Ethics statement and Animals
This study was approved by the bioethics committees of Southern Medical University and the Third Affiliated Hospital of Southern Medical University, Guangzhou, China. All the rats we need[Male SD: 3 to 4 weeks old] were purchased from Laboratory animal center of southern medical university and kept under specific pathogen-free conditions. All animal experiments were approved by the Ethics Committee of Southern Medical University. Guidelines of the Institutional Animal Ethics Committee were followed when carrying out in vivo experiments. All efforts were made to minimize animal suffering.
Isolation of MSCs from mouse bone marrow
Healthy Male SD rats weighted about 80-120g were used to isolate bone marrow-derived MSCs which were killed by cervical dislocation. Under sterilized conditions ，The skin was disinfected with 75% alcohol and then The rats were anaesthetized using pentobarbital (50 mg/kg, intraperitoneal injection) to minimize suffering and sacrificed by cervical dislocation without recovery from anesthesia. Tibias and femurs then were removed under sterile conditions. In the laboratory and under sterilized conditions, the femurs and tibias were cleaned off from the remaining muscle tissues with sterile surgical tools and washed few times with normal saline solution. The tip of the bone was inserted into the spiral tip bottom centrifuge tube(15ml)after ends of femurs and tibias the were cut. Bone marrow cell suspension was obtained by flushing marrow cavity using dispensable 1-ml syringe with low-glucose dulbecco’s modified eagle medium (DMEM).Then bone marrow cell suspension was filtrated through 200-mesh sieve in order to remove bone debris. the sediment which Contains bone marrow cell was acquired after centrifugation of the filtered suspension at 1000 rpm/min for 5 min. Remove the supernatant, the sediment was suspended with A moderate amount of DMEM .bone marrow cells suspension were shifted into petri dishes On average and incubated at 37°C in a humidified atmosphere containing 5% CO2,adn 95% air with low-glucose DMEM plus 10% heat-inactivated fetal bovine serum (FBS, Gibco, Grand Island, NY, USA) and 1% penicillin–streptomycin (Beyotime Biotechnology, Shanghai, China).
Culturing and propagation of BMSCs
According to the report(16. Freshney RI. Culture of Animal Cells. A Manual for Basic Technique. 5th ed. New York, NY: John Wiley & Sons; 2005. [Ref list]),The proportion of adhere cells and nonadherent cells would change after overnight incubation with medium. adhere cells was the majority, and the little bit of nonadherent cells were washed out with medium changes in MEM media with 20% FBS.so The remaining nonadherent cells were removed by exchanging the culture medium within every 2-3 days until the cultures become the developing colonies of adherent cells (about 7 days) and then shift to monolayer cells. Cells were subculture after being monolayered using 0.25% trypsin ethylenediaminetetraacetic acid (EDTA) (United States Biological). The morphological changes and growth conditions were observed with inverted phase contrast microscope .The passage one (P1) cells began to proliferate and form a monolayer of cells in 3–5 days (with 3.0× 106).Cells at 4th to 8th passage were utilized for subsequent experiments.
In short, the exosomes were isolated using gradient centrifugation.in order to exclude exogenous exosome contamination, BMSCs at 4th to 8th passage was cultured with exosome-free serum and the cell culture medium was collected. the culture supernatants were cleared of cell debris and large vesicles by sequential centrifugation at 300g for 10 min, 1000g for 20 min, and 10,000g for 30 min, followed by filtration using 0.45um and 0.22um sterile filters， Then, the cleared sample was centrifugal at 100,000g for two hour to pellet the exosomes. To get a purer exosome, Re-speeding in the same condition after removing the and the precipitating are suspended with phosphate buffer solution (PBS).The recovery of exosomes was estimated by measuring the protein concentration using the BCA Protein Assay Kit( Beyotime Biotech, Jiangsu, China).The obtained exosomes fraction was re-suspended in PBS (500 ul, 1 mg/mL total protein).
