Quantification of peptides after enzymatic digestion using nanodrop DIA detection

Each component underwent separation using the nanoElute liquid chromatography system by Bruker. Mobile phase A consisted of the 0.1% formic acid solution in water, while mobile phase B contained 0.1% formic acid in acetonitrile. The gradient elution conditions were as follows: 0–45 min, 2–22% B; 45–50 min, 22–37% B; 50–55 min, 37–80% B; 55–60 min, 80%. Chromatographic separation was conducted by the ultra-high-performance liquid chromatography system, and the peptides were injected into the timsTOF Pro2 mass spectrometer (MS) manufactured by Bruker for analysis. The mass spectrometry parameters were set as follows: the capillary voltage was maintained at 1,400 V, with primary and secondary scan ranges spanning from 100 to 1,700 m/z, and ion mobility windows (1/K0) ranging from 0.7 to 1.4 Vs/cm2.

Qualitative and quantitative analysis, as well as functional analysis of proteins

For processing the original DIA data, the Spectronaut Pulsar software was employed with fixed Carbamidomethyl © modification, variable Oxidation (M), and Acetyl (N-term) modifications, which allowed for a maximum of 2 missed cleavage sites. Differentially expressed proteins were required to meet the criteria of P < 0.05 and Fold change ≥ 2.0 or Fold change ≤ 0.5. We applied the Benjamini-Hochberg (BH) method to adjust P-values. Differential protein analysis entailed assessment of Biological Processes (BP), Cellular Components (CC), and Molecular Functions (MF) utilizing the Gene Ontology (GO) database. The Kyoto Encyclopedia of Genes and Genomes (KEGG) database was employed to investigate the primary pathways related to differentially expressed proteins. Furthermore, the protein-protein interaction (PPI) analysis was performed based upon the STRING database, leading to the development of a differential protein interaction network.

SiRNAs and plasmids transfection

Human Sertoli cells were plated at 60% density in a 6-well plate, and transfections were performed the next day using Lipofectamine 3000 (Thermo Fisher Scientific, USA). Next, 20 µM SPARC/FGF2 siRNA (Tsingke, China) or 2.5 µg FGF2 overexpressing plasmid (MiaolingBio, China) was diluted with 125 µL of opti-MEM (Thermo Fisher Scientific, USA), and 3.75 µL of Lipofectamine 3000 was diluted with another 125 µL of opti-MEM. P3000 (Thermo Fisher Scientific, USA) was employed for transfection with the FGF2 overexpressing plasmid. The two solutions were carefully mixed and subsequently incubated at room temperature for 15 min. The mixture was then added to the 6-well plate with 250 µL of the mixture per well. The medium needed to be changed for plasmid-transfected cells at 6 h after transfection. The siRNA sequences were listed in Table S1. The human FGF2-Flag-tagged plasmids were obtained from MiaoLingBio Inc (China).

Immunocytochemistry

Human Sertoli cells were resuspended with PBS and fixed by 4% paraformaldehyde (Biosharp, China) for 15 min at room temperature. Permeabilization of cells was performed with TritonX-100 (Beyotime, China) for 10 min, and blocking of cells was conducted with Blocking Buffer for Immunol Staining (Beyotime, China) at room temperature for 15 min. Cells were incubated with primary antibodies at 4 °C overnight and then with the corresponding secondary antibodies at room temperature for 1 h. The primary antibodies utilized in this study were shown as follows: SOX9 (1:100, AB5535, Sigma-Aldrich, USA), BMP4 (1:50, ab1124715, Abcam, UK), GATA4 (1:25, sc1237, Santa, USA), FGF2 (1:50, YP-Ab-15832, UpingBio, China; 1:50, 11234-1-AP, Proteintech, China), WT1 (1:50, 12609-1-AP, Proteintech, China), GDNF (1:50 26179-1-AP, Proteintech, China), SPARC (1:100, 15274-1-AP, Proteintech, China), ZO-1 (ab216880, Abcam), GJA1 (ab11370, Abcam), and CDH1 (20874-1-AP, Proteintech, China). DAPI was employed to label the nuclei of cells, and immunostaining was observed under a confocal microscope (Leica TCS SP8 SR, Germany) or fluorescence microscope (Leica DM3000, Germany).

Immunohistochemistry

Human testicular tissue sections were placed in a 65 °C oven for 10 min and immersed in Environmental Dewaxing dip wax Transparentize Solution (Biosharp, China) for 15 min twice. Next, these sections underwent hydration in a series of graded alcohols (Anhydrous Ethanol I, Anhydrous Ethanol II, 95% Ethanol, 90% Ethanol, 80% Ethanol, and 70% Ethanol) for 5 min and followed by immersion in distilled water for 5 min. Antigen retrieval was performed by 1x citrate antigen retrieval solution (Beyotime, China), and endogenous peroxidase was removed by incubation with Endogenous Peroxidase Blocking Buffer (Beyotime, China) for 10 min. Permeabilization was performed with TritonX-100 (Beyotime, China) for 10 min, and blocking was conducted with Blocking Buffer for Immunol Staining (Beyotime, China) at room temperature for 30 min. These sections were incubated with primary antibodies overnight at 4 °C and followed by incubating with the secondary antibodies at room temperature for 1 h. Primary antibodies utilized in this study were listed as follows: SPARC (1:2,000, 15274-1-AP, Proteintech, China), MAGEA4 (1:100, a gift by Professor Giulio C. Spagnoli of the University Hospital Basel, Switzerland). DAPI (Beyotime, China) was used to label cell nuclei. Finally, images were captured using a fluorescence microscope (Leica DM3000, Germany).

