Chemical compound

Metformin (PHR1084) purchased from Sigma-Aldrich (Louis, MO, USA) was diluted directly in PBS to various concentrations.

Animals

Female ICR mice (12 and 28 weeks old) were purchased from SLAC Laboratory (Shanghai, China) and maintained in an auto-controlled temperature and humidity environment with 12-h/12-h light/dark cycle and ad libitum access to food and water. This study was performed in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals and relevant Chinese laws and regulations. All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Shanghai Jiao Tong University. (Title: Mechanism for transdifferentiation of spermatogonia into germline stem cells and germ cells; Number: A2016084; Date: Oct 18, 2016). We declared that our manuscript reporting adheres to the ARRIVE guidelines (http://www.nc3rs.org.uk/page.asp?id=1357) for the reporting of animal experiments.

Culture of FGSCs

The FGSC line was cultured as described previously [23, 45, 46]. In brief, FGSCs were cultured on mitotically inactivated STO (SIM mouse embryo-derived thioguanine and ouabain-resistant) feeder cells at 37 °C with 5% CO2. The culture medium was minimum essential medium alpha (12000022, Gibco), supplemented with 10% fetal bovine serum (FBS03ES-5001, Front), 10 ng/mL human basic fibroblast growth factor (10018b, PeproTech), 10 ng/mL mouse glial cell line-derived neurotrophic factor (45044, PeproTech), 10 ng/mL mouse epidermal growth factor (31,509, PeproTech), 10 ng/mL mouse leukemia inhibitory factor (sc4378, Santa Cruz Biotechnology), 1 mM non-essential amino acids (11140050, Gibco), 2 mM L-glutamine (A2916801, Gibco), 50 U/mL penicillin and 50 μg/mL streptomycin (15070063, Gibco), 1 mM pyruvate (11360070, Gibco), and 100 μM β-mercaptoethanol (M3148, Sigma-Aldrich). FGSCs were subcultured every 3 or 4 days by digestion with TrypLE™ Express (12604021, Gibco) at a 1:4 or 5 split ratio. The culture medium was changed every 2 days. FGSCs were identified by RT-PCR and immunofluorescence (Additional file 1: Fig. S1).

Metformin treatment of mice

Female ICR mice (12 and 28 weeks old, n = 20 each) were allowed to acclimate for 1 week and under the premise of 4R (reduction, refinement, replacement, responsibility) principle in animal experiments. Then, mice were weighed and recorded in order of weight from lowest to highest, and four rearing rooms were assigned, with 5 mice in each rearing room. Random grouping was adopted as follows: mice were assigned according to rearing rooms from 1 to 4 and weight from lowest to highest, starting from rearing room 1 in the first round. In the second round, the mice were allocated from rearing room 2, and rearing room 1 was completed. In the third round, the mice were allocated from rearing room 3, and rearing rooms 1 and 2 were completed until the end of the four rounds. A new round was allocated from rearing room 1 in the same way as above until the mice were allocated. The experimental groups (12 and 28 weeks, n = 10 each) were treated with metformin intraperitoneally (i.p. 200 mg/kg) [20, 21] and the remaining mice as the control group were treated with PBS for 7 days. Then, 5-ethynyl-2ʹ-deoxyuridine (EdU, C00053, RiboBio) injection (i.p., 10 mg/kg, refer to the instruction of test kits) was performed at 1 and 3 weeks after metformin treatment (12 and 28 weeks, n = 5 each) to mark proliferating FGSCs. The injection procedure was operated at a sterilized workstation, and disposable syringes were used. The injection site was adequately disinfected before injection to prevent infection of the injection wound. The veterinarian was responsible for daily mental state and wound checks. The mice were euthanized by carbon dioxide at 24 h after EdU injection. Avoid the process to other mice for suffering from fear and mental pain.

Hormone analysis

Sera were separated from collected blood samples after euthanasia, immediately. Serum levels of estradiol and progesterone were measured by an Access Immunoassay System (Beckman Coulter, UniCelDxl 800, USA) in accordance with the manufacturer’s instructions.

