2.1 Cell culture
Human ovarian granulosa cell lines KGN (Procell CL-0603) were kindly provided by Procell Life Science＆Technology Co., Ltd (Wuhan, China). Cells were culture in DMEM/F12 with 10% (FBS) in an incubator at 37℃ with 5% CO2. The medium was changed every two days.
2.2 Isolation of menstrual-derived stem cells (MenSCs)
This study was approved in advanced by the Ethical Committee of The First Affiliated Hospital of Xi’an Jiaotong University, and all the participants have written an informed content. MenSCs were isolated and cultured as our previous study described. Briefly, collecting the menstrual blood from 6 healthy women (about 25-30 years old), then transferring the blood into a 50 ml centrifuge tube, which pre-contained 10 ml of phosphate-buffered saline (PBS), 100 U/ml penicillin, 100 mg/ml streptomycin, amphotericin B (0.25 mg/ml), and ethylenediaminetetraacetic acid (EDTA) (2 mM) (Gibco, Grand Island, NY, USA). Ficoll-Paque Plus (GE Healthcare Amersham, UK) was used to separate and purify the MenSCs. Finally, cells were culture in DMEM/F12 (Hyclone, USA) with 10% fetal bovine serum (FBS) (Sijiqing, China), in a T25 flask (Corning, New York, USA) at 37℃in 5% CO2.
2.3 establishing granulosa cell injury model and MenSCs-injured granulosa cells co-culture model
For the granulosa cell injury model, KGN was removed from the T25 flasks and seeded in a 96-well plate at a density of 1ⅹ104. CDDP (Sigma–Aldrich, St. Louis, MO) was used to make the granulosa cell injury model. After overnight culture, removed the medium and replaced the fresh medium contained CDDP (0-20 µM), and the 50% inhibitory concentration (IC50) was chosen for the injury model in the later experiments.
For the MenSCs-injured granulosa cells co-culture model, we need to pre-seed the KGN (2ⅹ105) in a 6-well plate and incubate for 24 h. Then, the CDDP was added for another 48 h. Finally, a 6-well transwell insert (pore size 4µm) was applied, and the MenSCs were added in the up chamber (the numbers of MenSCs: KGN were 3:1) for another 48h culturing at 37℃ with 5% CO2.
2.4 Experimental animals, POF model establishment
To establish the POF models, C57BL/6 female mice, aged 6-8weeks, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. All experimental procedures were approved by the Ethical Committee and the Institutional Animal Care and Use Committee of Xi’an Jiaotong University. All animals were housed in a relatively stable environment with a cycle of lights on at 8 a.m. and off at 8 p.m., maintained room temperature (21-25℃) with water and food available ad libitum.
To establish the POF model, mice were injected with CDDP (2mg/kg) intraperitoneally for 7 consecutive days, according to our previous study.
2.5 Experimental design
2.5.1 experiment 1
To demonstrated the protective effects of MenSCs transplantation on POF ovarian function, the POF mice were divided into two groups and administered either treatment of MenSC (passage 3-5, 200µl cell suspensions containing 2ⅹ106) by tail vein injection or an equal volume of the medium. Sacrifice the animals after 7 days of treatment, and collect the serum and ovaries
2.5.2 experiment 2
To explore the effects of FTO inhibitor MA on POF ovarian function, the mice were randomly divided into 4 groups: (1) DMSO group (dimethyl sulfoxide, a solvent of MA); (2) MA group; (3) cisplatin group; (4) cisplatin + MA group. The POF models were established as mentioned earlier. The MA was administered simultaneously with cisplatin injection at the dose of 10mg/kg. After 7 days of treatment, the animals were sacrificed and the serum and ovaries were collected.
2.6 Enzyme Linked Immunosorbent Assay (Elisa)
To assess the ovarian function, we collected the blood samples by eyeball extracting. Then, samples were coagulated for 2 h at room temperature and centrifuged at 3000r/min for 10 minutes at 4 ℃ to acquire the serum samples. Then, mice Elisa panel kits (Meimian Biotechnology, Jiangsu, China) were used to measure serum oestradiol (E2), FSH, and AMH levels according to the kit instructions.
