Mutation frequency declines during spermatogenesis in young mice but increases in old mice

Abstract

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.

Figure 1

Mutation frequencies and standard errors in somatic cells/tissues and enriched seminiferous tubule cell preparations obtained from 60-day-old male mice. The mutation frequencies are significantly lower for seminiferous tubule cells versus somatic cells (P ≤ 0.0001). A test for differences between brain, liver, Sertoli cells (6 days old), and Sertoli cells (8 days old) revealed no significant differences (P = 0.1094). Br, brain; Li, liver; SC, Sertoli cells; SAvg, somatic average; STC, seminiferous tubule cells.

Figure 2

Mutation frequencies and standard errors for specific spermatogenic cell types. All cell types tested had a significantly lower mutation frequency than the average somatic mutation frequency (P ≤ 0.001). Furthermore, there was a significant decline in mutation frequency during spermatogenesis between primitive type A spermatogonia and type B spermatogonia (P = 0.02). Prim. A, primitive type A spermatogonia; A, type A spermatogonia; B, type B spermatogonia; Pre-L, preleptotene spermatocytes; L+Z, leptotene plus zygotene spermatocytes; Pach., pachytene spermatocytes; Rnd., round spermatids; Sprm, spermatozoa.

Figure 3

Spontaneous mutation frequencies and standard errors are shown for spermatogenic cells obtained from 60-day-old, 15-month-old, and 28-month-old mice. The mutation frequency of old pachytene spermatocytes is greater than that of young spermatocytes (P < 0.0001) and greater than middle-aged pachytene spermatocytes (P < 0.0001). Similarly, the mutation frequency of old round spermatids is greater than young (P < 0.0001) and middle-aged (P < 0.0001) round spermatids. Furthermore, the mutation frequency of old spermatozoa is greater than that of young spermatozoa (P < 0.0001). The mutation frequency was greater in old spermatozoa than in old pachytene spermatocytes (P = 0.0314), but there were no significant differences among young pachytene spermatocytes, round spermatids, and spermatozoa (P = 0.4585). Y. Pach., young pachytene spermatocytes; Y. Rnd., young round spermatids; Y. Sprm., young spermatozoa; M. Pach., middle-aged pachytene spermatocytes; M. Rnd., middle-aged round spermatids; O. Pach., old pachytene spermatocytes; O. Rnd., old round spermatids; O. Sprm., old spermatozoa.