Effects of cordycepin on antioxidant capacity and proliferative potential of buffalo spermatogonial cells in vitro.

Abstract

Buffalo spermatogonial cells (bSCs) are essential for maintaining spermatogenesis and male fertility. Oxidative stress impairs bSCs proliferation through ROS-induced damage and apoptosis. Cordycepin, a bioactive compound from Cordyceps militaris, exhibits potent antioxidant effects but has not been systematically evaluated in bSCs. This study aimed to investigate the effects of cordycepin(0-5 μM) on the in vitro growth and survival of buffalo spermatogonial cells (bSCs) and to characterize associated changes in redox status and mitochondrial function.Cell proliferation was assessed by cell counting, and cellular metabolic activity was evaluated by CCK-8 assay. Proliferation markers (PCNA, ETV5, CDC25B) and reproductive markers (PGP9.5, DDX4, NANOS2, OCT4) were analyzed by qPCR and Western blotting. Antioxidant enzyme activities (SOD, CAT, GSH-Px) and oxidative stress markers (MDA) were measured by ELISA. Intracellular ROS, apoptosis, mitochondrial membrane potential, and mitochondrial dynamics proteins (MFN-1, OPA1, DRP-1, FIS-1) were assessed by DCFH-DA staining, flow cytometry, JC-1 probe, and immunofluorescence/Western blotting, respectively. At 1.25 μM, cordycepin significantly increased cell proliferation and upregulated PCNA and ETV5 expression. Cordycepin reduced intracellular ROS, enhanced antioxidant enzyme activities, and decreased MDA levels. Treatment increased JC-1 fluorescence intensity and was associated with selective mRNA- and protein-level changes in mitochondrial dynamics-associated factors; however, these data did not directly assess mitochondrial morphology or establish altered fusion/fission activity. Cordycepin decreased apoptosis and was associated with increased expression of selected germ cell- and spermatogonial cell-associated markers. At 1.25 μM, cordycepin treatment was associated with increased bSCs number, reduced intracellular ROS accumulation, enhanced antioxidant enzyme activities, altered mitochondrial membrane potential, reduced apoptosis, and increased expression of spermatogonial cell-associated markers. These findings support the potential utility of cordycepin as a culture supplement for improving the in vitro maintenance and expansion of bSCs.