Percoll gradient

The clinical and demographic information was recorded at the participant’s hospital visit. For patients, their semen examinations were performed at the first visit during the recovered period, and the recovered time for the seven patients was all ≤ 100 days. The serum Follicle−stimulating hormone (FSH), luteinizing hormone (LH), total T, and estradiol (E₂) concentrations were measured using a chemiluminescence immunoassay (Beckman Coulter, Fullerton, USA). Following the three to seven days of advised abstinence, freshly obtained semen was processed for 30 to 60 minutes at room temperature to liquefy it. Sperm examination was performed in strict accordance with the WHO laboratory manual (25 ). Purified sperm samples were obtained by centrifuging them for 20 minutes at 1000 g while using a Percoll gradient (Solarbio, Beijing, China). After washing them with PBS twice, the purified sperm samples were used for subsequent experiments.

The mouse cerebrum was removed and incubated for 30 min at 37°C in 10 ml of PBS containing 100 U/ml collagenase IV (Solarbio, C8160) and 20 U/ml DNase I (Solarbio, D8071) after anesthetization and intracardial perfusion. Brain tissue was passed through a tissue grinder and cells were recovered after centrifugation at 400 g for 10 min at room temperature and separated from myelin and debris in 70% and 30% isotonic Percoll gradient (Solarbio, P8370). Samples were centrifuged at 1000 g for 30 min without acceleration or brake. Cells were collected from the interface and washed once with PBS.²⁰ After sample preparing, lipid peroxidation (LPO) of the cerebrum was detected by BODIPY™ 581/591 C11 (Invitrogen, D3861) according to the manufacture's instruction. Cells were incubated at 4℃ for 30 min with the LPO sensor and washed with PBS for three times at 1000 g for 5 min. Data acquisition was carried out in a BD FacsCantoII cytometer (BD Biosciences) using the FacsDiva software (BD Biosciences) and detected at two separate wavelengths (PE for the reduced dye; FITC for the oxidized dye). The ratio of FITC/PE gives the read‐out for LPO in cells.

Cerebella from P7 animals were minced into small pieces using scissors and were digested at 37 °C for 30 min in 2 mg/ml papain (Solarbio) and 10 μg/ml DNase (Sigma). Single-cell suspension was obtained via trituration with glass pipettes and then centrifuged through a 35%–65% Percoll gradient (Solarbio). GCPs were harvested from the 35%–65% interface and centrifuged at 2000 rpm for 5 min at room temperature. Cells in high glucose DMEM medium (HyClone) were washed and suspended in the GCP culture medium, which is composed of Neurobasal A medium (Invitrogen) with 2 mM L-glutamine, 2% B27 supplement, and penicillin/streptomycin. GCPs were plated on poly-D-lysine (100 μg/ml)-coated dishes. Cultures were maintained in 5% CO₂ at 37 °C and analyzed 24 h after incubation. For vitro proliferation assays, GCP cells were treated with 1 μM Shh inhibitor Vismodegib (GDC-0449, Selleckchem) in DMSO or DMSO alone as control for 48 h, and then 10 μM BrdU for 2 h. For immunostaining analysis, GCPs in culture were immunostained with antibodies against BrdU and NeuN. The relative rate of BrdU⁺ cells among NeuN⁺ cells were quantified to examine GCP proliferation in vitro.