA NA NA NA NA C01081 Thiamine monophosphate NA NA NA NA NA NA NA NA NA C02890 Tetrahydropalmatine NA NA NA NA NA NA NA NA NA C03958 TRH NA NA NA NA NA NA NA NA NA C00255 Riboflavin NA NA NA NA NA NA NA NA NA C03451 S-Lactoylglutathione NA NA NA NA NA NA NA NA NA C11331 Leu-Leu-Tyr NA NA NA NA NA NA NA NA NA C01060 3,5-Diiodo-tyrosine NA NA NA NA NA NA NA NA NA C00440 5-Methyltetrahydrofolate NA NA NA NA NA NA NA NA NA C02465 3,3',5-Triiodothyronine NA NA NA NA NA NA NA NA NA In the case of lactate, there is a possibility that is detected higher than the actual concentration by elution from a plastic container used for the pre-processing and measurement. NA : not available

31 Dataset S2. Proteomic data of E13.5 male PGCs, Somas and ESCs for main energy metabolic pathways.

Glycolysis Proteome (Area value) Gene Uniplot P value P value GeneName PGC_Ave. Soma_Ave. ESC_Ave. Symbol GeneID Accession (PGCvsSoma) (PGCvsESC) Aldoa , fructose-bisphosphate 11674 P05064 2.51.E+08 1.79.E+08 8.62.E+08 0.421 0.002 Aldoc aldolase C, fructose-bisphosphate 11676 Bpgm 2,3-bisphosphoglycerate mutase 12183 P15327 NA 8.88.E+06 NA NA NA Eno1 1, alpha non- 13806 P17182 7.33.E+08 4.80.E+08 1.86.E+09 0.230 0.001 Eno2 , gamma neuronal 13807 Eno3 enolase 3, beta muscle 13808 P21550 4.10.E+08 2.58.E+08 9.92.E+08 0.190 0.001 Fbp2 fructose bisphosphatase 2 14120 Fbp1 fructose bisphosphatase 1 14121 G6pc glucose-6-phosphatase, catalytic 14377 G6pc2 glucose-6-phosphatase, catalytic, 2 14378 Gapdh -3-phosphate dehydrogenase 14433 P16858 9.82.E+08 7.56.E+08 2.60.E+09 0.524 0.002 Gapdhs glyceraldehyde-3-phosphate dehydrogenase, spermatogenic 14447 Q64467 NA NA 6.94.E+08 NA NA Gpi1 glucose phosphate 1 14751 P06745 1.23.E+08 5.06.E+07 1.83.E+08 0.064 0.056 Hk1 1 15275 P17710 1.09.E+07 5.38.E+06 2.17.E+07 0.118 0.009 Hk2 hexokinase 2 15277 O08528 6.37.E+06 5.01.E+06 7.91.E+07 0.620 0.000 Ldha A 16828 P06151 1.41.E+08 1.87.E+08 5.57.E+08 0.629 0.000 Ldhb lactate dehydrogenase B 16832 P16125 3.32.E+08 1.88.E+08 2.25.E+08 0.183 0.079 Ldhc lactate dehydrogenase C 16833 Pfkl phosphofructokinase, liver, B-type 18641 P12382 1.76.E+07 2.41.E+07 6.29.E+07 0.589 0.020 Pfkm phosphofructokinase, muscle 18642 P47857 NA 6.00.E+06 1.30.E+07 NA NA Pgam1 1 18648 Q9DBJ1 2.10.E+08 1.56.E+08 4.21.E+08 0.487 0.003 Pgk1 1 18655 P09411 1.90.E+08 2.03.E+08 6.14.E+08 0.843 0.000 Pgk2 phosphoglycerate kinase 2 18663 P09041 1.22.E+08 1.17.E+08 3.62.E+08 0.923 0.001 Pkm , muscle 18746 P52480 6.52.E+08 3.00.E+08 1.29.E+09 0.084 0.007 Pklr pyruvate kinase liver and 18770 E9Q509 1.24.E+08 NA NA NA NA Tpi1 triosephosphate isomerase 1 21991 P17751 8.47.E+07 1.78.E+08 3.28.E+08 0.133 0.000 Pgam2 phosphoglycerate mutase 2 56012 Pfkp phosphofructokinase, 56421 