Animals

Mouse care and experimental procedures were approved by the Animal Care and Use Committee of Kindai University (Application number: KAAT-22-001). All animal experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals. Mice were maintained on a 12:12 dark: light cycle at a constant temperature of 22–23 °C and euthanised with cervical dislocation immediately before oocyte collection. Muscle tissues of a female Asian elephant (Elephas maximus), named Haruko, were collected from a carcass at necropsy at Osaka Municipal Tennoji Zoological Gardens, and frozen until use. The animal welfare and ethics were in accordance with guidelines adopted by the Japanese Association of Zoos and Aquariums.

Dating of the Yuka mammoth

The dating was performed using tandem accelerator mass spectrometry (AMS) at the National Institute of Environmental Studies. Briefly, a frozen skin tissue derived from the Yuka mammoth was freeze-dried, homogenised and burned out at 850 °C for 8 h with a piece of copper and reduced copper in a vacuumed silica tube to produce CO 2 . Produced CO 2 was separated from water by ethanol-dry ice trap (−80 °C) in a vacuumed line. Next, CO 2 was reduced with H 2 via iron-catalysed reaction for 8 h and graphitised. The mixed graphite-iron powder was pressed into an aluminium cathode holder and analysed by AMS (NIES-TERRA, Tsukuba, Japan). The 14 C/C ratio was analysed as the percentage of Modern Carbon (pMC) based on the guaranteed value of HOXII standard value issued by the National Institute of Standards and Technology. This 14 C/C ratio was converted to conventional radiocarbon age. Finally, this conventional radiocarbon age was converted to calendar era using Intcal 13.

Protein extraction and trypsin digestion

Protein extraction from the mammoth tissues and following sample preparation were performed in a laboratory where modern elephantids have never been introduced. To avoid contamination, suits, gloves and facemasks were constantly used. During the procedures, protein investigations of modern animal species were not performed in the laboratory. Muscle samples from an Asian elephant were introduced into the laboratory after the LC-MS/MS measurements of the mammoth samples were completed.

The skeletal muscle and bone marrow tissues (100 mg) were homogenised on ice in 300 μl lysis buffer comprising 7 M urea, 2 M thiourea, 4% (w/v) CHAPS, 0.05% (v/v) tri-n-butylphosphine and protease inhibitor cocktail (Roche Diagnostics) using Sample Grinding Kit (GE Healthcare). The homogenates were centrifuged at 17,000 g for 10 min at 4 °C, and the supernatants were transferred to new tubes. This centrifugation step was performed twice to completely remove the tissue debris. Protein concentrations of the supernatants were measured by Bradford assay using γ-globulin as a standard. The protein solutions containing 300 μg proteins were transferred to new tubes, and six-times volume of ice-cold acetone was added to the tubes. After incubation at −20 °C for 2 h, proteins were precipitated by centrifugation at 17,000 g for 10 min at 4 °C and dried in vacuo. The precipitated proteins were dissolved in 100 μl of 8 M Urea in 200 mM triethylammonium bicarbonate (TEAB) solution. To reduce disulfide bonds, 10 μl of 50 mM Tris-(2-carboxyethyl) phosphine was added to the samples, followed by incubation at 37 °C for 2 h. Next, 5 μl of 200 mM methylmethanethiosulfonate in isopropanol was added to the samples, followed by incubation at room temperature for 30 min to block sulfhydryl residues of cysteines. Four hundred μl of digestion buffer (300 mM TEAB containing 1 mM CaCl 2 ) and 30 μl of sequence grade modified porcine trypsin (Roche Diagnostics) dissolved in the digestion buffer at 0.5 μg/μl were added to the samples, followed by incubation at 37 °C for 2 h. Subsequently, 50 μl of double distilled water and 30 μl of the trypsin solution were added to the samples, followed by incubation at 37 °C for 16 h. The reaction was stopped by adding 50 μl of 10% phosphoric acid and 1 ml of 0.1% formic acid. The resulting peptides were desalted with SepPak C18 columns (Merck Millipore) and vacuum-dried.

