Five percent of live-born human offspring will have a genetic disorder. Of these, 20% are because of germ-line de novo mutations. Several genetic diseases, such as neurofibromatosis and Duchenne muscular dystrophy, are associated with a high percentage of de novo germ-line mutations. Until recently, a direct analysis of spontaneous mutation frequencies in mammalian germ cells has been prevented by technical limitations. We have measured spontaneous mutation frequencies in a lacI transgene by using enriched populations of specific spermatogenic cell types. Similar to previously published results, we observed a lower mutation frequency for seminiferous tubule cell preparations, which contain all stages of spermatogenesis, relative to somatic tissues. We made the unexpected observation of a decline in mutation frequency during spermatogenesis, such that the mutation frequencies of type B spermatogonia and all subsequent stages of spermatogenesis are lower than the frequency for primitive type A spermatogonia. In addition, spermatogenic cells from old mice have significantly increased mutation frequencies compared with spermatogenic cells from young or middle-aged mice. Finally, the mutation frequency was observed to increase during spermiogenesis in postreplicative cell types when spermatogenic cells were obtained from old mice.From a genetic perspective, germ cells are profoundly different from somatic cells because they carry the genetic information that will direct the development of the next generation, not simply the next daughter cell. Thus, safeguarding the integrity of germ-line DNA might provide evolutionary advantages. Indeed, in mice, mutation frequencies obtained from mixed populations of germ cells are lower than for somatic tissues (1). This was demonstrated by using a transgenic system in which the bacteriophage genome carrying the lacI repressor gene and the ␣lacZ gene from the prokaryotic lac operon was introduced into the mouse genome as a transgene. DNA was recovered from genomic DNA preparations by packaging and used to infect a strain of Escherichia coli carrying a lacZ (␤-galactosidase) gene, but lacking a functional lacI gene. Mutation of the lacI gene renders a blue plaque on agarose containing the chromogenic substrate 5-bromo-4-chloro-3-indolyl-␤-D-galactopyranoside (X-gal). In contrast, other studies using a lacI transgenic mouse (2) or a lacZ transgenic mouse (3) did not report a significant difference in mutation frequencies for spermatogenic cells compared with somatic cells.Although provocative, interpretation of the results demonstrating a lower mutation frequency for male germ cells is complicated by the fact that adult seminiferous tubules contain a mixture of spermatogenic cell types encompassing all stages of spermatogenesis. Spermatogonia serve as the stem cells for spermatogenesis and undergo mitotic divisions that give rise to cells that will either retain their identity as spermatogonia to maintain the stem cell population or enter meiosis to become primary spermatocy...

To assess DNA repair activity relative to age, in vitro base excision repair assays were performed using brain and liver nuclear extracts prepared from mice of various ages. An 85% decline in repair activity was observed in brain nuclear extracts and a 50% decrease in liver nuclear extracts prepared from old mice compared with 6-day-old mice. Brain nuclear extracts prepared from old mice showed a decreased abundance of DNA polymerase-beta, but the addition of purified protein did not restore base excision repair activity. Abundances of other tested base excision repair proteins did not change relative to age. The conclusion is that, during aging, a decline in DNA repair could contribute to increased levels of DNA damage and mutagenesis.

