Production of Superoxide and Activity of Superoxide Dismutase in Rabbit Epididymal Spermatozoa

Abstract

Mature rabbit spermatozoa from the cauda epididymidis suspended in potassium Tris phosphate buffer at 24.degree. C produced .**GRAPHIC**. as measured by reduction of acetylated ferricytochrome c, with an intrinsic rate of 0.20 nmol/min per 108 cells. this rate increased to 1.80 nmol/min per 108 cells in the presence of 10 mM cyanide. These spermatozoa contain 2.8 units per 108 cells of superoxide dismutase activity 95% of which is sensitive and 5% of which is insensitive, to cyanide inhibition. These activities correspond to the cytosolic Cu-Zn form and the mitochondrial Mn form of the dismutase, respectively. Only the cyanide-sensitive form is released from the sperm on hyposmotic treatment or sonication. Hypo-osmotically treated rabbit epididymal spermatozoa produced .**GRAPHIC**. with an intrinsic rate of 0.24 nmol/min per 108 cells, which increased to 0.58 nmol/min per 108 cells in the presence of 10 mM cyanide. Both intact and hypo-osmotically treated cells react with .**GRAPHIC**. in a 2nd order reaction as inferred from the hyperbolic dependence on cell concentration of .**GRAPHIC**. production rate in both the absence and presence of cyanide. The 2nd order rate constant for this reaction with intact cells, kS, was calculated to be 22.9 .times. 10-8 (cells/ml)-1 min-1 in the presence of cyanide and 12.5 .times. 10-8 (cells/ml)-1 min-1 in its absence. For hypo-osmotically treated cells, the values of kS were 10.8 .times. 10-8 (cells/ml)-1 min-1 and 8.2 .times. 10-8 (cells/ml)-1 min-1, respectively. Since hypo-osmotically treated cells have lost much of their plasma membrane, the lower value of kS for the treated cells implies that this membrane is one site of reaction of .**GRAPHIC**. with the cells. The increase in kS in the presence of cyanide, which inhibits superoxide dimutase and so increases .**GRAPHIC**. production, suggests that the cells become more reactive with .**GRAPHIC**. as its production rate increases, as would be expected for the occurrence of radical chain oxidation. Apparently superoxide dismutase plays a major role in protecting rabbit sperm against damage from lipid peroxidation.