Reagents

The 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) and cordycepin were obtained from Solarbio Life Sciences (Beijing, China). Propidium iodide (PI), fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA), and HTF medium were bought from Sigma-Aldrich (St. Louis, MO, USA). SDS lysis buffer, the bicinchoninic acid (BCA) kit, and Fluo-3 AM were obtained from the Beyotime Institute of Biotechnology (Shanghai, China). The enhanced chemiluminescence (ECL) kit and prestained protein marker were bought from Thermo Fisher Scientific (Waltham, MA, USA). The protease and phosphatase inhibitor cocktails were provided by Roche (Mannheim, Germany). The PKA activity assay kit, antibodies to A2a AdoR (ab3461), A2b AdoR (ab229671), A3 AdoR (ab197350), and phosphotyrosine (ab179530), as well as the Alexa Fluor 488-conjugated goat anti-rabbit (ab150077), Alexa Fluor 555-conjugated goat anti-mouse (ab150118), horseradish peroxidase (HRP)-conjugated goat anti-rabbit (ab6721), and HRP-conjugated goat anti-mouse (ab6789) secondary antibodies were obtained from Abcam (Cambridge, UK). The anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (BK7021) was purchased from Baoke Biotechnology (Hangzhou, China). The anti-A1 AdoR antibody (sc-66193) was provided by Santa Cruz Biotechnology (Dallas, TX, USA). A sperm chromatin dispersion (SCD) kit was provided by BRED Life Science Technology (Shenzhen, China). The inhibitors to the A1 (DPCPX; HY-100937), A2b (MRS1754; HY-14121), and A3 (MRS1191; HY-124543) AdoRs were purchased from MedChemExpress (Shanghai, China). The A2a AdoR inhibitor (DMPX; S37466) was purchased from Yuanye Biotechnology (Shanghai, China).

Sperm capacitation culture

The study was performed in accordance with the Declaration of Helsinki and approved by the Medical Ethics Committee of Hangzhou Medical College (LL2022-16). We recruited 20 male semen donors (aged 20–38) who had passed physical examinations to exclude disease of the reproductive system or other serious conditions [23, 24]. All participants provided written informed consent. Their semen parameters are shown in Table S1. All donors met the criteria of the World Health Organization laboratory manual, with sperm survival rate ≥ 58%, sperm motility ≥ 40%, sperm concentration ≥ 15 × 106 sperm/mL, and normal sperm morphology ≥ 4%. After abstinence for 3–7 d, fresh samples from at least three donors were randomly obtained for each experiment. The semen samples were collected and liquefied in a 5% CO2 incubator at 37℃ for 30 min. Then, the samples were mixed. Dead spermatozoa and cell debris were removed by 40% and 80% discontinuous Percoll gradient centrifugation. The sperm precipitate was washed with phosphate-buffered saline, and then some sperm samples were collected as the control group at 0 h. Subsequently, the residual samples were adjusted to 20 × 106 sperm cells/mL using HTF and were divided into different groups: cordycepin (20, 100, and 500 µM), vehicle control (dimethyl sulfoxide [DMSO] 0.1% v/v), A1 AdoR inhibitor (DPCPX 0.001, 0.01, and 0.1 µM), A2a AdoR inhibitor (DMPX 0.001, 0.01, and 0.1 µM), A2b AdoR inhibitor (MRS1754 0.001, 0.01, and 0.1 µM), A3 AdoR inhibitor (MRS1191 0.001, 0.01, and 0.1 µM), and PKA inhibitor (H-89 50 µM). Finally, samples were cultured in a 5% CO2 incubator at 37℃ for 3 h.

Acrosome reaction evaluation

As only capacitated sperm can undergo AR, the sperm AR ratio was evaluated using FITC-PSA staining. Before capacitation culture, spontaneous AR was analyzed to serve as a control. After incubation with cordycepin for 3 h, the AR of the other groups was induced with progesterone (15 µM) for 15 min. Additionally, we tested whether cordycepin could directly induce the AR. After a 3-hour capacitation incubation (without cordycepin), sperm AR was induced using DMSO (vehicle control), different concentrations of cordycepin, progesterone, or a combination of cordycepin and progesterone for 15 min, respectively. Next, PI (0.03 µM) was used to stain dead sperm cells. After washing, sperm were fixed with 95% ethanol and then smeared on slides. Subsequently, sperm were stained using FITC-PSA (25 mg/L) at 4℃ overnight. After washing, the slides were observed by fluorescence microscopy. For each group, at least 600 sperm cells were counted. AR sperm showed only weak fluorescence in the equatorial zone or almost no fluorescence at the sperm head. The acrosome integrity (AI) sperm did not undergo AR, and bright fluorescence was observed at the sperm head. Bright red fluorescence can be observed at the head of dead sperm. The AR ratio and nonviable cell percentage (NVC%) were calculated.

