Isolation of EnSCs
Human endometrial tissues were acquired from the Department of Obstetrics and Gynecology, the First Affiliated Hospital of Xi’an Jiaotong University (Xi’an, Shaanxi, China). This study was approved by the Ethical Committee of the First Affiliated Hospital of Xi’an Jiaotong University and the participants have written an informed content. Human endometrial stem cells (EnSCs) were cultured and identified as our previous study described (24). The tissue is placed in glassware under aseptic conditions and cut into 1mm3 with ophthalmic scissors and then digested by collagenase type I for 1 hours in a 37℃ rotating shaker. The digestion was terminated using Dulbecco's Modified Eagle Medium/Nutrient Mixture F12 (DMEM/F12, HyClone, USA) complete media and filtered with 200 mesh screen, 400 mesh screen. Finally, the cell-debrispellet was obtained by centrifugation at 800g for 5 minutes. Images of representative fields were visualized via a microscope (Olympus Corporation, Tokyo, Japan).
KGN cells were obtained from Wuhan Procell (Procell Life Science&Technology Co., Wuhan, China). Cell lines (KGN, EnSCs) were cultivated in DMEM/F12 supplemented with 10% fetal bovine serum (FBS, SiJiqing, China). Cells were cultured in a 5% CO2 humidified incubator at 37℃. Human EnSCs were not maintained in culture for longer than 5 passages to ensure passage number remained fit for purpose.
Exosomes isolation, characterization and labeling
Exosomes were obtained from human EnSCs supernatants by differential centrifugation. The medium was discarded when EnSCs reached 80% confluency. Then, the cells were cultured in DMEM/F12 with 10% exosome-depleted FBS (VivaCell, cat. no.: C3801-0050, VivaCell Biosciences, Shanghai, China) for another 48 h. The supernatants were collected and then cleared by sequential centrifugation at 300 g for 10 min, 2000 g for 10 min and 10,000 g for 30 min at 4°C to filter cells and debris. The supernatants were ultracentrifuged at 120,000 g for 90 min to remove the supernatant (Beckman OptimaTM L-80 XP, Beckman Coulter, USA). Then precipitate was washed using phosphate-buffered saline (PBS) after which we centrifuged the supernatant at 120,000 g for 90 min at 4°C. Then, the precipitate was suspended in pre-cooled PBS to filtrate through 0.22-mm filters (Millipore, Billerica, MA, United States) (25). The exosome concentrations were determined with a BCA protein assay kit (Proandy, cat. no.:10136-1, Proandy Biotechnology, Shaanxi, China).
To confirm the successful isolation of exosomes, western blotting was performed to detect the exosome marker proteins anti-HSP70 (dilution 1:500, cat. no.: sc-32239), anti-Alix (dilution 1:500, cat. no.: sc-53540) and anti-CD81 (dilution 1:500, cat. no.: sc-166029) from Santa Cruz (Santa Cruz Biotechnology, United States), anti-CD9 (D8O1A) (dilution 1:1000, cat. no.: #13174) and β-Actin (8H10D10) (dilution 1:5000, cat. no.: #3700) antibodies from Cell Signaling Technologies (Beverly, MA). Transmission electron microscopy (TEM, hitachi H-7650, Japan) was performed to verify the presence of exosomes. Exosomes were dissolved in PBS buffer, dropped onto a carbon-coated copper grid, and then stained with 2% uranyl acetate.
The EnSCs-Exos were labeled with PKH26 (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer’s instructions. In short, EnSC-Exos were incubated with red fluorescent dye (PKH26) for 4 min and treated with 0.5% BSA to neutralize redundant dye. Then, the labeled exosomes were obtained after centrifuged at 120,000 g for 90 min at 4°C to remove contaminating dye.
