Male Sprague-Dawley rats (200–250 g) at 8–10 weeks of age were purchased from the Animal Experiment Center of Harbin Medical University. All experiments involving animals were approved by the Ethics Committee of Harbin Medical University (no. Ky2018-135).
Isolation and identification of ADSCs and ADSC-Exos
The ADSCs and ADSC-Exos were isolated as in our previous study . Briefly, ADSCs were isolated from subcutaneous fat and cultured in DMEM/F12 (Sigma, MO, USA), supplemented with 10% FBS (Sigma), and 1% penicillin and streptomycin (Beyotime, Haimen, China) at 37 ℃ with 5% CO2. The culture medium of ADSCs at passage three was replaced with exosome-depleted medium for 24 h and collected to isolate ADSC-Exos by ultracentrifugation, as previously reported . The surface markers (CD90, CD105, CD34, CD45, CD11b), and multilineage differentiation capacity (adipogenic, osteogenic, and chondrogenic) of ADSCs have been demonstrated in our previous study . Nanoparticle tracking analysis, transmission electron microscopy (TEM), and western blotting were used to identify the collected exosomes.
Experimental protocols and surgical procedures
Sixty rats were randomly divided into three groups: (1) Control (n = 12): animals received surgery for scrotal incision and suturing; (2) I/R (n = 24): animals received surgery for testicular torsion and local injection of 100 μL PBS before detorsion; (3) ADSC-Exos (n = 24): animals received a local injection of 100 μL PBS containing 400 μg ADSC-Exos before detorsion.
All surgical procedures were performed under aseptic conditions by anesthesia with ketamine (50 mg/kg). The left testis was rotated 720° in the anticlockwise direction for 3 h. After that, the testis was detached via the same surgical approach. Half an hour before detorsion, 100 μL ADSC-Exos or PBS was injected into the testis. The animals were sacrificed on the third and seventh postoperative days to collect the testes and epididymides for further study.
Determination of spermatozoal parameters
Rat epididymal tissues were cut into 1 mm3 cubes and immersed in 0.9% NaCl at 37 ℃ for 20 min to extract the spermatozoa. Sperm quality (quantity, morphology, and motility) was assessed using the WHO sperm analysis method . The morphology and motility of 200 sperm in each group were evaluated.
Histopathological and immunohistological analyses
Testicular tissues were fixed in Davidson's fixative (Beyotime) and the tissue sections were stained with hematoxylin and eosin (H&E). Johnsen’s score (Table 1) was used to evaluate the spermatogenic function . Fifty seminiferous tubules were examined in each testis.
The tissue sections were immunohistochemically stained by primary antibody anti-Ki-67 (Affinity Biologicals, ON, Canada), followed by incubation with goat anti-rabbit IgG secondary antibody (Abcam, Cambridge, UK). For immunofluorescence analysis, the tissue sections were incubated with primary antibodies anti-cleaved Caspase-3 (Abcam), anti-CCR7 (Abcam), anti-CD163 (Abcam), anti-IL-6 (Origene), and anti-IL-10 (Abcam), followed by incubation with secondary antibodies (Abcam). Nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI). Positive signals were quantified using Image J software.
The malondialdehyde (MDA) in testicular tissue was determined by colorimetry measurement using the Lipid Peroxidation MDA Assay Kit (Beyotime). Superoxide dismutase (SOD) in testicular tissue was detected using the Cu/Zn-SOD, and Mn-SOD assay kits with WST-8 (Beyotime).
Cell culture and treatment
The GC-1 spg cell line was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were cultured in DMEM with high glucose (Sigma) supplemented with 10% FBS, and 1% penicillin and streptomycin at 37 ℃ with 5% CO2. To establish the I/R model in vitro, 1 × 106 cells were cultured in glucose-free DMEM (Sigma) in a 1% O2 environment for 18 h, followed by reoxygenation with normal O2 in complete medium with or without exosomes.