The concentrations of all the protein we extracted from cells , exosomes and tissues were determined using a BCA protein assay kit ( Beyotime Biotech, Jiangsu, China) according to manufacturer instructions. Subsequently, a certain amount of total protein[20ug-80ug] was heated to 95 °C for 10 min in 1 × DTT‐containing sodium dodecyl sulfate (SDS) sample buffer and separated by 10% SDS‐polyacrylamide gel electrophoresis, followed by transfer onto polyvinylidene fluoride membranes (BioTrace; Bio‐Rad, Hercules, CA, USA). Membranes were blocked with 5% bovine serum albumin for 1 h, and polyclonal antibodies, including anti‐CD63,anti‐CD81, anti-TSG101,anti-AKT,anti-p-AKT anti‐ALIX, anti‐HMGB1, anti‐Caspase-3,anti‐Bcl2 (Abclonal, Woburn, MA, USA), anti‐ACTB, anti‐GAPDH (Ray Antibody, Beijing, China), and anti‐Calnexin (Bioworld, Dublin, OH, USA), were used for immunoblotting at 4 °C overnight. Each specific horseradish peroxidase‐conjugated secondary antibody (Ray Antibody) was added accordingly after the membranes were washed in TBS‐Tween solution (TBS-T) three times for 30 min, and signals were detected by enhanced chemiluminescence (Pierce, Rockford, IL, USA).
Nanoparticle tracking analysis (NTA)
Particle size and concentration distribution of the isolated exosomes stemmed from BMSCs were measured using NTA (v2.3; Malvern Instruments, Malvern, UK) according to manufacturer's instructions. Briefly, exosomes samples were vortexed and diluted to a final dilution of 1 : 2000 in filtered molecular‐grade H2O. Blank‐filtered H2O was run as a negative control. Each sample analysis was conducted for 60 s and measured three times using Nanosight automatic analysis settings.
Transmission electron microscopy (TEM)
TEM analysis was performed to confirm BMSCs-derived exosomes morphology. Briefly, exosomes samples were diluted with PBS to the appropriate concentration, and ~20–40 μL of PBS solution containing exosomes was transferred to a copper grid for incubation at room temperature for 5 min. Filter paper was used for absorbing unevaporated solution. Exosomes samples were negatively stained with 4% paraformaldehyde acid solution at room temperature for 5 min and dried at 65 °C for 10 min. Images of exosomes samples were obtained using a Hitachi H‐7650 transmission electron microscope (Hitachi, Tokyo, Japan).
BMSCs-derived exosomes uptake by GC-1 cells
Purified BMSC-Exo were labeled with 1 μM PKH67 (Invitrogen) as previously described. Briefly, BMSC-Exo were mixed with 1 μM PKH67, and the exosome-dye suspension was incubated for 5 min with regular mixing. Excess dye from the labeled exosomes was removed by ultracentrifugation at 100,000 g for 1 h at 4°C using a 70Ti rotor (Beckman Coulter), and the exosome pellets were washed three times by resuspending them in PBS. The final pellets were resuspended in PBS. PKH67-labeled exosomes were co-cultured with Spermatogonium cells(GC-1) for 6 h, then GC-1 were washed with PBS, and fixed in 4% paraformaldehyde (PFA).The nucleus of cells were stained using medium containing 4,969-diamidino-2-phenylindole (DAPI; Vector Laboratories, USA). The uptake was observed by fluorescence microscopy.
Under the condition of Specific pathogen Free(SPF) animal laboratory, Altogether 30 male Sprague -Dawley rats were randomly divided into 3 groups of 10 each :sham-operated control group ,experimental group(I/R) and Treated group(I/R+EXO).In order to induce the ischemia-reperfusion model of testis, rats were anesthetized with xylazine (20 mg/kg) and ketamine (50 mg/kg) after Overnight fasting。In the sham-operated control group, rats underwent left scrotal exploration only with similar surgical procedures without the torsion and detorsion. And the experimental and Treated group were through a sub-inguinal incision the left testis was brought out and was rotated 720°clockwise and then inserted and fixed into the scrotum with a 4/0 silk suture placed through the tunica albuginea and subcutaneous tissue. The incision was primarily closed with a 4/0 silk suture. After 4 hours, by using the same incision line, testis was counter rotated to the natural position and reinserted into the scrotum to induce reperfusion for 2 hours.After that ,the two groups ,experimental and Treated group, were respectively injected from the tail vein with the same amount of saline and 100ug/ml exosomes. At the end of the reperfusion period rats were decapitated and testis tissues were taken for biochemical analyses and histological evaluations. Biochemical and histological samples were blindly examined.