Western blots

Human Sertoli cells were lysed by RIPA lysis buffer (Beyotime, China) containing 1mM PMSF (Beyotime, China) for 30 min and followed by centrifugation at 12,000 rpm for 10 min. Proteins were denatured with boiling water at 99 °C for 5 min. Electrophoresis of proteins was performed using SDS-PAGE gels and transferred to PVDF membranes (Millipore, USA). The membranes were blocked for 30 min at room temperature using a blocking buffer (Beyotime, China) and incubated with primary antibodies at 4 °C overnight and secondary antibodies for 1 h at room temperature. The primary antibodies utilized in this study were as follows: SPARC (1:1,000, 15274-1-AP, Proteintech, China), ACTB (1:1,000, 2146, CST, USA), GDNF (1:1,000, 26179-1-AP, Proteintech, China), WT1(1:1,000, 12609-1-AP, Proteintech, China), SOX9 (1:1000, AB5535, Sigma-Aldrich, USA), FGF2 (1:1,000, 11234-1-AP, Proteintech, China), CDH1 (1:10,000, 20874-1-AP, Proteintech), and Occludin (1:1,000, 91131, CST, USA). The blots were detected using Chemiluminescent Imaging System (MiniChemi 610, Sinsage, China).

RT and qPCR

Total RNA was extracted from human Sertoli cells using Trizol and isopropanol (Aladdin, China). After measuring the concentration and purity of the RNA using Nanodrop, total RNA was reverse-transcribed (RT) by the Evo M-MLV RT Master Mix (Agbio, China), and then cDNA was subjected to qPCR using the SYBR Green Pro Taq HS Premix ( Agbio, China). Real-time qPCR was performed on the LightCycler® 480 Instrument II (Roche, Switzerland) using a two-step PCR amplification method. The relative fold change in mRNA expression of the target genes was calculated using the ΔΔCt method, and the value was expressed as 2−ΔΔCt. Primers of genes used for RT-PCR and qPCR were presented in Table S2.

Flow cytometry

The APC Annexin V Apoptosis Detection Kit with PI (Biolegend, USA) was utilized to detect apoptosis of human Sertoli cells without or with SPARC siRNAs. The cells were washed twice with PBS, and 100 µL of staining buffer was added to the cells. Next, 5 µL of APC and 10 µL of PI were added to stain the cells, and the staining was terminated with 400 µL of staining buffer after 15 min of incubation at room temperature. The apoptosis of human Sertoli cells was subsequently detected by flow cytometry (BD Bioscience, USA).

Cell counting kit-8 (CCK8) assay.

On the first day after transfection of SPARC siRNAs, human Sertoli cells were washed with PBS, and cell counting kit-8 (CCK8) (APExBIO, USA) working solution was added to each well (10 µL CCK8 reagent in 90 µL DMEM/F12). The cells were incubated in a 37 °C incubator for 3 h, and then OD values were measured on a microplate reader at a wavelength of 450 nm. The CCK8 assay was carried out for 5 consecutive days to observe the cell proliferation.

TUNEL assay

Human Sertoli cells were cultured and transfected without or with SPARC siRNAs. These cells were fixed with 4% paraformaldehyde for 30 min and incubated with 0.2% Triton X-100 in PBS for 5 min at room temperature. The TUNEL Cy3 Apoptosis Detection Kit (APExBIO, USA) solution was added to cells and incubated for 60 min at 37 °C. The cells were washed with PBS, and the nuclei were labeled with DAPI. The apoptotic cells were observed under a fluorescence microscope (Leica DMi8, Germany).

Cell adhesion assay

Human Sertoli cells were seeded at 60% density in a 6-well plate, and the cells were transfected with SPARC siRNAs for 48 h. The cells were then transferred to a 24-well plate coated with 10 µg/mL type I collagen (Sigma-Aldrich, USA), and after 12–24 h of culture, the cells were washed three times with PBS to remove non-adherent cells. Cells were fixed with 4% paraformaldehyde at room temperature for 15 min, followed by the addition of 100 µL of crystal violet solution for staining, and incubated at room temperature for 15 min. After staining, the cells were washed three times with PBS, and they were observed under an inverted fluorescence microscope (Leica DMI8, Germany). The number of adherent cells was quantitatively analyzed by Image J.

Transmission electron microscope (TEM)

The Sertoli cells were collected by centrifugation and fixed with 2.5% glutaraldehyde (Biosharp, China) at 4 °C for 2 h. After washes three times with PBS (pH 7.4), fixation was performed with 1% osmium tetroxide in 0.1 M PBS at room temperature for 2 h. Following the graded ethanol dehydration, the cells were embedded, sectioned with ultrathin, and stained with 2% uranyl acetate and lead citrate. The cells were then air-dried overnight and examined under the JEM-1400 (JEOL, Japan) for imaging and analysis.

Mitochondrial membrane potential (MMP) measurement

MMP of Sertoli cells was measured by using fluorescent probe JC-1 (Beyotime, China). Sertoli cells from OA and SCOS patients were seeded at 50% density in 24-well plates, while 500 µL of culture medium and 500 µL of JC-1 working solution were added to each well. After incubation at 37 °C in the dark for 30 min, the cells were washed 2–3 times with JC-1 staining buffer at 4 °C and then observed under a fluorescence microscope (Leica DMi8, Germany).

Statistical analysis

The experiments were repeated three times and the results were presented as mean ± SEM. GraphPad Prism 6.01 and two-tailed unpaired t-test were used for statistical analysis, and p values less than 0.05 were considered statistically significant.