LC-MS/MS

Nano LC-MS/MS measurements were performed using a TripleTOF 5600 plus system (AB SCIEX) equipped with NanoSpray III ion source (AB SCIEX) and Eksigent nanoLC system (Eksigent Technologies). An ion-spray voltage of 2300 V, curtain gas of 20 psi, nebulizer gas of 20 psi, an interface heater temperature of 150 °C and Decluster Potential of 80 were applied. MS scans were performed for 0.25 s in the m/z range of 400–1250. Overall, 20 product ions were selected in each MS scan for subsequent MS/MS scan if they exceeded a threshold of 150 counts per sec (counts/s). To minimise repeated scanning, previously scanned ions were excluded for 14 s. Each MS/MS scan was performed for 100 ms at the m/z range of 100–1600. A sweeping collision energy setting of 35 ± 5 eV was applied for all precursor ions. A ChromXP C18-CL 3 μM 120 A, 75 μm × 150 mm capillary column was connected to a Picochip emitter (New Objective), at which the spray voltage was applied. The flow rate was set at 300 nl/min. Mobile phases comprised (A) 0.1% formic acid (FA) and (B) 0.1% FA in 100% acetonitrile. A three-step linear gradient of 2–5% B in 5 min, 5–20% B in 95 min, 20–32% B in 20 min, 90% B for 5 min was employed. Peptide samples were re-suspended in 0.1% FA and 2% acetonitrile at the final concentration of 1.0 μg/μl and 1, 3 and 6 μl of the solution were injected. Blank runs were inserted between the sample runs to avoid potential carryover contamination.

Database search for identification of proteins and post-translational modifications

Identification of mammoth tissue proteins was performed using Proteinpilot 4.5 software (AB SCIEX). The raw MS spectrometry data was processed using ParagonTM algorithm in the ProteinPilot software to search against UniProt Mammuthus primigenius database (134 entries) first and subsequently against UniProt mammalian database (1,375,125 entries), which contains 25,876 proteins of Loxodonta africana, 223 of Elephas maximus, and 134 of Mammuthus primigenius. Trypsin was selected as the digestion enzyme and methane-thiosulfonation as the cysteine alkylation method. All combinations (380 patterns) of one amino acid substitutions and all the described modifications (295) were taken into consideration. False discovery rate (FDR) analyses were performed using a reverse decoy database to reduce false positive hits. Proteins with FDR rates under 1% were considered as significantly identified proteins in this study. Protein-Pilot automatically clustered the identified proteins into groups that share common peptides so that the minimal set of justifiable identified proteins was listed. The protein within each group that can explain more spectral data with confidence is shown in the results as the primary protein of the group. Common potential contaminant proteins such as trypsin and keratins were rejected from the identified protein lists. For annotation analysis, each identified protein was replaced with the homologous human protein using Blast search against UniProt human protein database, and a set of the homologous human proteins was subjected to the database for Annotation, Visualization and Integrated Discovery (DAVID) analysis (http://david.abcc.ncifcrf.gov/).

Comprehensive identification of post-translationally modified peptides was also performed using Proteinpilot 4.5 software. To enhance confidence of the identification, database search was performed under more restricted conditions. Specifically, the raw MS spectrometry data was searched against UniProt database taxonomically restricted to L. Africana. Amino acid substitutions were not taken into consideration. Peptides identified with confidence lower than 95% were rejected. The other parameters or data processing were the same as those of the protein identification.

Estimation of post-mortem protein damage

The extent of non-enzymatic deamidation of glutamine and asparagine is related to time and environmental conditions22 and potentially useful as a marker of bone collagen deterioration16. To compare preservation states of our samples between the tissues and with those of the previously reported mammoth remains8,17, we checked all the identified collagen sequences with various modifications one by one and calculated the deamidation rates according to the methods of Orlando et al.17. We counted a sequence as deamidated when a deamidated version of the sequence was identified even once.