Induction of GC apoptosis in vitro and coculture of GCs and EnSC-Exos
Cisplatin (Sigma-Aldrich, St. Louis, MO) was used to make the cisplatin-induced granulosa cell injury model as previous (26). In order to clarify the function of exosomes to repair granulosa cells from cisplatin injury, 1 × 105 GCs were seeded into the of six-well plates and incubate for 24 h. Then the cisplatin was added to the GC culture medium at 10 μM to induce apoptosis. After exposure to cisplatin for 24 h (10 μM), the GCs were cocultured with EnSC-exosomes (200 μg/mL) or EnSC-exosomes (200 μg/mL) pretreated with Verteporfin (27) (1 μM, VP, a YAP inhibitor, catalog no.: HY-B0146, MedChemExpress, China) in the system for another 72 h.
For uptake of labeled exosomes, the cisplatin-induced granulosa cell injury model incubation with 10 μg/mL, 200 μg/mL PKH26-labeled exosomes for 24 h. Cells were washed twice with PBS and fixed in 4% paraformaldehyde for 10 min, thereafter, the nucleic was stained with 4′, 6-diamidino-2-phenylindole (DAPI, Beyotime, cat. no.: P0131, Beyotime Biotechnology, Shanghai, China) and the cytoskeleton was stained with Actin-Tracker Green-488 (Beyotime, cat. no.: C2201S, Beyotime Biotechnology, Shanghai, China) according to the manufacturer’s instructions. The uptake of PKH26-labeled exosomes by cisplatin-damaged GCs was detected by a fluorescence microscope (Nikon Ti-S, Nikon Corporation, Japan) and confocal laser scanning microscope (Leica TCS SP5 II, Leica Biosystems, Germany).
Plant the cells on the slides and give them different treatments. Cells were washed twice with PBS, and fixed with 4% paraformaldehyde for 15 min. Then, cells were washed in PBS for three times and in 0.5% Triton X-100 (Beyotime, cat. no.: P0096, Beyotime Biotechnology, Shanghai, China) for 30 min. After that they were blocked with 5% BSA for 1 h. Next, they were incubated with primary antibodies (anti-YAP, dilution 1:100, cat. no.: 13584-1-AP, Proteintech, USA), secondary antibodies (Invitrogen) and DAPI (Beyotime, cat. no.: P0131, Beyotime Biotechnology, Shanghai, China). Using a fluorescence microscope (Nikon Ti-S, Nikon Corporation, Japan), images were captured.
Western blot analysis and antibodies
Cell lysates (total protein) were collected using RIPA lysis buffer and detected the protein concentration with a BCA kit. According to the manufacturer’s instructions, cell lysates were separated by 10% Bis-Tris Gels, under 72 V electrophoresis for 40 min, followed by 90 V electrophoresis for 90 min. After electrophoresis, proteins were transferred to NC membranes (PALL, Germany), under 320 mA for 100 min. After blocking with 5% non-fat milk or 5% BSA in TBS with 0.1% Tween-20 for 90 min at room temperature, the membranes were incubated with primary antibody overnight at 4℃. The next day, the membrane was then washed with Tris-Buffered Saline and Tween (TBST) for 30 min, the membranes were incubated with HRP-labeled secondary antibodies (dilution 1:3,000, cat. no.: ZB-2301, ZSGB-BIO, China) for 1.5 h, then the membrane was washed three times with TBST, chemiluminescence detection reagent was used to develop Chemiluminescent Imager (Tanon-5200). Gel image system was used to analyze the band density (Bio-Rad Laboratories, Inc). All protein expression levels were normalized to the level of the internal standard control GAPDH (dilution 1:4,000, cat. no.: AP0063, Bioworld Technology, USA). The following antibodies were used for immunoblotting: anti-Bcl-2 (dilution 1:1,500, cat. no.: 12789-1-AP), anti-Bax (dilution 1:1,500, cat. no.: 50599-2-Ig), anti-MST1 (dilution 1:1,000, cat. no.: 22245-1-AP), anti-LATS1 (dilution 1:1,500, cat. no.: 17049-1-AP) from Proteintech Group (Proteintech, USA), anti-Phospho-MST1 (Thr183)/MST2 (Thr180) (E7U1D) (dilution 1:1,000, cat. no.: #49332) antibody and anti-YAP (D8H1X) (dilution 1:1,000, cat. no.: #14074), anti-Phospho-YAP (Ser127) (D9W2I) (dilution 1:1,000, cat. no.: #13008), anti-Phospho-LATS1 (Ser909) (dilution 1:1,000, cat. no.: #9157), anti-CD9 (D8O1A) (dilution 1:1000, cat. no.: #13174) and β-actin (8H10D10) (dilution 1:5000, cat. no.: #3700) antibodies from Cell Signaling Technologies (Beverly, MA), anti-PCNA (dilution 1:500, cat. no.: sc-56), anti-Caspase-3 (dilution 1:500, cat. no.: sc-7272), anti-HSP70 (dilution 1:500, cat. no.: sc-32239), anti-Alix (dilution 1:500, cat. no.: sc-53540) and anti-CD81 (dilution 1:500, cat. no.: sc-166029) from Santa Cruz (Santa Cruz Biotechnology, United States).