Q9WUA3 2.44.E+06 NA 3.21.E+07 NA NA Pgm1 phosphoglucomutase 1 66681 Q7TSV4 NA 5.98.E+06 6.18.E+06 NA NA Pdhb pyruvate dehydrogenase (lipoamide) beta 68263 Q9D051 3.88.E+07 1.09.E+07 3.89.E+07 0.032 0.993 G6pc3 glucose 6 phosphatase, catalytic, 3 68401 Adpgk ADP-dependent 72141 Q8VDL4 1.29.E+07 7.42.E+06 7.77.E+06 0.164 0.031 Pgm2 phosphoglucomutase 2 72157 Q9D0F9 1.42.E+07 1.11.E+07 1.50.E+07 0.539 0.668 Gck glucokinase 103988 Ldhal6b lactate dehydrogenase A-like 6B 106557 Hk3 hexokinase 3 212032 Hkdc1 hexokinase domain containing 1 216019

32] Gene Entrez Uniplot P value P value GeneName PGC_Ave. Soma_Ave. ESC_Ave. Symbol GeneID Accession (PGCvsSoma) (PGCvsESC) Aldob , fructose-bisphosphate 230163 Galm galactose mutarotase 319625 Q8K157 NA NA 1.57.E+06 NA NA Aldoart1 aldolase 1 A, retrogene 1 353204 Eno1b enolase 1B, retrotransposed 433182

Pentose Phosphate Pathway Proteome (Area value) Gene Entrez Uniplot P value P value GeneName PGC_Ave. Soma_Ave. ESC_Ave. Symbol GeneID Accession (PGCvsSoma) (PGCvsESC) G6pd2 glucose-6-phosphate dehydrogenase 2 14380 G6pdx glucose-6-phosphate dehydrogenase X-linked 14381 Q00612 9.35.E+06 1.36.E+07 1.88.E+07 0.519 0.007 Prps1 phosphoribosyl pyrophosphate synthetase 1 19139 Q9D7G0 1.96.E+07 2.45.E+07 8.05.E+07 0.512 0.000 Rgn regucalcin 19733 Rpia ribose 5-phosphate isomerase A 19895 P47968 9.07.E+06 NA NA NA NA Taldo1 transaldolase 1 21351 Q93092 1.17.E+08 3.06.E+07 1.09.E+08 0.029 0.793 Tkt transketolase 21881 P40142 1.01.E+08 9.57.E+07 1.15.E+08 0.871 0.407 Pgls 6-phosphogluconolactonase 66171 Q9CQ60 4.00.E+07 3.82.E+07 1.34.E+07 0.907 0.035 Rpe ribulose-5-phosphate-3-epimerase 66646 Rbks ribokinase 71336 Tktl2 transketolase-like 2 74419 Prps1l1 phosphoribosyl pyrophosphate synthetase 1-like 1 75456 Q8C5R8 1.17.E+07 2.11.E+07 5.53.E+07 0.087 0.019 Idnk idnK gluconokinase homolog (E. coli) 75731 Tktl1 transketolase-like 1 83553 H6pd hexose-6-phosphate dehydrogenase (glucose 1-dehydrogenase) 100198 B2KGW9 2.32.E+07 1.18.E+07 4.72.E+07 0.073 0.006 Pgd phosphogluconate dehydrogenase 110208 Q9DCD0 4.54.E+07 2.89.E+07 5.06.E+07 0.362 0.562 Prps2 phosphoribosyl pyrophosphate synthetase 2 110639 Q9CS42 NA 1.93.E+07 7.44.E+07 NA NA Ppat phosphoribosyl pyrophosphate amidotransferase 231327 Q3TKC5 1.98.E+07 9.03.E+05 3.14.E+07 NA 0.139 Dera 2-deoxyribose-5-phosphate aldolase homolog (C. elegans) 232449 Q91YP3 8.70.E+06 4.51.E+06 NA 0.485 NA Glyctk glycerate kinase 235582 Prps1l3 phosphoribosyl pyrophosphate synthetase 1-like 3 328099

