The oocyte is a major locus of reproductive aging in women, as evidenced by the ability of oocyte donation from younger women to abrogate the effects of age on fertility. Oocytes also provide a model to study aging in large, metabolically active, long-lived cells. The experiments outlined in the proposal test the hypothesis that deletions in mtDNA, which increase with age in a number of long-lived cells, underlie oocyte senescence, and that reactive oxygen species contribute to mtDNA deletions and cellular damage. The experiments employ a number of molecular rand cell biology techniques which can be performed on single cells, including a polymerase chain reaction (PCR) -based strategy, which has been widely used to identify mtDNA deletion """"""""hot spots"""""""" in brain and heart of aged individuals. It depends on mtDNA deletions approximating otherwise widely-separating primers. Deletions within the mtDNA deletion """"""""hot spots"""""""" allow amplification, while undeleted mtDNA prevents amplification, because the primers are too widely separated under the specified reaction conditions. Levels of mtDNA deletions cia be estimated in single oocytes and embryos and correlated with markers of cellular oxidative damage, numbers of mitochondria and morphology. a reactive oxygen species generator will be used to test the hypothesis that reactive oxygen species induce mtDNA deletions and oxidative damage, deplete mitochondria and impair oocyte developmental potential. Finally, nuclear transfer between oocytes exposed to reactive oxygen species generators and control oocytes will be used to test the hypothesis that mtDNA is more sensitive to reactive oxygen species than nuclear DNA. This technique also hold promise as a possible therapy to treat women with age-related infertility.
