A total of 24 semen samples were collected from clinical patients by masturbation after 2–7 days of abstinence. All patients did not have obvious bad habits such as smoking, excessive drinking, staying up late, sauna, etc., use some medications, and suffer from basic diseases. After routine semen analysis was performed by a computer-aided sperm analysis (CASA) system (Beijing Suijia Medical Instrument Co., Ltd., Beijing, China), the remaining semen was used for the analysis of sperm DFI and preparation of sperm samples. The profiles of these semen samples are shown in Additional file 1: Table S1. These samples were divided into the experimental group and control group according to sperm DFI values. The patients’ ages in the experimental group and control group were 30.42 ± 3.68 and 29.17 ± 3.97 years old, respectively, and there was no significant difference between them (P = 0.432). The values of DFI in the experimental group (n = 12) were more than 30% (DFI ≥ 30%), while those in the control group (n = 12) were below 30% (DFI < 30%). Then, sperm were isolated from each semen sample by the density gradient centrifugation (DGC) method. Every 4 sperm samples in the experimental group and control group formed one replicate for sperm protein analysis, respectively. Three replicate samples in the experimental group were labeled as EXP1, EXP2 and EXP3, respectively, and 3 replicate samples in the control group were labeled as CON1, CON2 and CON3, respectively. This study has been approved by the Northern Jiangsu People’s Hospital ethics committee (Approval number: 2021ky068), and all patients provided informed written consent.
Detection of sperm DFISperm DFI was detected by the sperm chromatin structure assay (SCSA) [11, 23], and the corresponding kit was purchased from Zhejiang Cellpro Biotech Co., Ltd. (Ningbo, China). First, appropriate volume of semen were added into 0.1 ml of solution A (TNE buffer, sperm dilution) and mixed. Then, 0.2 ml of solution B (acid solution of 0.1% Triton X-100, 0.15 mol/l NaCl, and 0.08 mol/l HCl, pH 1.2) were added and mixed. After standing for 30 s, 0.6 ml of acridine orange (AO) staining solution (6 μg/ml AO, 37 mmol/l citric acid, 126 mmol/l Na2HPO4, 1 mmol/l Na2EDTA, 0.15 mol/l NaCl, pH 6.0) was added and mixed. After sperm were stained for 3 min, sperm DFI was detected by a flow cytometer (FACS Calibur, BD Bioscience, San Jose, CA, USA). A minimum of 5,000 sperm were acquired, and the data were analyzed by the software (DFIView 2010 Alpha11.15, CellPro Biotech, Ningbo, China). Sperm DFI was expressed as the percentage of sperm with fragmented DNA compared to the total number of sperm. The variability of the replicate DFI measures was less than 5%.
Since sperm used for protein analysis were selected by DGC to remove non-sperm cells, was there still a difference in sperm DFI after DGC between the experimental group and control group? To verify this, we compared sperm DFI of 6 sperm samples in each group before and after DGC.
Preparation of sperm samplesSperm samples were prepared by the DGC method according to the report of de Mateo et al. [24]. In brief, SpermGrad lower layer (90%), upper layer (45%) and SpermRinse solutions (Vitrolife, Sweden) were taken out from a refrigerator and recovered to room temperature for further use. First, 1 ml of SpermGrad lower layer (90%) solution was added into a 15-ml centrifuge tube, and then 1 ml of SpermGrad upper layer (45%) solution was gently added on the surface of SpermGrad lower layer (90%) solution. Next, normally liquefied semen was slowly added, and a clear interface between semen and gradient solutions could be seen. After 20 min of centrifugation at 400g, the upper liquids were carefully aspirated away using a pipette, and sperm sediments were transferred into a new centrifuge tube with the help of 3 ml of SpermRinse solution. The mixture was blown up and down slowly, and then centrifuged for 10 min at 200g. Next, the upper liquids were carefully aspirated away using a pipette. The obtained sperm were stored at − 80 °C and used for the extraction of proteins.
Preparation of sperm proteins and peptidesThe process of sperm protein library building mainly includes protein extraction, protein quantification, desalting, mass spectrometry, database retrieval, etc.
First, sperm samples were incubated in lysis buffer (7 mol/l urea, 2 mol/l thiourea, 4% sodium dodecyl sulfate, 40 mmol/l Tris–HCl, pH 8.5) containing 1 mmol/l phenylmethylsulfonyl fluoride (PMSF) and 2 mmol/l ethylene diamine tetraacetic acid (EDTA) for 5 min, and then 10 mmol/l dithiothreitol (DTT, final concentration) was added to the sample. The suspension was sonicated for 10 min on ice and then centrifuged at 16,000g for 20 min at 4 °C. The obtained supernatant was mixed with 4 volumes of precooled acetone and incubated for 2 h at − 20 °C. Then, the solution was centrifuged at 16,000g for 20 min at 4 °C, and the obtained protein pellets were air-dried and resuspended in 8 mol/l urea/100 mmol/l tetraethylammonium bromide (TEAB) solution (pH 8.0). The sperm protein samples were reduced for 30 min with 10 mmol/l DTT at 56 °C, and alkylated for 30 min in the dark with 50 mmol/l iodoacetamide (IAM) at room temperature. Next, four volumes of precooled acetone were added and incubated for 2 h at − 20 °C. Then, the solution was centrifuged at 16,000g for 20 min at 4 °C, and the obtained protein pellets were air-dried and resuspended in 8 mol/l urea/100 mmol/l TEAB solution (pH 8.0). The total protein concentration of the obtained solution was measured using the Bradford method. The protein precipitates were collected and dried, and then stored at − 80 °C until for further analysis.