Histological analyses and ovarian follicle counting

Mouse ovaries collected at the two time points were fixed with 4% paraformaldehyde, embedded in paraffin, and sectioned into 6-μm-thick slices. Sections were dewaxed, hydrated, and stained with hematoxylin and eosin (HE). Experimental procedures were performed in accordance with previous studies with some modification [23, 46]. Ovarian follicles were counted as described previously [47, 48]; in brief, the number of all types of follicles in the left and right ovarian slices of each mouse was recorded, and the repeated sections in the same position were counted only once. Finally, the total number of follicles in each mouse ovary in different treatment groups was counted and analyzed.

Immunofluorescence of ovarian tissue

Immunofluorescence staining was performed using a Cell-Light Apollo 567 Stain Kit (C10371-1, RiboBio) in accordance with the manufacturer’s protocol. Briefly, section dewaxing and rehydration were conducted as described for HE staining, and antigen retrieval was performed in citric acid buffer (10 mM sodium citrate, pH 6.0) by microwaving. Sections were permeabilized in 0.5% Triton X-100 at room temperature for 20 min, and EdU staining was performed according to the manufacturer’s protocol. Then, 10% goat serum was applied as a blocking regent at room temperature for 30 min, followed by incubation with the primary anti-Mvh antibody at 4 °C overnight and then with a CoraLite488-conjugated secondary antibody (1:200; SA00013-2, Proteintech) at room temperature for 1 h. Nuclei were counterstained with Hoechst 33342 at room temperature for 30 min. Sections were mounted as described for HE staining. Images were captured under a Leica fluorescence microscope (Leica, DM2500, Germany).

Cell counting

FGSCs were cultured in 24-well plates at 104 cells/well overnight and then treated for 24 and 48 h with various concentrations of metformin. Then, the cells were digested, resuspended in PBS, and counted using a hemocytometer.

Cell viability assay

FGSCs were seeded at 2000 cells/well in 96-well plates and cultured overnight at 37 °C with 5% CO2. The cells were treated with various concentrations of metformin for 24 h, and then, cell viability was measured by Cell Counting Kit-8 (CCK-8, C0038, Beyotime) in accordance with the manufacturer’s instructions. Optical density was measured at 450 nm using a microplate reader (Bio-Tek, Thermo Fisher Scientific, USA).

Cell proliferation assay

FGSCs were cultured in 48-well plates at 5000 cells/well overnight. After treatment with various concentrations of metformin for 24 h, a Cell-Light EdU Apollo 567 in vitro kit (C10310-1, RiboBio) was used to evaluate cell proliferation in accordance with the manufacturer’s protocol. The cells were incubated with 50 μM EDU for 2 h, fixed with 4% paraformaldehyde for 30 min at room temperature, neutralized in 2 mg/mL glycine for 5 min, and incubated for 10 min on a shaking table in PBS containing 0.5% Triton X-100) for permeabilization. Then, the cells were treated with 1 × Apollo staining solution for 30 min, followed by washing with PBS containing 0.5% Triton X-100 three times. And 1 × Hoechst 33342 solution was used for counterstaining. Finally, an inverted fluorescence microscope (Leica, DMI3000B, Germany) was used to capture images that were analyzed by ImageJ. In brief, three visual fields were taken from each well in each treatment group, and the red fluorescence (EDU) and blue fluorescence (Hoechst 33,342) in each visual field were counted. The cell proliferation index was determined as the ratio of EDU to Hoechst 33342.

