The primary goal of this study is to determine if mitochondrial DNA in human sperm is more sensitive to oxidative damage than nuclear DNA. The long-term goals of this research are to further our understanding of role of oxidative stress in human male infertility, and ultimately, to establish a link between oxidative damage in male germ cells, germ-line mutagenesis, and birth defects. Recent research on etiology of male infertility indicates that oxidative stress is a primary cause for sperm dysfunction. Excessive ROS production by spermatozoa has been associated with abnormal semen profiles, and results in loss of motility, lipid peroxidation, and DNA damage. However, effects of oxidative stress on sperm mitochondrial DNA has not been examined. Mitochondrial DNA of somatic cells is known to be more sensitive to oxidative damage than nuclear DNA. The experiments in this proposal will determine if the same is true in human sperm. If so, mitochondrial DNA may serve as a sensitive biomarker for DNA damage in human sperm. In separate experiments, human sperms ill be exposed to oxidative stress by treatment with hydrogen peroxide, the H2O2 generating system glucose/glucose oxidase, 4-hydroxynonenal, activated leukocytes, and NADPH (which causes increased ROS production by sperm). After treatment, total DNA from sperm will be isolated, and subjected to a quantitative PCR assay. Using this assay, gene- and mitochondria-specific DNA damage will be measured.