TCA cycle Proteome (Area value) Gene Entrez Uniplot P value P value GeneName PGC_Ave. Soma_Ave. ESC_Ave. Symbol GeneID Accession (PGCvsSoma) (PGCvsESC) Aco1 aconitase 1 11428 P28271 6.24.E+06 1.52.E+07 7.42.E+06 0.171 0.288 Aco2 aconitase 2, mitochondrial 11429 Q99KI0 1.45.E+08 7.81.E+07 6.50.E+07 0.182 0.047 Cs citrate synthase 12974 Q9CZU6 9.66.E+07 4.54.E+07 6.39.E+07 0.115 0.134 Dld dihydrolipoamide dehydrogenase 13382 O08749 3.51.E+07 2.43.E+07 2.62.E+07 0.341 0.189 Fh1 fumarate hydratase 1 14194 P97807 5.52.E+07 2.27.E+07 2.77.E+07 0.088 0.050 Gclc glutamate-cysteine ligase, catalytic subunit 14629 P97494 8.06.E+06 NA NA NA NA Gclm glutamate-cysteine ligase, modifier subunit 14630 O09172 1.76.E+07 2.60.E+06 2.35.E+07 0.015 0.207

33] Gene Entrez Uniplot P value P value GeneName PGC_Ave. Soma_Ave. ESC_Ave. Symbol GeneID Accession (PGCvsSoma) (PGCvsESC) Glul glutamate-ammonia ligase (glutamine synthetase) 14645 P15105 1.97.E+07 6.42.E+06 9.18.E+06 0.026 0.050 Gls glutaminase 14660 D3Z7P3 NA NA 6.67.E+06 NA NA Glud1 glutamate dehydrogenase 1 14661 P26443 3.66.E+07 1.86.E+07 6.15.E+07 0.084 0.026 Gsr glutathione reductase 14782 P47791 4.59.E+07 4.64.E+06 2.29.E+07 0.002 0.018 Gss glutathione synthetase 14854 A2AQN9 NA NA 6.77.E+06 NA NA Idh1 isocitrate dehydrogenase 1 (NADP+), soluble 15926 O88844 4.52.E+07 7.15.E+07 1.71.E+07 0.312 0.004 Idh3g isocitrate dehydrogenase 3 (NAD+), gamma 15929 P70404 1.27.E+07 3.95.E+06 1.86.E+07 NA 0.269 Mdh2 malate dehydrogenase 2, NAD (mitochondrial) 17448 P08249 4.48.E+08 1.23.E+08 1.95.E+08 0.021 0.032 Mdh1 malate dehydrogenase 1, NAD (soluble) 17449 P14152 1.44.E+08 1.09.E+08 9.29.E+07 0.421 0.059 Ogdh oxoglutarate (alpha-ketoglutarate) dehydrogenase (lipoamide) 18293 Q60597 9.14.E+07 1.61.E+07 1.12.E+07 0.238 0.213 Pck1 phosphoenolpyruvate carboxykinase 1, cytosolic 18534 Pcx 18563 Q05920 1.04.E+07 6.72.E+06 NA 0.213 NA Pdha1 pyruvate dehydrogenase E1 alpha 1 18597 P35486 6.78.E+07 1.59.E+07 5.28.E+07 0.020 0.553 Pdha2 pyruvate dehydrogenase E1 alpha 2 18598 P35487 2.02.E+07 NA 1.06.E+07 NA NA Sucla2 succinate-Coenzyme A ligase, ADP-forming, beta subunit 20916 Q9Z2I9 3.58.E+07 1.40.E+07 1.89.E+07 0.068 0.057 Suclg2 succinate-Coenzyme A ligase, GDP-forming, beta subunit 20917 Q9Z2I8 2.17.E+07 1.01.E+07 2.19.E+07 0.062 0.972 Suclg1 succinate-CoA ligase, GDP-forming, alpha subunit 56451 Q9WUM5 5.82.E+07 2.35.E+07 3.47.E+07 0.050 0.071 Sdhc succinate dehydrogenase complex, subunit C, integral membrane protein 66052 Q9CZB0 1.77.E+07 2.26.E+07 1.45.E+07 NA 0.465 Sdhd succinate dehydrogenase complex, subunit D, integral membrane protein 66925 Q9CXV1 6.56.E+06 NA NA NA NA Sdha succinate dehydrogenase complex, subunit A, flavoprotein (Fp) 66945 Q8K2B3 4.01.E+07 2.71.E+07 3.48.E+07 0.415 0.555 Sdhb succinate dehydrogenase complex, subunit B, iron sulfur (Ip) 67680 Q9CQA3 3.25.E+07 2.21.E+07 2.05.E+07 0.376 0.073 Idh3a isocitrate dehydrogenase 3 (NAD+) alpha 67834 Q9D6R2 2.24.E+07 1.61.E+07 