Quantitative analyses of the collagen peptides were performed using PeakView 2.2 software (AB SCIEX). Extracted ion chromatograms (XIC) were generated for non-deamidated and amidated forms of five collagen peptides, COL1A1-0912-0 (GPAGPQGPR), COL1A1-0510-1 (GVQGPPGPAGPR with oxidation at P6), COL1A1-0441-0 (DGEAGAQGPPGPAGPAGER), COL1A1-0441-1 (DGEAGAQGPPGPAGPAGER with oxidation at P10) and COL1A1-0939-3 (GFSGLQGPPGPPGSPGEQGPSGASGPAGPR with oxidation at P9, P12 and P15), whose deamidation rates were reportedly correlated with the thermal age of the collagen16. Although a deamidated form of a peptide (+0.984 Da) shows almost the same m/z value as one of isotopic variants of the non-deamidated form (+1.0 Da from a monoisotopic peak); the two peptides can be separately quantified because of their different retention times.

Whole-genome sequencing

Sample preparation and DNA extraction were performed in a dedicated ancient DNA laboratory into which modern elephantids have never been introduced. To avoid contamination, laboratory bench surfaces and tools were frequently sterilised by bleaching or UV-irradiation and suits, gloves and facemasks were constantly used during all steps. The Yuka mammoth’s genomic DNA derived from skeletal muscle tissues (50 mg) or bone marrow tissues (50 mg) were purified and collected using QIAGEN DNeasy Blood & Tissue Kit (69504, QIAGEN). DNA samples of 3.7 μg and 1.2 μg were collected from the skeletal muscle tissue and the bone marrow tissue, respectively. Both DNA samples were resuspended in TE (pH 8.0) and preserved at 4 °C. Cytosine deamination is a major post-mortem damage of DNA. The resulting product (uracil) directs the incorporation of adenine during library preparation, which eventually results in C-to-T or G-to-A substitutions, particularly in terminal regions of DNA fragments. To exclude this type of DNA lesions, we constructed Illumina DNA libraries using NEB next DNA Sample Prep Master Mix containing Phusion polymerase (New England Biolabs), which is unable to replicate through uracil23. Quality and quantity of the derived libraries were assessed by 2100 Bioanalyzer using DNA 1000 kit (Agilent Technologies) and qPCR with KAPA library Quantification Kit (Kapa Biosystems), respectively. After the cluster generation on the Illumina cBot (Illumina), sequencing was performed on Hiseq. 2500 (Illumina).

The same mammoth DNA samples, each of 10 ng, and DNA from Asian elephant were analysed by 2100 Bioanalyzer using High sensitivity DNA kit (Agilent Technologies).

Bioinformatic analysis

Obtained sequence data of 100 bp (paired end), 102 bp and 50 bp (single read) lengths were filtered to remove PCR duplicates, reads with low base quality score (QV < 20), reads containing ambiguous nucleotide ‘N’ and the sequences from phix control library (Illumina), which were spiked into each lane. Adapter sequence was also trimmed. Quality filtered reads were used for the alignment against the African elephant genome sequence (loxAfr3) by BWA (ver. 0.7.5a)24 with default settings. After alignment, sequence reads aligned uniquely and properly (for paired end) to the reference genome were used for SNP calling, which was performed using SAMtools (ver. 0.1.19)25 with default settings except for adding following criteria: minimum read depth = 3 and minimum SNP = 2. Identified SNPs were annotated according to the gene sets downloaded from the Ensembl website [http://www.ensembl.org/Loxodonta_africana/Info/Index]. First, they were classified into intergenic or genic regions. Those among genic regions were further separated into exons (synonymous or non-synonymous) and introns. Insertions/deletions were also identified as described above. They were classified into intergenic and genic (intron or CDS) regions. Consensus sequence was reconstructed using SAMtools and custom perl script. To infer the sequence of mitochondrial genome, using Bowtie226 with the default option, we aligned the filtered reads of a sequence run (KM2_SR100) to the known mammoth M19 mitochondrial genome (EU153448), the end of which was added to the first 100 bp to enable alignment effectively, resulting in the generation of an average 346-fold coverage over the entire length. We used sequence reads that aligned to mitochondrial DNA to assess the sequence substitution by comparing the individual reads with their consensus. The number of different or common bases was calculated in each site. DNA damage was assessed by investigating substitution patterns, which have been found to be elevated at 5′- and 3′-ends using mapDamage 2.027.