In vitro, we randomly sorted GC-1 spg cells into six groups as follows: (1) Control: cells cultured under normal conditions; (2) I/R: induction of cellular I/R injury only; (3) ADSC-Exos (100): induction of cellular I/R injury, and reoxygenation with 100 μg/mL ADSC-Exos; (4) ADSC-Exos (200): reoxygenation with 200 μg/mL ADSC-Exos; (5) ADSC-Exos+LY: pretreated with 50 μM LY294002 (PI3K/AKT inhibitor; MedChemExpress, NJ, USA) for 30 min before reoxygenation with 200 μg/mL ADSC-Exos; (6) ADSC-Exos+PD: pretreated with 50 μM PD98059 (MAPK/ERK1/2 inhibitor; MedChemExpress) for 30 min before reoxygenation with 200 μg/mL ADSC-Exos. Cells from each group were collected after 30 min or 24 h for western blotting, after 3 h for flow cytometric analysis or the TUNEL assay, and after 24 h for EdU, transwell, and scratch assays.
ADSC-Exos internalization analysis
ADSC-Exos were labeled using PKH26 dye (Sigma). After ultracentrifugation, PKH-26 labeled ADSC-Exos were added to GC-1 spg cells cultured in exosome-depleted medium. The nuclei were counterstained with DAPI after 24 h, and the internalization of ADSC-Exos was observed under a fluorescence microscope.
Proliferation of GC-1 spg cells
For cell proliferation analysis, 10 μM 5-ethynyl-2-deoxyuridine (EdU) was added to the GC-1 spg cell culture medium for 30 min. Then, the cells were fixed and stained using an EdU assay kit (UE, China). Next, cell proliferation was observed under a fluorescence microscope after DAPI counterstaining of the nuclei.
Migration of GC-1 spg cells
For cell migration analysis, we chose the scratch test and transwell assays. Briefly, after hypoxic injury with a serum-free culture medium, a scratch was made through the cultured cells. The extent of cell migration was measured after 0 and 24 h.
Each group of cells after hypoxic injury was digested for transwell assays. A total of 1 × 105 GC-1 spg cells were cultured in the upper chambers. The reoxygenation medium was added to the lower chambers. The upper chamber was fixed with paraformaldehyde and stained with crystal violet after 24 h.
Western blot analysis
Cells were lysed in RIPA buffer (Beyotime) to extract the proteins. Immunoblotting was performed with the primary antibodies, anti-Hsp70, anti-TSG101, anti-CD9, anti-AKT, anti-phospho-AKT, anti-ERK1/2, anti-phospho-ERK1/2, anti-Bcl-2, anti-Bax, and anti-β-actin (all from Abcam), according to the manufacturer’s instructions. Goat anti-rabbit IgG (Abcam) was used as the secondary antibody. Image J software was used for quantification of protein bands.
Flow cytometry analysis
To analyze the apoptosis, cells were collected and stained with the FITC Annexin V Apoptosis Detection Kit (Becton-Dickinson, USA), and then analyzed by flow cytometry. Becton-Dickinson in-house FACSDiva software was used to analyze the data.
Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay
In vivo, apoptosis of spermatogenic cells was detected using a TUNEL apoptosis assay kit (Wanleibio, Shenyang, China). Briefly, paraffin sections of testicular tissues were incubated with 50 μL TUNEL reaction mixture. The sections were dehydrated and fixed after hematoxylin counterstaining of the nuclei. Apoptosis of spermatogenic cells was evaluated under a light microscope.
In addition, we performed a TUNEL assay in vitro. Briefly, GC-1 spg cells from each group were fixed and stained with a TUNEL Assay Apoptosis Detection Kit (UE, China). Next, apoptosis was observed under a fluorescence microscope after DAPI counterstaining of the nuclei.
Micro RNA (miRNA) sequencing and data analysis
The sequencing of miRNA in ADSC-Exos was performed by OE Biotech Company (Shanghai, China). Briefly, 20 ng of exosomal RNA was extracted and sequenced using an Illumina HiSeq 2500 instrument (Illumina, CA, USA) (n = 3). The top 50 highly expressed miRNAs in ADSC-Exos were predicted to target genes using miRanda software. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses of target genes were performed using DAVID (https://david.ncifcrf.gov/), and KOBAS 3.0 (http://kobas.cbi.pku.edu.cn/kobas3/), respectively. The results of the analyses were visualized using R software.
Data were expressed as the mean ± standard deviation (SD). Statistical analysis for multiple groups were conducted by the Tukey-Kramer t-test. P < 0.05 was considered statistically significant.