Ionizing Radiation Mediates a Greater Mutagenic Response in Spermatogenic Cells from Middle-Aged and Old Mice Compared to Young Adult Mice.

Spermatogenic cells maintain and package DNA that will direct the development of the next generation. A high level of genetic integrity is important for insuring successful reproduction. Genotoxic agents in the environment can exacerbate mutagenesis in the germline and increase the risk of siring children with a genetic disorder. The purpose of the present study was to determine if there is an age-related decline in the ability to respond to a genotoxin. LacI transgenic mice were sham irradiated or irradiated with 1.2Gy of ionizing radiation at 4-, 15- or 26-months-old. The lacI transgenic mouse model was designed to measure in vivo mutagenesis. Approximately 40 head-to-tail copies of lambda DNA are carried by hemizygotes and 80 copies by homozygotes. The lacI gene is inserted into the lambda DNA and serves as the mutation reporter. Spermatogenic cells were collected at 15 and 49 days post irradiation. Pachytene spermatocytes and round spermatids were separated using a StaPut apparatus. High molecular weight DNA was isolated from the cells and packaged with appropriate phage packaging extracts. The phage were then used to infect E. coli engineered to carry lacZ, the beta-galactosidase gene. E. coli was grown as a lawn on agarose containing X-gal which serves as a chromogenic substrate for beta-galactosidase. Mutations in the lacI gene result in expression of the lacZ gene and functional beta-galactosidase produces blue plaques. When the lacI gene is not mutated, it suppresses expression of lacZ and the plaques appear colorless because beta-galactosidase is not made. Blue plaques are cored and re-tested to confirm their mutant status. The infection of E. coli with lambda phage effectively produces single clones of mutated lacI genes that can then be sequenced to identify the mutagenic event. Mutant clones were sequenced by the University of Victoria sequencing facility. Spontaneous mutant frequency was increased in spermatogenic cells recovered from old mice and was particularly evident in the round spermatids obtained from sham irradiated 26-month-old mice. Ionizing radiation resulted in an increased mutant frequency in pachytene spermatocytes regardless of age at 15 and 49 days post irradiation. Mutant frequency was not elevated beyond the spontaneous level in round spermatids obtained from old mice, however there was an increase in mutagenesis in round spermatids obtained from middle-aged mice at 15 and 49 days after irradiation. In conclusion, middle-aged and old mice displayed a greater mutagenic response to ionizing radiation than did young mice. Environmental and therapeutic exposures to ionizing radiation may exacerbate naturally occurring increases in mutagenesis in the spermatogenic lineage as males age.

(poster)