Phylogenetic analysis

The complete mitochondrial sequences from the Yuka mammoth, 19 other woolly mammoths and 2 extant elephants and mastodon, used as an outgroup, were used for phylogenetic analysis. The mitochondrial sequences were aligned using MUSCLE and the resulting multiple alignments were subjected to the construction of a phylogenetic tree by maximum likelihood using MEGA628.

Preparation of mammoth nuclei for nuclear transfer

Buffer A was prepared by mixing 98 μl of the nuclear isolation media (731086; Beckman Coulter) and 2 μl of 0.05% trypsin-EDTA solution (25300-054; Thermo Fisher Scientific). Mammoth muscle tissue (approximately 30 mg) was minced with forceps and scissors in a culture dish. Next, the minced muscle tissue was transferred into a BioMasher column, followed by the addition of 100 μl Buffer A and homogenisation using the BioMasher III (320302; Nippi). After centrifugation at 900 g for 4 min, the supernatant was transferred into a new 0.2 ml tube, the same volume of TE buffer (pH 8.0) was added and mixed well, and the mixed solution was centrifuged at 400 g for 4 min. The supernatant was removed and the pellet was suspended into 30 μl of CZB medium. DAPI (D3571; Thermo Fisher Scientific) was added to the nuclei suspension at a final concentration of 0.1 µg/ml. The suspension was transferred into a new culture dish and somatic cell nuclei with intrinsic fluorescence were picked up under a fluorescent microscope for nuclear transfer.

γH2A.X monoclonal antibody

To generate monoclonal antibodies directed against γH2A.X, serine 139-phosphorylated form of histone H2A.X, mice were immunised with a synthetic peptide 163-6 [CGGKKATQA(phospho-S)QEY] as described previously29. After generating hybridomas, clones were screened by ELISA using peptides; 163-5 [CGGKKATQASQEY], 163-6 [CGGKKATQA(phospho-S)QEY], 163-7 [CGGKKA(phopho-T)QASQEY], 163-8 [CGGKKA(phopho-T)QA(phospho-S)QEY] and H3S10P [ARTKQTARK(phospho-S)TGGKAPRKQC]. Clone CMA281 specifically reacted with the peptides containing phospho-S139 and isotyped as IgG1-κ using IsoStrip Mouse Monoclonal Antibody Isotyping Kit (Roch). Antibody purification, Fab preparation and fluorescent dye conjugation were performed as described previously30. To validate the reactivity of CMA281 to cellular γH2AX in a damage-dependent manner, HeLa cells were untreated or treated with etopside (20 μg/ml; 20 min) and stained with purified IgG followed by Alexa Fluor 488-conjugated donkey anti-mouse IgG (Jackson Immunoresearch) and Alexa Fluor 488-labelled Fab. DNA was counterstained with Hoechst3334230.

Immunofluorescence

Immunofluorescence of cell nuclei was performed using a micromanipulator system (Fig. S5). Individual nucleus-like structures were fixed with 3.7% paraformaldehyde (w/v) in phosphate buffered saline (PBS) for 15 min and permeabilised with 2% Triton X-100 in PBS for 2 h at room temperature. After fixation and permeabilisation, the samples were incubated in the blocking buffer (3% BSA + 0.02% Tween in PBS) for 1 h at room temperature and incubated with primary antibodies against histone H3 (1:50) and lamin B2 (1:50) in blocking buffer at 4 °C overnight. The samples were washed with blocking buffer for 15 min at room temperature; this step was performed three times. The samples were then incubated with secondary antibodies (1:200) for 1 h at room temperature. The DNA was stained with 0.05 µg/ml of 4′, 6-diamidino-2-phenylindole (Thermo Fisher Scientific., D3571) in blocking buffer for 1 min. Finally, the samples were mounted on a slide glass and examined with a laser-scanning confocal microscope (Zeiss LSM800 Axio Observer Z1) and imaging software (Zeiss ZEN 2).

Nuclear transfer (NT)

NT was performed as described previously31. Metaphase II (MII) oocytes were collected from B6D2F1 female mice (Japan SLC). Briefly, nuclei of the donor muscle tissue (DAPI positive nuclei) were injected into MII oocytes using Piezo Micro Manipulator (Prime Tech). After NT, morphologically normal oocytes were incubated for 0.5–1 h in CZB medium before live-cell imaging analysis. After 3 h of NT, the reconstructed oocytes were chemically activated by 5 mM SrCl 2 and 2 mM EGTA containing KSOM medium in presence of 5 µg/ml cytochalasin B for 6 h31.

