Distinct requirements for energy metabolism 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-glutamine (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 uniprot protein 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.