The combined observations of elevated DNA repair gene expression, high uracil-DNA glycosylase-initiated base excision repair, and a low spontaneous mutant frequency for a lacI transgene in spermatogenic cells from young mice suggest that base excision repair activity is high in spermatogenic cell types. Notably, the spontaneous mutant frequency of the lacI transgene is greater in spermatogenic cells obtained from old mice, suggesting that germ line DNA repair activity may decline with age. A paternal age effect in spermatogenic cells is recognized for the human population as well. To determine if male germ cell base excision repair activity changes with age, uracil-DNA glycosylase-initiated base excision repair activity was measured in mixed germ cell (i.e., all spermatogenic cell types in adult testis) nuclear extracts prepared from young, middle-aged, and old mice. Base excision repair activity was also assessed in nuclear extracts from premeiotic, meiotic, and postmeiotic spermatogenic cell types obtained from young mice. Mixed germ cell nuclear extracts exhibited an age-related decrease in base excision repair activity that was restored by addition of apurinic/apyrimidinic (AP) endonuclease. Uracil-DNA glycosylase and DNA ligase were determined to be limiting in mixed germ cell nuclear extracts prepared from young animals. Base excision repair activity was only modestly elevated in pachytene spermatocytes and round spermatids relative to other spermatogenic cells. Thus, germ line shortpatch base excision repair activity appears to be relatively constant throughout spermatogenesis in young animals, limited by uracil-DNA glycosylase and DNA ligase in young animals, and limited by AP endonuclease in old animals.Germ line genomic stability is an important factor in reproductive health, with approximately 20% of genetic diseases attributed to new germ line mutations (12,13,46). Several autosomal dominant genetic diseases due to de novo male germ line mutations are associated with increased paternal age (18,21,45,49), suggesting that male germ line genomic stability is compromised with age. Analysis of chromosomes in spermatozoa revealed an increased frequency of aberrations with age (24, 34, 41, 54), providing additional evidence that male germ line genomic stability decreases with age. Similarly in mice, the spontaneous mutant frequency in a lacI transgene recovered from pachytene spermatocytes, round spermatids, and epididymal spermatozoa obtained from old animals was approximately 10-fold higher than the mutant frequency observed for young mice (60).In contrast to the decline in germ line genetic integrity with old age, the germ line DNA of young mice appears to be well maintained. Compared to somatic tissues, a lower spontaneous mutant frequency in the male germ line for a lacI transgene has been reported for fish (65), mice (32, 60), and rats (16). The murine mitotically proliferating male germ cell pool is comprised of primitive type A spermatogonia, which sequentially give rise to mitotically active, type ...

Spermatogenic cells exhibit a lower spontaneous mutation frequency than somatic tissues in a lacI transgene and many base excision repair (BER) genes display the highest observed level of expression in the testis. In this study, uracil-DNA glycosylase-initiated BER activity was measured in nuclear extracts prepared from tissues obtained from each of three mouse strains. Extracts from mixed spermatogenic germ cells displayed the greatest activity followed by liver then brain for all three strains, and the activity for a given tissue was consistent among the three strains. Levels of various BER proteins were examined by western blot analyses and found to be consistent with activity levels. Nuclear extracts prepared from purified Sertoli cells, a somatic component of the seminiferous epithelium, exhibited significantly lower activity than mixed spermatogenic cell-type nuclear extracts, thereby suggesting that the high BER activity observed in mixed germ cell nuclear extracts was not a characteristic of all testicular cell types. Nuclear extracts from thymocytes and small intestines were assayed to assess activity in a mitotically active cell type and tissue. Overall, the order of tissues/cells exhibiting the greatest to lowest activity was mixed germ cells > Sertoli cells > thymocytes > small intestine > liver > brain.

O 6 -methylguanine (O 6 mG) is a potent mutagenic and procarcinogenic DNA lesion. Organisms have evolved with a DNA repair mechanism that largely ameliorates the deleterious effects of O 6 mG through a direct reversal mechanism by a protein termed O 6 -methylguanine-DNA methyltransferase (MGMT). However, the contribution of O 6 mG to carcinogenesis, in the absence of known exposure to agents that produce it, has not been defined. Nontransgenic C3HeB male mice have a high frequency of spontaneous liver tumors. Transgenic CeHeB͞FeJ mice expressing human MGMT (hMGMT) were generated that had elevated hepatic MGMT activity. The spontaneous development of hepatocellular carcinoma was significantly reduced in those mice expressing hMGMT compared with nontransgenic C3HeB͞FeJ male mice. No differences were detected in spontaneous mutant frequencies in lacI transgenes in mice carrying hMGMT compared with that without hMGMT but the proportion of GC to AT transition mutations was lower in the transgenic mice carrying hMGMT as well as lacI. Tumors that arose in C3HeB͞FeJ transgenic mice were largely deficient in hMGMT protein as determined by immunohistochemistry with a monoclonal antibody directed against hMGMT. Together these data indicate that spontaneous O 6 mG lesions induced hepatocellular carcinogenesis in C3HeB͞FeJ male mice. These transgenic mice represent a rare example of reduced spontaneous carcinogenesis.