3.56.E+07 0.409 0.093 Pdhb pyruvate dehydrogenase (lipoamide) beta 68263 Q9D051 3.88.E+07 1.09.E+07 3.89.E+07 0.032 0.994 Csl citrate synthase like 71832 Pck2 phosphoenolpyruvate carboxykinase 2 (mitochondrial) 74551 Q8BH04 NA 1.08.E+07 2.11.E+07 NA NA Dlst dihydrolipoamide S-succinyltransferase (E2 component of 2-oxo-glutarate complex) 78920 Q9D2G2 1.62.E+07 1.00.E+07 6.71.E+06 0.368 0.222 Idh3b isocitrate dehydrogenase 3 (NAD+) beta 170718 Q91VA7 6.56.E+06 1.06.E+07 1.06.E+07 0.440 0.093 Dlat dihydrolipoamide S-acetyltransferase (E2 component of pyruvate dehydrogenase complex) 235339 Q8BMF4 3.22.E+07 1.07.E+07 1.62.E+07 0.010 0.008 Ogdhl oxoglutarate dehydrogenase-like 239017 B2RXT3 2.74.E+07 NA 9.28.E+06 NA 0.010 Idh2 isocitrate dehydrogenase 2 (NADP+), mitochondrial 269951 P54071 7.32.E+07 1.43.E+08 1.36.E+07 0.114 0.000

OXPHOS Proteome (Area value) Gene Entrez Uniplot P value P value GeneName PGC_Ave. Soma_Ave. ESC_Ave. Symbol GeneID Accession (PGCvsSoma) (PGCvsESC) Atp4a ATPase, H+/K+ exchanging, gastric, alpha polypeptide 11944 Atp4b ATPase, H+/K+ exchanging, beta polypeptide 11945 Atp5a1 ATP synthase, H+ transporting, mitochondrial F1 complex, alpha subunit 1 11946 Q03265 4.29.E+08 2.67.E+08 3.79.E+08 0.244 0.507 Atp5b ATP synthase, H+ transporting mitochondrial F1 complex, beta subunit 11947 P56480 4.78.E+08 2.44.E+08 3.05.E+08 0.050 0.088 Atp5c1 ATP synthase, H+ transporting, mitochondrial F1 complex, gamma polypeptide 1 11949 Q91VR2 6.82.E+07 4.05.E+07 6.56.E+07 0.173 0.763 Atp5f1 ATP synthase, H+ transporting, mitochondrial F0 complex, subunit B1 11950 Q9CQQ7 2.46.E+07 1.48.E+07 5.31.E+07 0.176 0.019

34] Gene Entrez Uniplot P value P value GeneName PGC_Ave. Soma_Ave. ESC_Ave. Symbol GeneID Accession (PGCvsSoma) (PGCvsESC) Atp5g1 ATP synthase, H+ transporting, mitochondrial F0 complex, subunit C1 (subunit 9) 11951 Atp5j ATP synthase, H+ transporting, mitochondrial F0 complex, subunit F 11957 P97450 4.71.E+07 2.43.E+07 5.53.E+07 0.141 0.550 Atp5k ATP synthase, H+ transporting, mitochondrial F1F0 complex, subunit E 11958 Q06185 5.33.E+07 1.27.E+07 2.76.E+06 NA 0.286 Atp6v1a ATPase, H+ transporting, lysosomal V1 subunit A 11964 P50516 3.93.E+07 2.42.E+07 2.57.E+07 0.374 0.387 Atp6v1b2 ATPase, H+ transporting, lysosomal V1 subunit B2 11966 P62814 1.88.E+07 1.24.E+07 2.07.E+07 0.238 0.498 Atp6v0d1 ATPase, H+ transporting, lysosomal V0 subunit D1 11972 P51863 6.15.E+06 7.28.E+06 9.29.E+06 0.821 0.469 Atp6v1e1 ATPase, H+ transporting, lysosomal V1 subunit E1 11973 P50518 9.09.E+06 NA 1.29.E+07 NA 0.081 Atp6v0e ATPase, H+ transporting, lysosomal V0 subunit E 11974 Atp6v0a1 ATPase, H+ transporting, lysosomal V0 subunit A1 11975 Atp6v0c ATPase, H+ transporting, lysosomal V0 subunit C 11984 