RNA extraction and quantitative real-time PCR (qRT-PCR)
Total RNA was extracted using TRIzol reagent (Invitrogen, USA), and only highly pure RNAs (1.7 < A260/A280 < 2.2) were used. RNA (1 µg) was converted into cDNA using the PrimeScript™ RT reagent Kit with gDNA Eraser (Takara, Japan). After 10-fold dilution, 1000μg of cDNA was subjected to PCR amplification using TB Green® Premix Ex Taq™ II (Takara) according to the manufacturer’s protocol in a Bio-Rad CFX Manager. The following thermocycling conditions were used for qPCR: 95℃ for 1 min, then 39 cycles with 95℃ for 20 sec, 60℃ for 20 sec, 72℃ for 30 sec. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. The expressions of genes were quantified using the 2-∆∆Cq method. The primer sequences were as follows:
Flow cytometry assay
Cisplatin-induced granulosa cells apoptosis and EnSC-Exos repair of the cisplatin-induced granulosa cell injury model were determined by flow cytometry using a Annexin V-FITC Apoptosis Detection Kit (BD Biosciences, CA, USA) according to the manufacturer’s protocol. Briefly, cells were washed two times with cold PBS and stained with Annexin V-FITC (5 μL) and propidium iodide (5 μL) for 15 min at room temperature (in darkness). The stained cells were analyzed using flow cytometry (FC 500, MCL, CA).
5-ethynyl-2'-deoxyuridine (EdU) labelling staining
To assess appropriate concentration for EnSC-Exos contribute to cisplatin-damaged GCs repair and the proliferation rate of the cisplatin-induced granulosa cell injury model in different groups, BeyoClickTM 5‐ethynyl‐2‐deoxyuridine (EdU)-594 kit (Beyotime, cat. no.: C0078S, Beyotime Biotechnology, Shanghai, China) was used according to the manufacturer’s instructions. In brief, KGN cells (1ⅹ104) were cultured in a 96-well plate for 24 h, the cisplatin was added to the GCs culture medium at 10 μM for 24 h to induce apoptosis (the cisplatin-induced granulosa cell injury model), then 100, 200, 400 μg/mL EnSC-Exos were suspended in cisplatin-damaged GCs for 72 h to detect the appropriate concentration for EnSC-exos contribute to cisplatin-damaged GCs repair. To detect the effect of Hippo pathway inhibitor on KGN proliferation in different groups, the GCs were cocultured with EnSC-exosomes (200 μg/mL) or EnSC-exosomes (200 μg/mL) pretreated with Verteporfin (1 μM) in the system for 72 h.