2.7 Hematoxylin-eosin staining
The ovarian tissues were embedded in paraffin and then cut into 5-µm serial section. Then the tissue sections were rehydrated by incubating in xylene and subjecting to an alcohol gradient of 100%-70%. After deparaffinization, the section was stained with hematoxylin and eosin (HE).
Immunohistochemistry (IHC) was performed as previously described. The ovarian tissues of mice were fixed in 4% formaldehyde and paraffin-embedded using standard procedures. First, consecutive 4-mm sections were cut, deparaffinized, rehydrated, and retrieved the antigen in sodium citrate solution (pH 6.0) for 20 minutes. Followed by 3% hydrogen peroxide and 1% bovine serum albumin blocking for 30 minutes, separately. Then, tissue sections were incubated with anti-FTO (Abcam, Cambridge, USA; 1:150) overnight at 4 °C and a biotinylated secondary antibody (1:1000, Santa, Cruze) for another 30 minutes. Finally, a 3, 3-diaminobenzidine tetrahydrochloride (DAB) (Beyotime, Wuhan, China) substrate kit was applied to detect peroxidase reactivity.
2.9 Plasmids and small interfering RNA transfection
Overexpression and inhibition of FTO were achieved by transfection of pCAG-FTO (Miaoling, Wuhan, China) and FTO small interfering RNAs (siRNAs) (Ribobio, Guangzhou, China) separately. The empty vector for the plasmids and siRNAs were also included. The transient transfection of KGN was performed using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. In brief, a total of 2ⅹ105 cells per well were seeded into a 6-well plate. Until 60-80% confluence, 3.0µg vector DNA or 50nM siRNA were transfected by 3.0µL Lipofectamine 2000 per well for 6 h, then the medium was switched to fresh DMEM/F12. The overexpression and knockdown efficiency of the target gene was measured by qRT-PCR and western blotting.
2.10 Western blotting analysis
Cells were harvested after 48 h incubation. The total protein was extracted by RIPA buffer, which was pre-added with protease inhibitor cocktail and PMSF. BCA kit (Beyotime, China) was used to detect the protein concentration. 30 µg of protein was subjected to 10% SDS - PAGE gels and then transferred polyvinylidene difluoride (PVDF) membrane. The membranes were blocked in 0.1% TBST (Tris – HCl buffer saline with 0.1% Tween – 20) contained 5% skimmed milk. Then, these membranes were incubated with corresponding primary antibodies in 4℃ overnight. The primary antibodies are as follows: FTO (1:1000, ab126605 Abcam, USA), BNIP3(1:1000, ab109362 Abcam, USA), BAX (1:1000, 50599-2-Ig, Proteintech, China), Bcl-2(1:1000, 12789-1-AP, Proteintech, China), β-actin(1:1000, 66099-1-Ig, Proteintech, China). The next day, these membranes were incubated with peroxidase - conjugated secondary antibodies at room temperature for 1 h. Finally, these membranes were visualized by enhanced chemiluminescence (ECL) procedure.
2.11 RNA extraction and quantitative real-time PCR (qRT-PCR)
Total RNA from cells was isolated using RNAiso Plus (Takara, Japan) according to the reagent instruction. Genes primers used in this study were as follows: FTO (forward, 5’ – CTTCACCAAGGAGACTGCTATTTC - 3’, reverse, 5’ - CAAGGTTCCTGTTGAGCACTCTG - 3’), METTL3 (forward, 5’ – TTGTCTCCAACCTTCCGTAGT – 3‘， reverse， 5’ – CCAGATCAGAGAGGTGGTGTAG – 3’), METTL14 (forward, 5’ – ACCTTGGAAGAGTGTGTTTACGA – 3’, reverse, 5’ – TGTGAGCCAGCCTTTGTTCT – 3’), WTAP (forward, 5’ – TGTGCTGTGTAAGGGCATTCGTACTCATGC – 3’, reverse, 5’ – ACTGGGCAAACTTGGCAGTCATAAACCCAC – 