Cox17 cytochrome c oxidase assembly protein 17 12856 P56394 6.93.E+06 2.95.E+06 1.96.E+07 NA 0.017 Cox4i1 cytochrome c oxidase subunit IV isoform 1 12857 P19783 9.93.E+07 3.98.E+07 5.16.E+07 0.077 0.111 Cox5a cytochrome c oxidase subunit Va 12858 P12787 9.51.E+07 3.64.E+07 5.50.E+07 0.119 0.244 Cox5b cytochrome c oxidase subunit Vb 12859 P19536 4.82.E+07 4.57.E+07 5.05.E+07 0.925 0.874 Cox6a1 cytochrome c oxidase subunit VIa polypeptide 1 12861 Cox6a2 cytochrome c oxidase subunit VIa polypeptide 2 12862 Cox6c cytochrome c oxidase subunit VIc 12864 Q9CPQ1 3.36.E+07 8.36.E+06 1.17.E+07 0.156 NA Cox7a1 cytochrome c oxidase subunit VIIa 1 12865 P56392 4.25.E+06 NA NA NA NA Cox7a2 cytochrome c oxidase subunit VIIa 2 12866 P48771 5.50.E+07 1.55.E+07 1.70.E+07 0.077 0.045 Cox7c cytochrome c oxidase subunit VIIc 12867 P17665 NA NA 2.86.E+07 NA NA Cox8a cytochrome c oxidase subunit VIIIa 12868 Cox8b cytochrome c oxidase subunit VIIIb 12869 ATP6 ATP synthase F0 subunit 6 17705 ATP8 ATP synthase F0 subunit 8 17706 P03930 1.31.E+08 7.05.E+07 8.66.E+07 0.164 0.136 COX1 cytochrome c oxidase subunit I 17708 COX2 cytochrome c oxidase subunit II 17709 P00405 6.31.E+07 4.10.E+07 3.78.E+07 0.511 0.456 COX3 cytochrome c oxidase subunit III 17710 CYTB cytochrome b 17711 P00158 3.88.E+07 NA NA NA NA ND1 NADH dehydrogenase subunit 1 17716 ND2 NADH dehydrogenase subunit 2 17717 ND3 NADH dehydrogenase subunit 3 17718 P03899 1.14.E+07 5.72.E+06 NA 0.072 NA ND4 NADH dehydrogenase subunit 4 17719 ND4L NADH dehydrogenase subunit 4L 17720 ND5 NADH dehydrogenase subunit 5 17721 ND6 NADH dehydrogenase subunit 6 17722 Ndufa2 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 2 17991 Q9CQ75 2.36.E+07 1.18.E+07 1.15.E+07 0.140 0.045 Ndufa4 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 4 17992 Q62425 1.21.E+08 6.79.E+07 9.02.E+07 0.117 0.198 Ndufs4 NADH dehydrogenase (ubiquinone) Fe-S protein 4 17993 Q9CXZ1 3.73.E+07 1.70.E+07 7.97.E+07 0.042 0.239 Ndufv1 NADH dehydrogenase (ubiquinone) flavoprotein 1 17995 Q91YT0 2.04.E+07 1.09.E+07 1.70.E+07 0.082 0.193 Cox7a2l cytochrome c oxidase subunit VIIa polypeptide 2-like 20463 Q61387 8.90.E+06 4.49.E+06 6.08.E+06 0.279 0.256 Atp6v0a2 ATPase, H+ transporting, lysosomal V0 subunit A2 21871

35] Gene Entrez Uniplot P value P value GeneName PGC_Ave. Soma_Ave. ESC_Ave. Symbol GeneID Accession (PGCvsSoma) (PGCvsESC) Uqcrq ubiquinol-cytochrome c reductase, complex III subunit VII 22272 Q9CQ69 2.96.E+07 1.85.E+07 1.95.E+07 0.381 0.396 Uqcrc1 ubiquinol-cytochrome c reductase core protein 1 22273 Q9CZ13 6.43.E+07 2.49.E+07 3.04.E+07 0.044 0.041 Tcirg1 T cell, immune regulator 1, ATPase, H+ transporting, lysosomal V0 protein A3 27060 Atp5l ATP synthase, H+ transporting, mitochondrial