Sperm motility parameters evaluation

Capacitated sperm exhibit hyperactivated motility; therefore, a computer-assisted sperm analyzer (CASA) was used to evaluate sperm motility parameters, including progressive motility, sperm motility, amplitude of lateral head displacement (ALH), beat-cross frequency (BCF), straight-line velocity (VSL), average path velocity (VAP), curvilinear velocity (VCL), straightness (STR, VSL/VAP × 100), linearity (LIN, VSL/VCL × 100), and hyperactivated sperm. Hyperactivated sperm have the following characteristics: VCL ≥ 150 μm/s, LIN ≤ 50%, and ALH ≥ 7.0 μm [25].

PKA activity assessment

Human sperm PKA activity was detected using a PKA activity ELISA kit. Sperm were lysed using NP40 lysis buffer according to the manufacturer’s instructions. Next, the lysate protein concentration was analyzed using a BCA kit. The sperm lysate was then added to a microplate coated with PKA-specific substrates. The PKA in sperm lysate can phosphorylate the substrates. Next, a specific primary antibody was added to detect phosphorylated substrates. HRP-conjugated secondary antibodies were used to interact with the primary antibodies. The color reaction was performed using 3,3′,5,5′-tetramethylbenzidine (TMB). A microplate reader was used to measure the color intensity.

Western blotting

Protein tyrosine phosphorylation levels and adenosine receptor expression were detected by western blotting. After capacitation, sperm samples were collected and ultrasonically lysed in SDS lysis buffer supplemented with phosphatase and protease inhibitor cocktails. Protein concentrations were detected using a BCA kit. Subsequently, 10% SDS-PAGE was used to separate different group proteins. Proteins were then transferred to polyvinylidene fluoride (PVDF) membranes. After blocking with 3% bovine serum albumin (BSA) for 1 h, the membranes were then incubated with antibodies to phosphotyrosine, A1 AdoR, A2a AdoR, A2b AdoR, or A3 AdoR at 4℃ overnight. After washing with tris-buffered saline, HRP-conjugated secondary antibody was used to incubate the membranes for 2 h at 25℃. An ECL kit was used for the color reaction. The membranes were observed using a gel imaging system. Thereafter, antibodies were removed. The membranes were blocked using BSA again, and incubated with an antibody to GAPDH. The gray intensity was analyzed using ImageJ software.

Sperm chromatin dispersion assay

Sperm DNA fragmentation was analyzed using an SCD assay kit according to the manufacturer’s instructions. After mixing with fused agar, the sperm samples were pipetted onto a slide and covered. The samples were solidified at 4℃. After removing the coverslips, the slides were immersed in solution A (acidic solution) and solution B for 7 min and 25 min, respectively. After dehydrating in an ethanol series (70-90-100%) and air drying, the sperm samples were stained using Wright’s dye solution. At least 500 sperm cells from each group were observed under a microscope. Sperm with halo widths greater than the diameter of the core were considered to show DNA integrity (DI), whereas others were regarded as having DNA fragmentation (DF).

ROS level detection

Sperm ROS levels were detected using the fluorescent probe DCFH-DA. First, sperm were labeled using 10 µM DCFH-DA for 30 min in the dark. After washing, the free DCFH-DA was removed. Sperm were then added to a 96-well plate (106 cells/mL). The fluorescence signals were recorded using a microplate reader at 488 nm. Initial recordings were acquired before the addition of DMSO or different concentrations of cordycepin. Data were recorded every 3 min for 30 min. After capacitation for 3 h, data were acquired again. The initial values were normalized.

Indirect immunofluorescence assay

Sperm samples before or after capacitation for 3 h were fixed in 4% paraformaldehyde for 1 h. Subsequently, they were smeared on slides and air-dried. After permeabilizing with 0.1% Triton X-100, sperm samples were blocked using 10% goat serum. Thereafter, sperm were incubated with antibodies to the A1, A2a, A2b, or A3 AdoRs overnight at 4 ℃. Negative control samples were incubated with normal mouse or rabbit IgG. After washing to remove uncombined primary antibodies, Alexa Fluor 488-conjugated anti-rabbit secondary antibody or Alexa Fluor 555-conjugated anti-mouse secondary antibody was incubated for 1 h at 25℃. The nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Finally, the sperm were observed using fluorescence microscopy.

Molecular docking

A1, A2a, and A2b AdoRs protein structure files (PDB ID: 5N2S, 5NLX, and 8HDO, respectively) were obtained from the PDB database. The A3 AdoR protein structure file (AF-P0DMS8-F1) was downloaded from UniProt. As there is no published crystal structure for A3 AdoR, the structure was predicted using AlphaFold. All protein structures were preprocessed using PyMol to remove water, ligands, and other proteins. The SDF file of the cordycepin 3D structure was obtained from the PubChem database and converted to mol2 using Open Babel. Molecular docking of the cordycepin and AdoRs was performed using AutoDock 4. The protein-ligand conformation with the lowest binding energy was obtained. PyMol was used to visualize the final images.

Statistical analysis

Statistical analyses were carried out using the Social Science Statistics Software Package (SPSS). Statistical significances among groups were analyzed using one-way analysis of variance (ANOVA). Dunnett’s T3 test was used if the homogeneity test for variance was P < 0.05; otherwise, the least significant difference test was used. Data were presented as mean ± standard deviation (SD). Statistical significance was set at P < 0.05.