3’), ZC3H13 (5’ – AAAGGAGGTTTCACCAGAAGTG – 3’, reverse, 5’ – CGCTTCGGAGATTTGCTAGAC – 3’), KIAA1429 (5’ – AAGTGCCCCTGTTTTCGATAG – 3’, reverse, 5’ – ACCAGACCATCAGTATTCACCT – 3’), RBM15 (5’ – AGCCGCGAGTATGATACCG – 3’, reverse, 5’ – GCCCGAAGAATTTTTGGTGCTC – 3’), YTHDF1 (forward, 5’ – AACAATGAGGGCGAACCAGT – 3’, reverse, 5’ – GACACACTGGAGCTGACCAA – 3’), YTHDF2 (forward, 5’– TAGCCAACTGCGACACATTC – 3’, reverse, 5’ – CACGACCTTGACGTTCCTTT – 3’), YTHDF3 (forward, 5’ – TGACAACAAACCGGTTACCA – 3’, reverse, 5’ – TGTTTCTATTTCTCTCCCTACGC – 3’), YTHDC1 (forward, 5’ – TCATCTTCCGTTCGTGCTGT – 3’, reverse, 5’ – TACAGGGAGCGTGGACCATA – 3’), GAPDH (forward, 5’ – AAAATCAAGTGGGGCGATGCT – 3’, reverse, 5’ - TGGTTCACACCCATGACGAAC), BNIP3 (forward, 5’ – TGAGTCTGGACGGAGTAGCTC – 3’, reverse, 5’ – CCCTGTTGGTATCTTGTGGTGT – 3’), BAX (forward, 5’ – AGTGGCAGCTGACATGTTTT – 3’, reverse, 5’ – GGAGGAAGTCCAATGTCCAG – 3’), Bcl-2 (forward, 5’ – TTCCACGCCGAAGGACAGCG – 3’, reverse, 5’ - GGCACTTGTGGCGGCCTGAT – 3’). Real-time quantitative PCR was performed using SYBR Premix ExTaq™ (Takara) on a StepOne Real – Time PCR System (7300 Real – Time PCR system, Applied Biosystems, USA). The reaction conditions were as follows: 95℃ for 30s, 40 cycles at 95℃ for 15s, 60℃ for 30s, and extension at 72℃ for 60s. The target genes relative expression levels were analyzed by the 2-ΔΔCt method.
2.12 Cell counting kit-8 (cck-8) assay
The cell proliferation was determined by using the Cell Counting Kit-8 (CCK8) assay. In brief, 1ⅹ104 HTR8/SVneo cells per well were plated into a 96-well plate. Then, cells were cultured at 37℃ with 5% CO2 for 24 h. Next, 10ul of CCK8 reagent (Dojindo, Kumamoto, Japan) and 90ul culture medium were added to per well and incubated at 37℃ for 2 h. Finally, measured the absorbance at 450nm at 24 h, 48h, and 72 h.
2.13 EdU labeling
To assess the KGN proliferation, 5‐ethynyl‐2‐deoxyuridine (EdU) Apollo567 kit (Ribobio Co., Ltd.) was used. First, KGN (1ⅹ104) were cultured in a 96-well plate and incubated with EdU (1:1000, 50 μmol/L) for 2 h. Then, KGN was fixed with 4% formaldehyde for 30 minutes at room temperature, followed by incubation with glycine (2mg/ml) and PBS for 5 minutes respectively. Next, permeabilized the cells in 0.5% Triton X‐100 (100 μL) for 10 minutes and added the Apollo® reaction cocktail (100 μL) for 30 minutes under light‐shading conditions at room temperature. Finally, 4′,6′‐diamidino‐2‐phenylindole (DAPI) was used to counterstain the nuclei for 30 minutes at room temperature. After 3 times washes with PBS, the images were acquired using fluorescent microscopy (Olympus, Japan).
2.14 Flow cytometry analysis
Annexin V – FITC Apoptosis Detection Kit (BD Biosciences, CA, USA) was used to assess the KGN cell apoptosis. Briefly, KGN was harvested and washed in ice-cold PBS. Then, resuspended the cells in binding buffer (200 μL) and added the Annexin V – FITC (5 μL) and propidium iodide (5 μL) in it at room temperature for 10 minutes (in darkness). Finally, added 300 μL binding buffer to each tube and the percentage of apoptotic KGN cells were analyzed using flow cytometer (FC 500, MCL, CA).
2.15 Statistical analysis
All the experiments were carried out in triplicate. The data were represented as mean ± SEM and statistical analysis was conducted with GraphPad Prism 5. Student's t-test and one-way ANOVA were applied to compare the two experimental groups and multiple groups, respectively. P value ＜ 0.05 was considered statistically significant.