The protocol of experimental animals was approved by the Ethics Committee and Institutional Animal Care and Use Committee. 36 healthy male mice (C57BL/6, 5-week-old; body weight, 16–18 g) were purchased from the Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences (Lanzhou, China) and adaptively fed for one week before experimentation. The mice were housed in cages (n = 3/cage) according to the previous condition, randomly and equally assigned to the control group and irradiated group.
Animals were enterocoelia irradiated with X‐rays performed at the Medical Electron Linear Accelerator (EDGE, Varian, CA, USA) of the Gansu Provincial Hospital with A 6MV flattening filter (FF) beam (LET = 8 GeV/µm, dose rate 6 Gy/min) at a dose of 4 Gy under the same irradiation conditions, the testes were separated at 6, 12, and 24 h for analyzing after irradiation.
The GC-1 cells derived from mouse spermatogonia (no. BNCC338398) (BeNa, Culture Collection, Beijing, China) were cultured in the complete DMEM high-glucose medium (with 10% fetal bovine serum, Hyclone, MA, USA) under controlled conditions (37 °C, 5% CO2). GC-1 cells were irradiated with X‐rays performed at the X‐ray apparatus (precision, X-RAD 225, CT, USA) of the Institute of Modern Physics, Chinese Academy of Sciences with 225 KV (dose rate 150 mGy/min).
The proteins of testes and GC-1 cells were extracted with lysis buffer (Solarbio science & technology, Beijing, China), and the protein quantitation was detected by the bicinchoninic acid assay (Solarbio). Forty µg of proteins were separated by 10% and 12% (w/v) polyacrylamide gels, and transferred to polyvinylidene difluoride membranes, then the membranes were blocked in 5% nonfat milk (in TBST) and incubated with primary antibodies (Abs) diluted to 1:1,000 in TBS against β-actin (ab8227), p21 (ab109199) and RPL23a (ab223089) (Abcam, Cambridge, UK), MDM2 (GTX100531) and p53 (GTX70214) (GeneTex, TX, USA) at 4 °C overnight. Afterward, the membranes were incubated with a secondary Ab labeled with horseradish peroxidase (ZB2301; Beijing Zhongshan Jinqiao Biotechnology, Beijing, China) for 1 h at room temperature (RT). Protein bands were detected with a horseradish peroxidase substrate kit and a FluorChem 2 imaging system (Alpha Innotech Corporation, San Leandro, CA, USA) was used to capture images and analyze the band intensities.
The TUNEL kit (Beyotime Biotechnology, Shanghai, China) was used to detect the apoptotic spermatogonia after IR in vivo. The testicular tissues were dehydrated with gradient alcohol, fixed with dimethylbenzene, and embedded in paraffin. The embedded testicular tissues were cut into sections at a thickness of 4 µm, which were permeabilized with proteinase K at 37 °C for 15 min, and then dropwise added the reaction mixture for 2 h at RT. After the reaction, the sections were washed and then stained with 5 μg/mL of 4, 6-diamidino-2-phenylindole (DAPI; solarbio) for 10 min. At last, the sections were sealed with glycerin and scanned, then analyzed by Panoramic MIDI software (3DHISTECH, Budapest, Hungary).
The sections and GC-1 cells were permeabilized with 0.5% Triton X-100 in phosphate-buffered saline (PBS) at RT for 15 min, and blocked at RT for 30 min (5% bovine serum albumin in PBS), then incubated with the primary Abs against RPL23a, p53, MDM2, RPL11 and Nucleolin (dilution, 1:500 in PBS) at 4 °C overnight. The sections were incubated with a secondary Alexa Fluor 555 Ab (bs-0295G-AF555; BoAoSeng Company, Beijing, China) for 1 h at 37 °C. Afterward, the sections and cells were washed three times with TBS for 10 min each time and then stained with 5 μg/mL DAPI for 10 min. Finally, the sections with were sealed with glycerin, then observed under a biomicroscope (BX53, Olympus, Tokyo, Japan) or a confocal laser microscope (LSM, Carl Zeiss AG, Germany). Image-J 1.46 system (Media Cybernetics Inc., Bethesda, MD, USA) was used for the semi-quantitative evaluation. Average optical density per area (AOD) (Integral optical density/Area) was applied to indicate the level of corresponding protein expression.
