Microcystin-leucine arginine mediates apoptosis and engulfment of Leydig cell by testicular macrophages resulting in reduced serum testosterone levels

Microcystin-leucine arginine (MC-LR) causes decline of serum testosterone levels resulting in impaired spermatogenesis; however, the underlying molecular mechanisms are not fully understood. In this study, we aimed to investigate the effects of MC-LR exposure on the number of Leydig cells (LCs) in testis. Following chronic low dose exposure to MC-LR, the number of LCs was markedly decreased while macrophages were significantly increased. Then, we established a co-culture system to study the interaction between macrophages and LCs in the presence of MC-LR. No significant apoptosis of LCs cultured alone was observed after MC-LR (< 5 000 nM) treatment; however, apoptosis was robustly increased when LCs were co-cultured with macrophages in the presence of MC-LR. Further studies identified that MC-LR could stimulate macrophage to produce TNF-α, and secreted TNF-α induced LC apoptosis by binding to the tumor necrosis factor receptor 1 (TNFR1) on the LCs and thus activating reactive oxygen species (ROS)-p38MAPK signaling pathway. Furthermore, we also examined increased expression of Axl receptor and growth arrest-specific 6 (Gas6) in macrophages after MC-LR treatment. GAS6 mediates phagocytosis of apoptotic LCs by binding to the Axl receptor on macrophages and phosphatidylserine (PtdSer) on apoptotic LCs. Together, these results suggested that reduced serum testosterone levels may be associated with decrease of LCs as a result of LC apoptosis and phagocytosis by immune cells in MC-LR-treated mice.