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). The studies involving human participants were reviewed and approved by Ethical Committee of The First Affiliated Hospital of Xi’an Jiantong University. The participants provided their written informed consent to participate in this study. 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. The representative images in this part were captured using a microscope (Olympus Corporation, Tokyo, Japan).
Cell culture
KGN cells were obtained from Wuhan Procell (Procell Life Science&Technology Co., Wuhan, China). The KGN and EnSCs cell lines were cultivated in DMEM/F12 supplemented with 10% fetal bovine serum (FBS, SiJiqing, China). Cells were cultured in 5% CO2 humidified incubator at 37℃. Human EnSCs were maintained for 3-5 passages in culture, in order to ensure the EnSC-exosomes was fit for experiments.
Exosomes isolation, characterization and labeling
Exosomes were acquired from human EnSCs supernatants by differential centrifugation. In the first place, the medium was thrown away when EnSCs reached 80% confluency. Second, 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 gathered and then sequential centrifugation through 300 g for 20 min, 2000 g for 20 min and 10,000 g for 40 min at 4°C in order to filter cells and debris. Then, the supernatants were ultracentrifuged using 120,000 g for 90 min to discard cell supernatant and precipitate was washed using sterile PBS using 120,000 g for 90 min to discard PBS wash solution (Beckman OptimaTM L-80 XP, Beckman Coulter, USA). Finally, the precipitate was suspended in pre-cooled PBS to filtrate through 0.22 μm 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 verify the successful isolation of exosomes, Western Blotting (WB) was performed to detect the exosome marker proteins, such as 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 examine the existence of human EnSC-Exos. Exosomes were dropped onto a carbon-coated copper grid and stained with 2% uranyl acetate for 1 min.
The human 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 equal PKH26 (4 μM) for 4 min at room temperature and treated with equal exosome-depleted FBS to neutralize redundant dye. Finally, the PKH26 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 the granulosa cell injury model as previous (26). In order to clarify the effect of exosomes to repair granulosa cells (GCs) from cisplatin injury, 1 × 105 GCs were seeded into the of six-well plates and incubate for 24 h. Then the cisplatin (10 μM) was added to the GCs culture medium to induce apoptosis. After exposure to cisplatin for 24 h (10 μM), the GCs were cocultured with human EnSC-exosomes (200 μg/mL) or EnSC-exosomes (200 μg/mL) pretreated with Verteporfin (27) (VP, 1 μM, MedChemExpress, China, catalog no.: HY-B0146) 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 sterile PBS and then fixed in 4% paraformaldehyde for 10 min. After that, 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 observed by the fluorescence microscope (Nikon Ti-S, Nikon Corporation, Japan) and confocal laser scanning microscope (Leica TCS SP5 II, Leica Biosystems, Germany).
Immunofluorescence
Plant the cells on the slides and provide them with different treatments. Cells were washed twice with sterile PBS, and fixed with 4% paraformaldehyde for 15 min. Then, cells were washed in sterile PBS for three times and then 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
Total protein of different groups was gathered using RIPA/PMSF/PI (100:1:2) lysis buffer according to the manufacturer’s instructions and the protein concentration in different groups was calculated using the BCA protein assay kit (Proandy Biotechnology, Shaanxi, China). Total protein was separated by 10% Gels, under 60 V electrophoresis for 40 min, followed by 100 V electrophoresis for 60 min. Then, total protein of different groups after electrophoresis were transferred into a 0.22 μm NC membrane (PALL, Germany) using 320 mA for 100 min. After blocking with 5% non-fat milk in Tris-Buffered Saline and Tween (TBST) for 120 min at room temperature shaker (50 rpm). The membranes were incubated with primary antibody overnight at 4℃ refrigerator. The membrane was then washed with TBST 3 times for 8 min next day, afterwards, the membranes were incubated with secondary antibodies (HRP-labeled, dilution 1:3,000, cat. no.: ZB-2301, ZSGB-BIO, China) for 1.5 h. The membrane was washed 3 times for 10 min with TBST to detect using chemiluminescence detection reagent and Chemiluminescent Imager (Tanon-5200, Shanghai, China). All protein expression levels were normalized to the level of the internal standard control Glyceraldehyde-3-phosphate dehydrogenase (GAPDH, dilution 1:5,000, cat. no.: AP0063, Bioworld Technology, USA). The following antibodies were used for Western blot: 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 of cells was collected using Trizol reagent (Invitrogen, USA) and standardized total RNA (1.8 < A260/A280 < 2.2) was used in experiments. Total RNA (1 µg) was converted into cDNA using the PrimeScript™ RT reagent Kit with gDNA Eraser (Takara, Japan). After that, cDNA was subjected to PCR amplification using TB Green® Premix Ex Taq™ II (Takara) in Bio-Rad CFX (Bio-Rad, United States). The following thermocycling conditions were used for qRT-PCR: 95℃ for 1 min, 39 cycles with 95℃ for 20 sec, 60℃ for 20 sec, 72℃ for 30 sec. GAPDH was used as an internal control. The expressions of genes were quantified using the 2-∆∆Cq method. The primer sequences were as following:
ANKRD1-F: 5’-GCCAAAGACAGAGAAGGAGATAC-3’;
ANKRD1-R: 5’-GAGATCCGCGCCATACATAAT-3’;
CYR61-F: 5’-CACACCAAGGGGCTGGAATG-3’;
CYR61-R: 5’-CCCGTTTTGGTAGATTCTGG-3’;
CTGF-F: 5’-GGAAATGCTGCGAGGAGTGG-3’;
CTGF-R: 5’-GAACAGGCGCTCCACTCTGTG-3’;
GAPDH-F: 5'-GTGAAGGTCGGAGTCAACGG-3’;
GAPDH-R: 5'-GAGGTCAATGAAGGGGTCATTG-3’.