RT-PCR and real-time qRT-PCR

Total RNA was extracted from FGSCs using Trizol reagent (15596026, Life Technologies). The RNA concentration was measured using a Nano Drop 2000 spectrophotometer (Thermo Fisher Scientific, USA). Approximately 1000 ng total RNA was reverse-transcribed into cDNA using a reverse transcription kit (11141ES60, YEASEN). RT-PCR was performed with 2 × HieffPCR Master Mix (10102ES03, YEASEN) in a Master cycler PCR (Eppendorf AG, 6331, Germany) using touchdown PCR mode (95 °C for 5 min, followed by 20 cycles at 95 °C for 30 s, touchdown annealing at 65–50 °C, 72 °C for 30 s, and then 15 cycles at 95 °C for 30 s, 50 °C for 30 s, 72 °C for 30 s, and final extension at 72 °C for 10 min). RT-PCR products were separated on 1.5% agarose gels and imaged with an AlphaImager EP bioimaging system (Alpha Innotech, USA). Real-time qRT-PCR analysis was carried out with a HifairIII One Step RT-qPCR SYBR Green Kit (11184ES08, YEASEN) in an Applied Biosystems Real-Time PCR System (Thermo Fisher Scientific, 7500, USA) with the following reaction conditions: 50 °C for 2 min, 95 °C for 10 min, and then 40 cycles of 95 °C for 15 s and 60 °C for 60 s. Data were analyzed by the 2-ΔΔCt method. Primers are shown in Additional file 6: Table S1 and Additional file 7: Table S2.

Western blotting

Cells were lysed in RIPA buffer (P0013B, Beyotime) containing InStabTMProtease Cocktail inhibitor (20123ES10, YEASEN), scraped into an EP tube using a BeyoGold cell scraper (FSCP023, Beyotime), and incubated for 5 min after 20 s of vortexing for six times in total. The whole procedure was conducted on ice. Protein concentrations were measured by a BCA protein quantification kit (YEASEN, 20201ES76). A total of 20–40 µg proteins was separated using a 12.5% SDS-PAGE gel fast preparation kit (PG113, Epizyme) and then transferred to Immobilon-P PVDF membranes (Millipore, IPVH00010, Merk, Germany). Membranes were blocked with 5% dry non-fat milk in Tris-buffered saline Tween 20 (TBST) for 1.5 h with gentle shaking at room temperature and then incubated at 4 °C overnight (16–18 h) in QuickBlock™ primary antibody dilution buffer (P0256, Beyotime) with the following primary antibodies: anti-Kbhb (1:2000; PTM-1201, PTM), anti-H2BK5bhb (1:2000; PTM-1230, PTM), anti-histone H3 (1:2000; 17168-1-AP, Proteintech), anti-Gata2 (1:2000; ab109241, Abcam, UK), or anti-GAPDH (1:50000; 60004-1-Ig, Proteintech). The membranes were washed with Tris-buffered saline Tween 20 three times for 10 min each, incubated for 2 h with secondary antibodies (1:2000; SA00001-1 or SA00001-2, Proteintech) diluted in blocking buffer, and then washed three times with TBST for 10 min each. Finally, protein brands were visualized using ECL reagent (MA0186, Meilunbio) and scanned by a Gel Imager System (Tanon, 4600SF, China). The density of protein bands was calculated by ImageJ.

Immunofluorescence

FGSCs were cultured to 80% confluence in 24-well plates. Then, the cells were fixed in 4% paraformaldehyde for 30 min at room temperature and washed with PBS three times. Samples were blocked in 10% goat serum at room temperature for 1 h, incubated with an anti-Mvh primary antibody (1:200; ab13840, Abcam) at 4 °C overnight (16–18 h), washed with PBS three times, and then incubated with a CoraLite594-conjugated secondary antibody (1:200; SA00013-4, Proteintech) for 1 h at room temperature. Nuclei was counterstained with 4′,6-diamidino-2-phenylindole (DAPI) for 3 min. After three washes, the cells were mounted in antifade mounting medium (P0126, Beyotime) to capture images under a fluorescence microscope (Leica, DMI3000B, Germany).

Sequencing of proteomic pan-β-hydroxybutyrylation

Protein extraction, fragmentation, enrichment, mass spectrometry, and sequencing analysis were performed by PTM Bio Company. Briefly, samples were mixed with pyrolysis buffer for ultrasonication. After centrifugation, the supernatant was collected to determine the protein concentration using the BCA kit and then subjected to trypsin digestion overnight, following the manufacturer’s instructions. After enzymatic hydrolysis, the peptides were dissolved in IP buffer and transferred to prewashed β-hydroxybutyrylated resin (PTM Bio, China) for incubation overnight. Then, the resin-bound peptides were washed and eluted three times, collected for desalinization in accordance with the C18 Zip Tip instructions and subjected to HPLC-MS/MS analysis.