F0 complex, subunit G 27425 Q9CPQ8 6.27.E+07 4.60.E+07 5.35.E+07 0.412 0.250 Atp5o ATP synthase, H+ transporting, mitochondrial F1 complex, O subunit 28080 Q9DB20 1.30.E+08 7.41.E+07 9.44.E+07 0.151 0.182 Ndufa1 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 1 54405 Atp6ap1 ATPase, H+ transporting, lysosomal accessory protein 1 54411 Atp5j2 ATP synthase, H+ transporting, mitochondrial F0 complex, subunit F2 57423 P56135 1.01.E+08 5.64.E+07 7.47.E+07 0.152 0.241 Atp5d ATP synthase, H+ transporting, mitochondrial F1 complex, delta subunit 66043 Q9D3D9 6.75.E+07 3.29.E+07 4.14.E+07 0.201 0.193 Ndufb5 NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 5 66046 Q9CQH3 5.60.E+06 1.16.E+07 1.03.E+07 NA NA Sdhc succinate dehydrogenase complex, subunit C, integral membrane protein 66052 Q9CZB0 1.77.E+07 2.26.E+07 1.45.E+07 NA 0.465 Ndufa3 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 3 66091 Ndufa9 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 9 66108 Q9DC69 1.28.E+07 9.70.E+06 1.15.E+07 0.496 0.396 Cox7b cytochrome c oxidase subunit VIIb 66142 Atp6v1f ATPase, H+ transporting, lysosomal V1 subunit F 66144 Q9D1K2 1.12.E+07 6.18.E+06 1.27.E+07 0.090 0.435 Uqcr10 ubiquinol-cytochrome c reductase, complex III subunit X 66152 Q8R1I1 7.65.E+07 3.34.E+07 3.57.E+07 0.046 0.016 Ndufb9 NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 9 66218 Q9CQJ8 2.91.E+07 8.98.E+06 1.40.E+07 0.035 0.159 Atp6v1g2 ATPase, H+ transporting, lysosomal V1 subunit G2 66237 Atp6v1g1 ATPase, H+ transporting, lysosomal V1 subunit G1 66290 Q9CR51 9.50.E+06 5.11.E+06 1.95.E+07 0.229 0.027 Atp6v1c1 ATPase, H+ transporting, lysosomal V1 subunit C1 66335 Q9Z1G3 7.28.E+06 NA 4.71.E+06 NA 0.291 Ndufc1 NADH dehydrogenase (ubiquinone) 1, subcomplex unknown, 1 66377 Ndufa12 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 12 66414 Q7TMF3 9.29.E+06 4.55.E+06 5.94.E+06 0.050 0.069 Ndufa7 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 7 (B14.5a) 66416 Q9Z1P6 8.62.E+06 9.24.E+06 2.12.E+06 0.886 NA Cyc1 cytochrome c-1 66445 Q9D0M3 8.73.E+07 3.54.E+07 3.46.E+07 0.023 0.004 Ndufb3 NADH dehydrogenase (ubiquinone) 1 beta subcomplex 3 66495 Q9CQZ6 NA 7.70.E+06 NA NA NA Uqcrh ubiquinol-cytochrome c reductase hinge protein 66576 P99028 1.62.E+07 6.57.E+06 NA NA NA Uqcr11 ubiquinol-cytochrome c reductase, complex III subunit XI 66594 Uqcrfs1 ubiquinol-cytochrome c reductase, Rieske iron-sulfur polypeptide 1 66694 Q9CR68 2.44.E+07 1.49.E+07 2.71.E+07 0.466 NA Ndufb7 NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 7 66916 Q9CR61 9.81.E+06 6.84.E+06 4.58.E+06 0.397 NA Sdhd succinate dehydrogenase complex, subunit D, integral membrane protein 66925 Q9CXV1 6.56.E+06 NA NA NA NA Sdha succinate dehydrogenase