The obtained sperm protein solution was further diluted with 5 volumes of 100 mmol/l TEAB (pH 8.0). Then, trypsin was added at an enzyme-protein ratio of 1:50 (w/w), and sperm proteins were digested overnight at 37 °C. The peptide sample was dissolved in 2% acetonitrile/0.1% formic acid solution and analyzed with Triple TOF 5600 plus mass spectrometer coupled with Eksigent nanoLC system (AB SCIEX, USA). First, peptide solution was added to the C18 capture column (3 μm, 300 μm × 0.5 mm, AB Science, USA). Then, gradient elution was performed on the C18 analytical column (3 μm, 75 µm × 150 mm, Welch Materials, Inc., USA) with a time gradient of 60 min and a flow rate of 300 nl/min. Their mobile phases were buffer A (2% acetonitrile/0.1% formic acid/98% H2O) and buffer B (98% acetonitrile/0.1% formic acid/2% H2O), respectively. For information-dependent collection (IDA), the first-order mass spectrum (MS1) was scanned with an ion accumulation time of 250 ms, and the second-order mass spectrum (MS2) of 30 precursor ions was collected using an ion accumulation time of 50 ms. The MS1 spectrum was collected in the range of 350–1200 m/z, and the MS2 spectrum was collected in the range of 100–1500 m/z. The dynamic elimination time of precursor ions was set as 15 s. The mass spectrometry data were analysed using ProteinPilot 4.5 software (July 2012; AB Sciex). Spectral library generation and SWATH data processing were performed with the Peakview version 2.2 software.
Western blottingThe reliability of proteomics could be validated by Western blotting. Two differentially expressed proteins (DFFA and RAD23B) and two major protein modifications (ubiquitination and acetylation) were selected for Western blotting.
Briefly, 20 µg of protein was separated by 12% polyacrylamide gel electrophoresis (concentrated gel at 80 V for 50 min and separated gel at 120 V for 2 h). Subsequently, the gels were stained with Coomassie blue dye or proteins were transferred to polyvinyl difluoride (PVDF) membranes (Millipore, Bedford, Mass, USA) at 30 V overnight at 4 °C using the Mini TransBlot transfer unit (Bio-Rad, Hercules, CA, USA). Then, the membranes were blocked in tris-buffered saline (TBS) containing 0.1% Tween-20 and 5% nonfat dry milk for 60 min at room temperature, and incubated with antibodies against acetyllysine (PTM-101, 1:1000, PTM BIO, Hangzhou, China), ubiquitins (PTM-1106, 1:1000, PTM BIO, Hangzhou, China), DFFA (ab108924, Abcam, USA) and RAD23B (12,121–1-AP, Proteintech, China) overnight at 4 °C, respectively. Subsequently, the membranes were washed with PBST (PBS, 0.05% Tween-20), and incubated with horseradish peroxidase-conjugated secondary antibody (1:10,000; Pierce, Rockford, IL, USA) for 1 h at room temperature. Last, the membranes were detected by the enhanced chemiluminescence.
Functional enrichment analysisProteomics analysis of sperm proteins was carried out by the SWATH-MS according to previous reports [25], and then differentially expressed proteins were performed Gene Ontology (GO) analysis by the link http://geneontology.org/. All of differentially expressed proteins were assigned to their GO annotations, including biological process (BP), cellular component (CC), and molecular function (MF). Furthermore, the Kyoto Encyclopedia of Genes and Genomes (KEGG) annotations of differentially expressed proteins were obtained by the link https://www.kegg.jp. The STRING database (https://cn.string-db.org/) was used to identify the functional enrichments, and the Cytoscape software 3.5.1 was used to visualize the interaction among proteins.
Statistical analysisThe quantitative values of proteins were mainly calculated by the peak area of the mass spectrum data. Then, the mean value of each protein in each sample group was calculated, and the median of the ratio of the sample value to the mean value was taken as the normalization factor of the sample. The differential expressions of sperm proteins between the experimental and control groups were analyzed by the DEqMS/Bioconductor package, and the candidates with a Q-value ≤ 0.05 and a |Fold change|≥ 2 were considered as differentially expressed proteins. The differences in sperm DFI and the expression levels of differential proteins between the experimental and control groups were analyzed using Student’s t-test of SPSS 22.0 statistical software (SPSS Inc., Chicago, IL, USA), and P < 0.05 was considered statistically significant.
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