Human Reproduction, Vol. 15, No. 7, 1552-1561,
July 2000
© 2000 European Society of Human Reproduction and Embryology
Differences in nuclear DNA fragmentation and mitochondrial integrity of semen and prepared human spermatozoa
Department of Obstetrics & Gynaecology, The Queen's University of Belfast, Northern Ireland
Sperm DNA integrity is essential for accurate transmission of genetic material to offspring. Fragmentation of genomic DNA is an initial hallmark of apoptosis (programmed cell death). The aim of this study was to determine sperm nuclear DNA integrity and mitochondrial function, to quantify possible apoptosis and to investigate any relationship between these parameters. Semen samples (n = 25) were prepared by discontinuous Percoll density centrifugation (95.0:47.5). DNA integrity was determined using a modified alkaline single cell gel electrophoresis (Comet) assay. DNA fragmentation, possibly indicative of apoptosis, was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL). Mitochondrial transmembrane potential was determined using the mitochondrial probe 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl carbocyanine iodide (JC-1). The DNA integrity of prepared spermatozoa was significantly greater than that of semen (P < 0.005). Further, the percentage of spermatozoa with fragmented DNA and the degree of fragmentation within these cells in prepared spermatozoa is significantly less than in semen (P < 0.005). There is a significant correlation between DNA damage quantified using the Comet assay and DNA fragmentation determined using TUNEL (R = 0.562, P < 0.01). The percentage of spermatozoa with dysfunctional, possibly apoptotic, mitochondria was significantly lower in prepared spermatozoa than in neat semen samples (P < 0.001). There was a negative correlation between the percentage of spermatozoa with dysfunctional mitochondria and the percentage of progressively motile spermatozoa (R = –0.67, P < 0.01).
Key words: apoptosis/CASA/COMET assay/mitochondria/TUNEL assay
1 To whom correspondence should be addressed at: Department of Obstetrics & Gynaecology, The Queen's University of Belfast, Institute of Clinical Science, Grosvenor Road, Belfast BT12 6BJ, UK. E-mail:e.donnelly{at}qub.ac.uk
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