Distribution of exosome bodies in vivo (tracking)
According to the manufacturer's instructions, exosomes derived from the BMSCs were isolated as described above were labeled using PKH67 Fluorescent Cell Linker kits (Sigma-Aldrich, St. Louis, MO).The washed exosomes pellets from the 100 ml culture media were resuspended in 700 µl of Diluent C(exosomes solution). PKH67 dye (1 µl) was diluted in 250 µl of Diluent C (PKH67 solution). Then, 250 µl exosomes solution and 250 µl PKH67 solution were mixed in a 4.7 ml centrifugation tube. Samples were mixed gently for 4 min, and 4.2 ml of 1% BSA was added to bind the excess PKH67 dye. PKH67-labeled exosomes were ultracentrifuged at 120,000 x g for 3 h at 4˚C using the Optima Ultracentrifuge (Beckman Coulter). Exosome pellets were washed three times in PBS by ultracentrifugation. Finally, PKH67-labeled exosomes were resuspended in D-MEM or RPMI-1640 medium. As the negative controls, no PKH67 control and no exosome control were prepared. Exosomes were collected by ultracentrifugation without PKH67 dye, and then D-MEM or RPMI-1640 medium were added to the centrifuged tubes (no PKH67 control). After PKH67 dye was washed by ultracentrifugation without exosomes, the supernatant was discarded and media described above were added to the centrifuged tubes (no exosome control).then the equal amount of exosome was injected into different mice through the tail vein.
For anoikic analysis, TM4 and GC1 cells were cultured in 80 µmol/L H202 for 8 h. Cells were then trypsin zed and stained with Annexin V- APC/7AAD and analyzed by flow cytometry using an Annexin V- APC/7AAD Apoptosis Detection kit (BD Biosciences) according to the manufacturer’s instructions. Data were collected on a BD Franciscan and analyzed using the FlowJo software
Testes were removed from the rats and fixed with 10% formaldehyde. Thereafter, the testes were dehydrated with subsequent 70, 90, 96 and 100% ethanol and cleared with toluene. After overnight incubation of paraffin in a 60 °C incubator, testes were embedded and blocked in paraffin at room temperature. Approximately 5 μm thick paraffin sections in midline area of the testis were stained with hematoxylin and eosin (H&E). In each section at least 30 seminiferous tubules were evaluated microscopically at × 200 magnification. The first seminiferous tubules were selected randomly and the others were taken by sliding the section towards the clockwise.
Biochemical indicators Analysis
An appropriate amount of testicular tissue and aseptic saline were prepared with a ratio of 1:9 to 10% tissue homogenate and then determination of the supernatant after centrifugation for various indicators. The testicular homogenates were assayed for total antioxidant capacity (T-AOC), superoxide dismutase (SOD),nitric oxide synthesis(NOS: tNOS and iNOS)，Catalase (CAT), Glutathione (GSH) and malondialdehyde (MDA) by using colorimetry for all the oxidation indicators. The specific determination principle of each index and the preparation method of reagent and the determination method of each indicator protein content were strictly carried out according to the instructions of the kit(Institute of Bioengineering，Nanjing，Jian Cheng，China).
For the all experiments, data are presented as the mean ± S.E.M. Tests for significant differences between the groups were performed using a t-test or one-way ANOVA with multiple comparisons (Fisher's pairwise comparisons) using GraphPad Prism 5.0 (GraphPad software, San Diego, CA, USA).A minimum p value of 0.05 was chosen as the significance level.