Germline mutations can impact substantially on human health by having debilitating effects on ensuing offspring. It is estimated that 5% of liveborn offspring will have a genetic disorder. Of these, 20% are due to germline de novo mutations. From a genetic perspective, germ cells are profoundly different from somatic cells because they carry the DNA for the next generation of the organism, not simply for the next daughter cell. It seems logical to assume that safeguarding the integrity of germ- line DNA would provide survival advantages. Notably, testis has the lowest mutation frequency among tissues in lacI transgenic mice. Thus, it is reasonable to speculate that multiple safeguarding mechanisms may have evolved with this tissue type. An approach is presented to test the hypothesis that modulation of DNA repair pathways that are potentially important in determining spontaneous mutation frequencies in spermatogenic cells. First, various DNA repair activities will be assayed in cells at defined stages of spermatogenesis to identify DNA repair pathways that are potentially important in determining spontaneous mutation frequencies in spermatogenic cells. Based on these results, transgenic mice that have reduced activity in an appropriate pathway(s) will be made doubly transgenic by crossing with mice carrying the lacI transgene. lacI mutation frequencies will then be measured for specific spermatogenic cell types to confirm which DNA repair pathways play a fundamental role in determining spontaneous mutation frequencies. In the next phase, lacI and lacZ transgenic male mice will treated with an environmentally significant genotoxin, ionizing radiation. Subsequently mutation frequencies will be measured in discrete populations of spermatogenic cells to directly determine: 1) if there is a differential mutability among spermatogenic cells and 2) if mutation frequencies correlate with DNA repair activities measured in the first phase. This study will advance an understanding of the fundamental mechanisms involved in mutagenesis in germ cells.
