Tumor Necrosis Factor α Phosphorylates c-Jun N-Terminal Kinase in Stallion Spermatozoa: Effect of Cryopreservation

The presence of tumor necrosis factor (TNF) α, TNF-R1, and TNF-R2 was investigated in lysates of fresh and frozen-thawed stallion sperm. Western blotting and immunohistochemistry were performed with available human and equine antibodies against TNFα. Positive controls were lysates of human white blood cells. The antibody used in our study identified two bands of approximately 28 and 17 kDa and also a third band of approximately 50 kDa. The first two bands were also detected in controls, whereas the third band showed a slight deviation of the molecular mass, appearing as a band of 62 kDa in the controls. However, when a specific equine antibody was used, both bands (50 and 62 kDa) were detected, suggesting that this band correspond to the TNFα homotrimer. The intensity of this latter band was significantly reduced in frozen-thawed sperm, suggesting that the freezing and thawing procedure disrupts the TNFα homotrimer. The antibody against TNFα-R1 did not reveal any signal in sperm lysates, whereas in positive controls (human white blood cells), a band of approximately 55 kDa appeared. The antibody against TNFα-R2 recognized two bands of approximately 50 and 72 kDa in human cells; also in stallion lysates, both bands were detected; however, only the band of 50 kDa was consistently present in all the replicates. Tumor necrosis factor α induced the phosphorylation of c-jun N-terminal kinase suggesting a role in the regulation of the survival of stallion spermatozoa.