complex, subunit A, flavoprotein (Fp) 66945 Q8K2B3 4.01.E+07 2.71.E+07 3.48.E+07 0.415 0.555 Uqcrc2 ubiquinol cytochrome c reductase core protein 2 67003 Q9DB77 8.31.E+07 3.74.E+07 5.63.E+07 0.074 0.098 Atp5e ATP synthase, H+ transporting, mitochondrial F1 complex, epsilon subunit 67126 P56382 1.84.E+07 1.43.E+07 6.73.E+06 0.766 0.391 Ndufa6 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 6 (B14) 67130 Q9CQZ5 1.80.E+07 7.43.E+06 7.81.E+06 0.014 NA Ndufa13 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 13 67184 Q9ERS2 3.62.E+07 1.07.E+07 1.09.E+07 0.295 0.292 Ndufb8 NADH dehydrogenase (ubiquinone) 1 beta subcomplex 8 67264 Q9D6J5 2.01.E+07 1.23.E+07 1.17.E+07 0.182 0.015 Ndufa10 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex 10 67273 Q99LC3 1.40.E+07 1.17.E+07 1.74.E+07 0.707 0.651 Uqcrb ubiquinol-cytochrome c reductase binding protein 67530 Q9D855 5.01.E+07 1.78.E+07 2.32.E+07 0.045 0.039 Sdhb succinate dehydrogenase complex, subunit B, iron sulfur (Ip) 67680 Q9CQA3 3.25.E+07 2.21.E+07 2.05.E+07 0.376 0.073 Ppa1 pyrophosphatase (inorganic) 1 67895 Q9D819 1.07.E+08 2.84.E+07 7.34.E+07 0.003 0.031 Atp5g2 ATP synthase, H+ transporting, mitochondrial F0 complex, subunit C2 (subunit 9) 67942

36] Gene Entrez Uniplot P value P value GeneName PGC_Ave. Soma_Ave. ESC_Ave. Symbol GeneID Accession (PGCvsSoma) (PGCvsESC) Ndufb4 NADH dehydrogenase (ubiquinone) 1 beta subcomplex 4 68194 Ndufc2 NADH dehydrogenase (ubiquinone) 1, subcomplex unknown, 2 68197 Q9CQ54 4.65.E+07 2.07.E+07 1.80.E+07 0.049 0.026 Ndufb2 NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 2 68198 Q9CPU2 NA 2.08.E+06 NA NA NA Ndufa5 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 5 68202 Q9CPP6 3.35.E+06 3.59.E+06 5.79.E+06 NA NA Ndufb10 NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 10 68342 Q9DCS9 1.82.E+07 3.08.E+06 NA NA NA Ndufs3 NADH dehydrogenase (ubiquinone) Fe-S protein 3 68349 Q9DCT2 3.51.E+07 1.56.E+07 1.88.E+07 0.046 0.032 Ndufa8 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 8 68375 Q9DCJ5 2.84.E+07 1.64.E+07 2.26.E+07 0.179 0.264 Atp6v1c2 ATPase, H+ transporting, lysosomal V1 subunit C2 68775 Cox11 cytochrome c oxidase assembly protein 11 69802 Ndufa11 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex 11 69875 Q9D8B4 1.67.E+07 NA 1.07.E+07 NA NA Ndufab1 NADH dehydrogenase (ubiquinone) 1, alpha/beta subcomplex, 1 70316 Q9CR21 2.89.E+07 1.90.E+07 1.15.E+07 0.318 0.028 Cox10 cytochrome c oxidase assembly protein 10 70383 Atp5h ATP synthase, H+ transporting, mitochondrial F0 complex, subunit D 71679 Q9DCX2 9.72.E+07 5.36.E+07 8.84.E+07 0.080 0.544 Ndufv2 NADH dehydrogenase (ubiquinone) flavoprotein 2 72900 Q9D6J6 1.98.E+07 1.13.E+07 8.28.E+06 NA NA Atp6v1d ATPase, H+ transporting, lysosomal V1 subunit D 73834 P57746 NA NA 4.44.E+06 NA NA Ppa2 pyrophosphatase (inorganic) 2 74776 Q91VM9 1.05.E+07 5.45.E+06 1.22.E+07 0.154 0.684 Atp6v1e2 ATPase, H+ transporting, lysosomal V1 subunit E2 74915 Q9D593 NA NA NA NA NA Ndufs7 NADH dehydrogenase (ubiquinone) Fe-S protein 7 75406 Cox8c cytochrome c oxidase subunit VIIIc 75483 Atp6v0e2 ATPase, H+ transporting, lysosomal V0 subunit E2 76252 Lhpp phospholysine phosphohistidine inorganic pyrophosphate phosphatase 76429 Q9D7I5 NA NA NA NA NA Cox7b2 cytochrome c oxidase subunit VIIb2 78174 Ndufv3 NADH dehydrogenase (ubiquinone) flavoprotein 3 78330 Q8BK30 2.10.E+07 1.22.E+07 1.16.E+07 NA NA Cox4i2 cytochrome c oxidase subunit IV isoform 2 84682 Ndufb11 NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 11 104130 Atp6v1h ATPase, H+ transporting, lysosomal V1 subunit H 108664 Q8BVE3 7.10.E+06 3.25.E+05 2.74.E+07 NA 0.122 Cox6b1 cytochrome c oxidase, subunit VIb polypeptide 1 110323 P56391 8.05.E+07 3.42.E+07 5.76.E+07 0.005 0.053 Atp6v1b1 ATPase, H+ transporting, lysosomal V1 subunit B1 110935 Atp6v0b ATPase, H+ transporting, lysosomal V0 subunit B 114143 Atp6v0a4 ATPase, H+ transporting, lysosomal V0 subunit A4 140494 Atp12a ATPase, H+/K+ transporting, nongastric, alpha polypeptide 192113 Ndufs8 NADH dehydrogenase (ubiquinone) Fe-S protein 8 225887 Q3UY05 NA 8.31.E+06 6.95.E+06 NA NA Cox15 cytochrome c oxidase assembly protein 15 226139 Ndufs2 NADH dehydrogenase (ubiquinone) Fe-S protein 2 226646 Q91WD5 9.36.E+07 4.05.E+07 4.05.E+08 0.290 NA Ndufs1 NADH dehydrogenase (ubiquinone) Fe-S protein 1 227197 Q91VD9 4.63.E+07 1.62.E+07 2.17.E+07 0.026 0.024 Atp5g3 ATP synthase, H+ transporting, mitochondrial F0 complex, subunit C3 (subunit 9) 228033 Ndufb6 NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 6 230075 Q3UIU2 9.58.E+06 9.86.E+06 6.92.E+06 0.878 NA Gm4943 predicted 4943 239760 Atp6v0d2 ATPase, H+ transporting, lysosomal V0 subunit D2 242341 Cox6b2 cytochrome c oxidase subunit VIb polypeptide 2 333182 Atp6v1g3 ATPase, H+ transporting, lysosomal V1 subunit G3 338375

37] Gene Entrez Uniplot P value P value GeneName PGC_Ave. Soma_Ave. ESC_Ave. Symbol GeneID Accession (PGCvsSoma) (PGCvsESC) Ndufs6 NADH dehydrogenase (ubiquinone) Fe-S protein 6 407785 P52503 1.20.E+07 1.71.E+07 1.68.E+07 0.466 0.403 Ndufa4l2 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 4-like 2 407790 Ndufs5 NADH dehydrogenase (ubiquinone) Fe-S protein 5 595136 Q99LY9 NA NA NA NA NA *MS/MS data were analyzed with Proteome Discoverer 1.4 (Mascot and Sequest HT). The node ‘Precursor Ions Area Detector’ was used for the blue n=2 calculation of area values of each protein peak. red n=1 not detected detected only one peptide NA not available

38]