For EdU labelling assay, cells were incubated with EdU (1:1000, 10 μmol/L) for 2 h. Then, KGN cells was fixed with 4% formaldehyde for 15 minutes. Next, permeabilized the cells in 0.5% Triton X‐100 (100 μL) for 15 minutes and added the BeyoClickTM Labeled-Azide reaction cocktail (100 μL) for 30 minutes under light‐shading conditions at room temperature. Finally, cells were counterstained with Hoechst 33342 (nuclear staining) for 30 minutes at room temperature. After 3 times washes with PBS, the images were acquired using luorescence microscope (Nikon Ti-S, Nikon Corporation, Japan). The proliferation rate of cells was assessed with the proportion of EdU-positive nucleus (red) to blue fluorescent nucleus.
Experimental animals, POF model establishment
To establish the POF model in mice, C57BL/6 female mice, aged 6-8weeks, body weight 16-18g, 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. The mice were bred in a free condition in which the temperature is at 22-25℃ with water and food available ad libitum.
To establish the POF model, mice were injected with cisplatin (2 mg/kg) intraperitoneally for 10 consecutive days (5). To demonstrat the therapeutic effect of EnSC-Exos on POF ovarian function through Hippo pathway, the mice were randomly divided into 4 groups: (1) Control group (n = 10), (2) Cisplatin group (n = 10), cisplatin (2 mg/kg) intraperitoneal injection daily for 10 days, (3) Exosome group (n = 10), cisplatin (2 mg/kg) intraperitoneal injection daily for 10 days, exosome (350 µg/mouse) tail vein injection every other day on 11th day for 12 days, (4) Verteporfin group (n = 10), cisplatin (2 mg/kg) intraperitoneal injection daily for 10 days, Verteporfin (75 mg/kg) intraperitoneal injection every other day on 11th day for 4 times and exosome (350 µg/mouse) tail vein injection every other day on 11th day at the same time for 12 days. The animals were euthanized by cervical dislocation after 22 days of treatment to collect serum and ovaries. We have recorded the body weight of each mouse every day (Figure 5A).
Enzyme Linked Immunosorbent Assay (ELISA)
After successful establishment of the POF mice model, the blood collected from the excised eyeball was transferred to a sterile tube for centrifugation at 3000 r/min for 10 minutes at 4℃, after which serum was obtained. Then, mice ELISA panel kits (Meimian Biotechnology, Jiangsu, China) were used to measure serum anti-Mullerian hormone (AMH), estradiol (E2) and follicle stimulating hormone (FSH) levels according to the kit instructions.
Hematoxylin-eosin (HE) staining
The ovarian tissues were fixed in 4% paraformaldehyde for 24 h and then embedded in paraffin, finally cut into a 4-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.
The ovarian tissues of mice were fixed in 4% formaldehyde and paraffin-embedded using standard procedures. First, consecutive 4-µm sections were cut, deparaffinized with xylenes, rehydrated, and retrieved the antigen in sodium citrate solution (pH 6.0) for 20 minutes. Then the slides were treated with 3% hydrogen peroxide to quench the endogenous peroxidase and blocked with 1% bovine serum albumin (BSA) for 30 minutes to block the nonspecific binding. Next, tissue sections were incubated with primary antibody anti-YAP (dilution 1:200, cat. no.: 13584-1-AP, Proteintech, USA) overnight at 4℃. Finally, wash the slides with PBS three times and incubate the slides with HRP-conjugated goat anti-rabbit IgG secondary antibody (1:1000, Santa, Cruze) for another 30 minutes. Finally, a 3, 3-diaminobenzidine tetrahydrochloride (DAB) (Beyotime, Shanghai, China) substrate kit was applied to detect peroxidase reactivity. According to the manufacturer instructions, we prepared DAB peroxidase substrate in 5 mL ddH2O in a glass vial. Then, drop the DAB substrate on top of the slides and watch the brown staining. Dip slides into ice plus tap water to stop the reaction and rinse under cold tap water for 5 min.
The statistical analyses were carried out using GraphPad Prism software. All experiments were performed in triplicate and all results are presented as the mean ± SD. Analysis of differences between the two groups were performed using Student’s t test, one-way ANOVA. Significance is indicated as follows: *P < 0.05, **P < 0.01.