Copper-induced apoptosis and autophagy through oxidative stress-mediated mitochondrial dysfunction in male germ cells.

Excess copper reduces sperm number and motility but the causes are unclear. We investigated the toxic effects of copper exposure on the immortalized male germ cell line GC-1. Copper addition to cells altered viability and morphology in a dose-dependent manner. Copper addition resulted in increased levels of reactive oxygen species (ROS), malonaldehyde (MDA) and lactate dehydrogenase (LDH) while catalase (CAT) activity and glutathione (GSH) declined. The mitochondrial transmembrane potential and ATP levels decreased in response to copper as did mitochondria fission that led to mitochondrial dysfunction. The apoptosis rate was also proportional to the level of copper in the growth medium. Copper also down-regulated Bcl2 and up-regulated Bax, Casp8 and Casp3 linking the effects of copper to increased apoptosis. The levels of mRNA for the autophagy-related genes (Atg3, Atg5, p62, Lc3b/Lc3a) and proteins (Lc3b/Lc3a, BECN1, Atg5, p62) all increased in copper-treated cells as were levels Lc3 determined by fluorescence microscopy. These results indicated that copper induces apoptosis and autophagy through oxidative stress-mediated mitochondrial dysfunction.