Hexavalent chromium inhibits testosterone synthesis in bovine testicular leydig cells through mitochondrial damage via BNIP3

This study investigated the molecular mechanisms underlying the inhibitory effects of hexavalent chromium (Cr(Ⅵ)) exposure on testosterone synthesis in bovine testicular Leydig cells. First, bovine Leydig cells were isolated and purified, cultured after purity identification, and the effect of different dose gradients of Cr(Ⅵ) on cell viability after 12 h of exposure was detected using the CCK-8 method. Cell proliferation was assessed using a cell proliferation assay kit. Testosterone levels were measured by ELISA. Intracellular reactive oxygen species (ROS) levels were detected using the DCFH-DA fluorescent probe. Cell autophagy was examined by the monodansylcadaverine (MDC) method. Changes in mitochondrial membrane potential were determined using the JC-1 probe. Ultrastructural changes in mitochondria were observed via transmission electron microscopy. The expression of key genes involved in testosterone synthesis, cell autophagy, mitochondrial fusion, mitochondrial fission, and mitophagy was detected by qRT-PCR. Differentially expressed genes were screened through RNA-seq. Our results demonstrate that Cr(Ⅵ) treatment significantly suppressed testosterone synthesis. Concurrently, Cr(Ⅵ) enhanced intracellular MDC levels and induced mitochondrial dysfunction, characterized by ultrastructural damage, reduced mitochondrial membrane potential, and disrupted mitochondrial dynamics. RNA sequencing analysis revealed that Cr(Ⅵ) activates BNIP3 recruitment and high expression within cells. Subsequent BNIP3 silencing using siRNA interference significantly attenuated Cr(Ⅵ)-induced abnormalities in the expression of mitochondrial genes and intracellular ROS accumulation. Moreover, BNIP3 expression knockdown markedly upregulated the mRNA expression of key steroidogenic genes and the level of testosterone in cell supernatant. These findings suggest that Cr(Ⅵ) inhibits testosterone synthesis in Leydig cells through BNIP3-mediated mitochondrial damage. This study identifies BNIP3 as a potential therapeutic target and provides a theoretical foundation for addressing Cr(Ⅵ)-associated male reproductive dysfunction.