Transplantation of human umbilical cord mesenchymal stem cells repairs radiation-induced reproductive injury in male mice via improving mitochondrial function and attenuating oxidative stress

To investigate the ameliorative effect of human umbilical cord mesenchymal stem cells (hUC-MSCs) on radiation-induced reproductive damage in male mice and its potential mechanisms.

After obtaining informed consent and signed consent forms from healthy full- term pregnant women in the Department of Obstetrics and Gynecology at Xijing Hospital of Air Force Medical University, umbilical cord tissue approximately from 6 to 8 cm in length from healthy full-term newborns was collected under sterile conditions in the operating room for isolating and culturing hUC-MSCs; 45 healthy male C57BL/6 mice aged from 6 to 8 weeks with a body weight of 22±2 g were divided into Control+PBS (n=3), Control+MSCs (n=6), Rad+MSCs (n=6), Control (n=10), Rad (n=10), and Rad+hUC-MSCs (n=10) groups using a random number table method; the Rad, Rad+MSCs, and Rad+hUC-MSCs groups were subjected to whole-body single -dose irradiation using ⁶⁰Co γ-ray at a dose rate of 1.28 Gy/min and a total dose of 6 Gy; immediately after irradiation, the Rad+MSCs and Rad+hUC-MSCs groups received a single tail vein injection of hUC-MSCs suspension (2×10⁶ cells/200 μL); the distribution of hUC-MSCs in testicular tissue was observed using a multimodal in vivo imaging system and confocal fluorescence microscopy; at 12 weeks post-irradiation, bilateral testes were harvested and weighed to calculate the testicular coefficient; sperm quality was assessed under a microscope; testicular morphology and structure were evaluated using Hematoxylin and Eosin (HE) staining; reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) levels in testicular tissue were measured by flow cytometry; malondialdehyde (MDA), total superoxide dismutase (T-SOD), and catalase (CAT) contents were detected by colorimetry; testicular cell apoptosis was assessed using TUNEL staining; and Western blot was used to detect the expression levels of Parkin RBR E3 ubiquitin-protein ligase (Parkin), PTEN-induced putative kinase 1 (PINK1), Beclin1, OPA1 mitochondrial dynamin-like GTPase (OPA1), dynamin-related protein 1 (Drp1), Bcl2-associated X protein (Bax), and B cell lymphoma-2 (Bcl-2).

At 24 h and 48 h post-irradiation, multimodal in vivo imaging showed no fluorescent signals in the testicular tissue of the Control+PBS group, while fluorescent signals were observed in the Control+MSCs and Rad+MSCs groups; further confocal fluorescence microscopy revealed red fluorescence in the interstitial regions of testicular tissue in the Control+MSCs and Rad+MSCs groups, detecting fluorescence dye labeled MSCs signals; at 12 weeks post-irradiation, compared to the Control group, the Rad group showed significantly decreased sperm count (P<0.05) and motility (P<0.01), significantly increased malformation rate (P<0.001), vacuolization and disordered arrangement of spermatogenic cells, reduced seminiferous tubule diameter and spermatogenic epithelium thickness (P<0.001), elevated testicular tissue ROS levels (P<0.05), significantly decreased MMP levels (P<0.001), increased MDA content (P<0.001), elevated T-SOD activity (P<0.05), decreased CAT activity (P<0.05), increased protein expression of Parkin (P<0.01), PINK1 (P<0.05), Beclin1 (P<0.05), OPA1 (P<0.01), and Drp1 (P<0.05), increased TUNEL-positive cells (P<0.001), and decreased Bcl-2/Bax ratio (P<0.01); compared to the Rad group, the Rad+hUC-MSCs group exhibited significantly increased sperm count and motility (P<0.05), significantly decreased malformation rate (P<0.001), improved vacuolization and arrangement of spermatogenic cells, increased spermatogenic epithelium thickness (P<0.01), reduced testicular tissue ROS levels (P<0.05), significantly increased MMP levels (P<0.01), decreased MDA content (P<0.001), reduced T-SOD activity (P<0.05), increased CAT activity (P<0.05), decreased protein expression of Parkin (P<0.05), PINK1 (P<0.05), Beclin1 (P<0.05), OPA1 (P<0.05), and Drp1 (P<0.05), reduced TUNEL-positive cells (P<0.001), and increased Bcl-2/Bax ratio (P<0.05).

hUC-MSCs may alleviate radiation-induced male reproductive damage by mitigating mitochondrial damage, reducing testicular tissue oxidative stress injury and cell apoptosis.