Flow cytometry assay
According to the manufacturer’s protocol, cisplatin-induced GCs apoptosis model and EnSC-Exos repair of the cisplatin-induced GCs injury model were determined by flow cytometry using the Annexin V-FITC Apoptosis Detection Kit (BD Biosciences, CA, USA). Briefly, cells were washed with cold PBS twice, then cells 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) in 1 h.
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 were 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, a total of 40 C57BL/6 female mice aged 6–8 weeks were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The animal study was reviewed and approved by Ethical Committee and the Institutional Animal Care and Use Committee of Xi’an Jiaotong University. The mice were bred in a free condition in a comfortable temperature.
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 from 11th day for 6 times; (4) Verteporfin group (n = 10): cisplatin (2 mg/kg) intraperitoneal injection daily for 10 days, Verteporfin (75 mg/kg) intraperitoneal injection every other day from 11th day for 6 times and exosome (350 µg/mouse) tail vein injection every other day from 11th day for 6 times at the same time. 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 22 days of treatment in different groups, blood collected from mice eyeball was transferred to sterile tube for centrifugation at 4500 r/min for 15 minutes at 4℃ (eppendorf, Germany) to obtain mice serum. Then, anti-Mullerian hormone (AMH), estradiol (E2) and follicle stimulating hormone (FSH) levels were detected using mice ELISA kit (Meimian Biotechnology, Jiangsu, China) according to the kit instructions.
Hematoxylin-eosin (HE) staining
First, mice ovarian tissues were fixed in 4% paraformaldehyde for 24 h, embedded in paraffin and cut into a 4 µm serial sections. Then, mice ovarian tissue sections were incubated in xylene, dehydration in alcohol gradient of 100%-70% and rehydrated. Finally, mice ovarian tissue sections were stained with hematoxylin and eosin (HE) after deparaffinization.
Immunohistochemistry (IHC)
First, mice ovarian tissues of different groups were fixed in 4% formaldehyde, paraffin-embedded and cut into a 4 µm serial sections. Then, serial sections were deparaffinized with xylenes, rehydrated and retrieved the antigen in sodium citrate solution (pH 6.0) for 20 minutes. After that the slides of the endogenous peroxidase were quenched using 3% hydrogen peroxide and blocked with 1% BSA to block the nonspecific binding for 30 minutes. Then mice ovary tissue sections were incubated with primary antibody anti-YAP (dilution 1:200, cat. no.: 13584-1-AP, Proteintech, USA) overnight at 4℃ refrigerator. The slides were washed using PBS for 5 times and incubate with Secondary antibody (HRP-conjugated, 1:1000, Santa, Cruze) for 1 h. Finally, the DAB substrate kit (Beyotime, Shanghai, China) was applied to detect peroxidase reactivity. DAB peroxidase substrate was prepared in 2 mL ddH2O in a clean bottle. Then, drop the DAB substrate on top of the slides and observe the brown staining using a microscope. Immerse the slides in tap water to stop the reaction and rinse under cold tap water for 5 min.
Statistical analysis
The statistical analyses were conducted through GraphPad Prism and SPSS. The cell experiments in this article were carried out in triplicate. All results are shown as the mean ± SD. Student's t-test and one-way ANOVA were applied to compare the two experimental groups and multiple groups, respectively. Significance is indicated as following: P < 0.05; **P < 0.01.