Differences in nuclear DNA fragmentation and mitochondrial integrity of semen and prepared human spermatozoa

Sperm DNA integrity is essential for accurate transmission of genetic mater ial to offspring. Fragmentation of genomic DNA is an initial hallmark of ap optosis (programmed cell death). The aim of this study was to determine spe rm nuclear DNA integrity and mitochondrial function, to quantify possible a poptosis and to investigate any relationship between these parameters. Seme n samples (n = 25) were prepared by discontinuous Percoll density centrifug ation (95.0:47.5). DNA integrity was determined using a modified alkaline s ingle cell gel electrophoresis (Comet) assay. DNA fragmentation, possibly i ndicative of apoptosis, was detected by terminal deoxynucleotidyl transfera se-mediated dUTP nick end labelling (TUNEL), Mitochondrial transmembrane po tential was determined using the mitochondrial probe 5,5',6,6'-tetrachloro- 1,1',3,3'-tetraethyl- benzimidazolyl carbocyanine iodide (JC-1). The DNA in tegrity of prepared spermatozoa was significantly greater than that of seme n (P < 0.005), Further, the percentage of spermatozoa with fragmented DNA a nd the degree of fragmentation within these cells in prepared spermatozoa i s significantly less than in semen (P < 0.005). There is a significant corr elation between DNA damage quantified using the Comet assay and DNA fragmen tation determined using TUNEL (R = 0.562, P < 0.01). The percentage of sper matozoa with dysfunctional, possibly apoptotic, mitochondria was significan tly lower in prepared spermatozoa than in neat semen samples (P < 0.001). T here was a negative correlation between the percentage of spermatozoa with dysfunctional mitochondria and the percentage of progressively motile sperm atozoa (R = -0.67, P < 0.01).