Use of SYBR14, 7-amino-actinomycin D, and JC-1 in assessing sperm damage from rats with spinal cord injury.

BACKGROUND: Although fluorescent dyes combined with flow cytometry have been used to confirm the viability of sperm in the past, methods to detect damage to spermatozoa following injury have been limited to use of dyes, which are often difficult to adequately compensate for in a single laser system. METHODS: In this article, we present what we believe is a better method to assess damage to sperm secondary to spinal cord injury in an in vivo model, for use with a standard Ar laser and flow cell. In this rat model of spinal cord injury leading to sperm damage, the spinal cords of the rats were injured, but the reproductive organs were not. To understand the origins of sperm injury, and to develop ways to overcome the loss of fertility, we used the viability dye SYBR-14 along with 7-amino actinomycin D to detect apoptosis. Additionally, we used the dye JC-1 to measure the changes in mitochondrial transmembrane potential that accompany the damage. RESULTS: We found that SYBR-14 plus 7-amino actinomycin D was a useful method for quantifying apoptosis, particularly when another dye, such as JC-1, was used simultaneously. By using these dyes in concert with motility studies, we were able to quantify the extent of damage to sperm and correlate it to the decrease in motility of sperm (r(2) = 0.99 for SYBR14 versus motility and r(2) = 0.98 for JC-1 versus motility by regression analysis). CONCLUSIONS: With a method established to measure injury to sperm, we hope to determine which treatment regimens of ones we will test are effective in restoring sperm to a more fertile state, in the future.