Chromatin immunoprecipitation sequencing and qPCR

Chromatin immunoprecipitation (ChIP) and input DNA fragment preparation were conducted following a published protocol (Chapter 17) [49]. In brief, cells were cross-linked with 1% formaldehyde for 8 min. The reaction was terminated by incubation in 125 mM glycine for 5 min at room temperature. Then, the cells were resuspended in lysis buffer (50 mM Tris-HCL, pH 8.0, 10 mM EDTA, pH 8.0, 1% SDS, 1 mM PMSF, 20 mM sodium butyrate, and 1 × protease inhibitors) and sheared with a BioruptorPico sonication device (Diagenode, Belgium) to 200–500 bp. Premixed Protein A + G Magnetic beads (Thermo Fisher Scientific, USA) were bound to 8–10 μg anti-H2BK5bhb at 4 °C for 2–4 h and then incubated with DNA fragment at 4 °C for 3 h or overnight for immunoprecipitation in the IP group. The input group stored at 4 °C. Finally, the DNA fragments were washed, isolated, and purified by relevant buffers and reagents. End repair and ligation with adapters were carried out using an NEBNext Ultra EndRepair/dA-Tailing Module (E7442, NEB) and NEBNext Ultra Ligation Module (E7445, NEB), respectively. High-throughput sequencing of ChIP fragments was performed using an Illumina NextSeq 500, following the manufacturer’s protocol after quality evaluation of ultrasonic fragment (Additional file 2: Fig. S2). To align the reads to the mm9 reference genome, bowtie2 was used. PCR duplicates were removed using SAMtools (version 2.0.1). Normalized genome coverage tracks were generated from uniquely mapped reads using deepTools2 (version 3.1).

DNA fragment preparation was performed as described in 2.12. ChIP-qPCR primers for Gata2, Psen2, and Ptgr2 promoters are shown in Additional file 8: Table S3. The data were normalized to the adjusted input sample and analyzed by the percent input method.

RNA extraction and sequencing

Total RNA was extracted from FGSCs using Trizol reagent (15596026, Life Technologies). cDNA libraries were constructed using a VAHTSTM mRNA seq v2 Library Prep Kit for Illumina1 (Vanzyme, China). Sequencing libraries were constructed in accordance with the manufacturer’s protocol. The library quality was determined by a Bioanalyzer 2100 (Agilent, Santa Clara, CA, USA). The Illumina HiSeq 2500 platform (Illumina, San Diego, CA, USA) was used for RNA-sequencing (RNA-seq). The quality of RNA-seq data was evaluated using FastQC (Additional file 3: Fig. S3).

Gene ontology and Kyoto encyclopedia of gene and genomic pathway analysis

Gene Ontology (GO) analysis was performed to identify the biochemical processes of differentially expressed mRNAs. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis was applied to identify significant pathways of differentially expressed mRNAs using DAVID (http://david.abcc.ncifcrf.gov/home.jsp). Fisher’s exact test was used to identify significant results, and the false discovery rate was applied to correct P-values (P < 0.05).

Infection by shRNA-carrying lentiviruses

FGSCs were cultured to 50% confluence in a 48-well plate and incubated with a 100-μL mixture of culture medium and lentivirus solution (1:1 volume ratio). After 24 h of infection, the cells were cultured in normal FGSC medium for 24–48 h. Then, the cells were selected with puromycin (5 µg/mL) for 5–7 days to obtain stable Gata2-knockdown FGSCs.

Statistical analyses

All experiments were performed at least three times. Data are expressed as means ± SD. Two-tailed Student’s t-test was used to analyze differences. Statistical analysis was performed by GraphPad Prism 8.0 software (San Diego, USA). P < 0.05 was considered statistically significant.