Cranial radiation-induced abscopal effects drive time-dependent testicular injury in mice

Objective: To investigate the time-dependent progression of cranial radiation-induced testicular injury in mice, focusing on apoptotic pathways and brain-testis axis involvement. Methods: Male mice were randomly divided into sham irradiation group (sham) and cranial irradiation group (IR) with 36 mice in each group. The IR group received a single dose of 20 Gy cranial X-ray irradiation, while peripheral organs were shielded. Assessments of body weight, testis weight, and sperm parameters were conducted at 1, 2, and 4 weeks post-irradiation. Histological changes and apoptosis were evaluated via H&E staining and TUNEL assay, respectively. Cell-specific markers SOX9, WT1, SYCP3 and PRND were quantified by qRT-PCR, while apoptotic proteins Bcl-2, Bax and Cleaved-caspase 3 were examined by Western blot. Serum levels of brain injury biomarkers NSE and S100B were measured using ELISA. Results: Compared with sham group, IR mice displayed distinct, time-dependent testicular and systemic alterations. At 1 week post-irradiation, no obvious testicular structural damage was observed, but significant reductions in body and testis weight were accompanied by elevated serum NSE (142.1 ± 16.5 vs. 102.1 ± 3.6, t = 7.1, P < 0.05) and S100B ( 178.2 ± 23.8 vs. 123.9 ± 11.5, t = 6.2, P < 0.05). At 2 weeks post-irradiation, disorganized spermatogonial stem cells (SSCs), downregulated SYCP3 and PRND expression, and a marked increase in sperm abnormalities were evident (52.45 ± 3.35 vs. 33.46±6.05, t = 7.77, P < 0.05), alongside sustained weight loss and persistent elevation of NSE (128.6±3.2 vs.110.6 ± 2.8, t =12.8 , P < 0.05) and S100B levels (163.1 ± 5.9 vs. 131.5 ± 10.9, t = 7.8, P < 0.05). At 4 weeks post-irradiation, severe testicular atrophy manifested, characterized by reduced seminiferous tubule diameter, further increased sperm abnormalities (64.61 ± 8.18 vs. 42.64 ± 3.13 , t = 7.10, P < 0.05), decreased sperm counts (vs. 3.08 ± 1.36 vs. 8.55 ± 2.05, t = 6.30, P < 0.05), upregulated Cleaved-caspase 3 protein expression (1.75 ± 0.15 vs. 1.00 ± 0.21, t = 7.10, P < 0.05), TUNEL-positive cells localized to spermatogonial stem cells (SSCs) niches. Concurrently, SOX9 (2.12 ± 0.96 vs. 1.00 ± 0.31) and WT1 (2.51 ± 1.34 vs. 1.00 ± 0.57) mRNA levels were significantly upregulated (t = 2.72, 2.55, P < 0.05). Persistent reductions in body and testis weight, as well as sustained elevations in NSE and S100B, were observed throughout the study. Conclusion: Cranial radiation induces progressive, time-dependent testicular injury in mice via mechanisms mediated by the brain-testis axis, primarily targeting SSCs for apoptosis. These findings identify the brain-testis axis as a novel therapeutic target for mitigating reproductive toxicity in male patients undergoing cranial radiotherapy.