Total RNA of GC-1 cells was isolated using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA). Polymerase chain reaction (PCR) was performed using the SYBR green dye on the StepOnePlusTM RealTime PCR System (Applied Biosystems). The primers of p53, MDM2 and p21 were shown in Supplementary File 1 (available in www.besjournal.com). The sequence of the RPL23a siRNAs was shown in Supplementary File 2 (available in www.besjournal.com) and synthesized by Genepharma (Shanghai, China), and the efficiency of knockdown was shown in Supplementary File 3 (available in www.besjournal.com). The overexpression plasmid was shown in Supplementary File 4 (available in www.besjournal.com).
Name Primer Sequence (5'-3') Size (bp) β-actin Forward CACGATGGAGGGGCCGGACTCATC 240 Reverse TAAAGACCTCTATGCCAACACAGT RPL23a Forward TGCTATCATCAAATTCCCAC 192 Reverse ACATACGCCTTCTTCTCTCC p53 Forward GGACAGCTTTGAGGTTCGTG 237 Reverse TCATTCAGCTCCCGGAACAT MDM2 Forward CGGTGGAACTTTGACTTCGT 212 Reverse AGAGTGCAAGACAGCGACAA P21 Forward CTTCGTGAGAACTGGCTTCC 225 Reverse CGATGGTTGGGAATAGTCGT
Table 1. The sequences of primers. Supplementary
Gene Sequence Sense (5'-3') Antisense (5'-3') Rpl23a-mus-47 GCCGAAAGCGAAGAAGGAATT UUCCUUCUUCGCUUUCGGCTT Rpl23a-mus-352 CCAAUAAGCAUCAGAUCAATT UUGAUCUGAUGCUUAUUGGTT Rpl23a-mus-455 UCGCUUGGCUCCUGAUUAUTT AUAAUCAGGAGCCAAGCGATT
Table 2. The sequences of RPL11 using three different siRNAs
GC-1 cells were transfected for 48 h, washed with PBS and harvested with lysis buffer (Solarbio), and homogenized on ice. After centrifugation at 10,000 rpm/ 10 min at 4 °C, the supernatant was divided as an input control for Western blot analysis. Briefly, 500 μg of protein was diluted in pre-cooled PBS (final volume, 500 μL), mixed with agarose protein A+G beads, washed twice with pre-cooled PBS, centrifuged at 3,000 rpm/ 5 min at 4 °C, and then formulated into a 50% concentration with pre-cooled PBS. The agarose protein A+G beads were divided into two portions, one for removing non-specific binding and one for binding to Abs. As a pre-treatment step, the sample and 30 µL of agarose A+G beads were added to individual tubes, which were then slowly shaken for 2 h at 4 °C to eliminate non-specific binding. The samples were centrifuged at 3,000 rpm/ 5 min at 4 °C to remove the protein A+G beads. Then, 5 μL of Ab RPL11 was added to 500 μL of total protein to react with the target protein (no Ab in the control group). The antigen-Ab mixture was slowly shaken overnight at 4 °C. The next day, 30 μL of 50% agarose protein A+G (30 μL/tube) were added and the reaction was carried out for 3–6 h at 4 °C. Afterward, the reaction mixture was centrifuged at 3,000 rpm/ 5 min at 4 °C. Then, the supernatant was discarded and the precipitate was washed three times with 800–1,000 μL of pre-cooled PBS. Next, the pellet was resuspended in loading buffer, boiled in water for 5 min, and then immediately placed on ice. After cooling to RT, the sample was centrifuged at 12,000 rpm/ 10 min at 4 °C for and collected the supernate for western blot analysis.
GC-1 cells were digested with 0.25% trypsin (Hyclone), washed with cooled PBS and fixed in 70% ethanol, then stained with 50 μg/mL propidium iodide (PI) and 50 μg/mL RNase A (Meilune Biotechnology, Dalian, China) for 30 min at RT. GC-1 cells were digested and washed with cooled PBS, resuspended in 500 μL buffer and then stained with 5 μL Annexin V and 1 μL propidium iodide (PI) in dark for 15 min at RT. The flowsight imaging flow cytometer (Amnis/Merck Millipore, Darmstadt, Germany) was used to collect data and analyze the apoptotic ratio, the modFit software program (Verity Software House, Topsham, ME, USA) was used to analyze cell cycle.
Prism 8 software (GraphPad Software, CA, USA) was used to analyze the data. Statistical differences were analyzed based on the Student's t test and on one-way analysis of variance test. P value of < 0.05 was represented statistically significant.
Cell Culture and Irradiation Procedure
TUNEL (Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling) Assay and Immunofluorescence
Analysing of mRNA Expression