Live-cell imaging

Synthesis and purification of mRNA for injection have been described previously32. MII oocytes collected from B6D2F1 mice (10–20 weeks old) were injected with a mixture of 10 μg/ml histone H2B-mCherry mRNA and 10 μg/ml EGFP-EB1 mRNA20 using Piezo Micro Manipulator to visualize DNA and microtubule. For measurement of DNA double-strand break, a mixture of 10 μg/ml histone H2B-mCherry mRNA and 0.2 mg/ml of Alexa488-labled anti-γH2A.X Fab fragment was injected. The γH2A.X is phosphorylated form of histone H2A variant H2A.X at serine 139 and a marker for DNA DSBs21. It was used for the quantification of DNA integrity based on the FabLEM technology30 in which the kinetics of protein modification could be captured in living cell32. H2B-mCherry was used for the normalization of injection volume in each embryo. After 4–5 h of incubation, the oocytes were injected with somatic cell nuclei and transferred to drops of KSOM medium on a glass-bottom dish (MatTek) and placed in an incubation chamber stage (Tokai Hit) at 37 °C under 6% CO 2 , 5% O 2 and 89% N 2 atmosphere on an inverted microscope (IX-71; Olympus), which was equipped with a spinning disk confocal system (CSU-W1; Yokogawa Electric), EM-CCD (iXon3-888; Andor Technology), plus a filter wheel and z-motor (Mac5000, Ludl Electronic), in a temperature-controlled dark room at 30 °C. Two-colour fluorescence images in 51 different focal planes with 2 μm intervals were captured every 4–5 min using UPLSAPO30XS (NA = 1.05) or UPLSAPO60XS (NA = 1.30) objective lens with 488- (Melles Griot) and 561-nm (Cobolt) laser lines employing MetaMorph software ver. 7.7.10 (Molecular Devices). Image editing was performed using the same MetaMorph software. Image processing and three-dimensional measurements of signal intensities of histone H2B and γH2A.X were performed using Volocity software ver. 6.3 (Perkin Elmer). Scheme for the image analysis and definition of DNA Damage Index (DDI) are summarised in Fig. S10. Normality and homoscedasticity of DDI in each sperm sample were examined by Shapiro–Wilk test and Bartlett test, respectively. After these analyses, significances with control (fresh sperm) were analysed by Steel test.

Sperm preparations and Intracytoplasmic Sperm Injection (ICSI)

For preparation of frozen-thawed sperm samples, cauda epididymal sperm were collected from B6D2F1 mice (12–24 weeks old), suspended in Hepes-CZB medium (2.0 × 107 sperm/ml) and frozen at −30 °C in a refrigerator. For DNase treated sperm, cauda epididymal sperm were suspended in DNase buffer containing 50 mM Tris-HCl (pH 7.5), 10 mM MnCl 2 /4H 2 O and 100 mM NaCl at the same sperm concentration (2.0 × 107 sperm/ml), and DNase (RQ1 RNase-free DNase, M6101, Promega) was added at the final concentration of 50 U/ml. Sperm suspensions were incubated at 37 °C for 15 min; subsequently, DNase was inactivated by incubation at 65 °C for 10 min. One microliter of each sperm suspension was mixed with 9 µl of Hepes-CZB medium containing 12% (w/v) polyvinylpyrrolidone (PVP) and was applied to intracytoplasmic sperm injection (ICSI). Protocols for ICSI have been described elsewhere. Briefly, the head of each sperm was separated from the tail by applying Piezo pulses to the head–tail junction using piezo-driven pipette. Only the sperm head was injected into each MII oocyte cytoplasm. After ICSI, the fertilised embryos were live-cell imaged, as described above, and further cultured in KSOM medium at 37 °C under 6% CO 2 , 5% O 2 and 89% N 2 until the blastocyst stage.

In vitro fertilisation and embryo transfer