Isolated spermatozoa as indicators of mutations transmitted to progeny.
Spermatozoa comprise a large and homogeneous population of cells that may serve as an alternative to resource-intensive assays of transmissible mutations based on progeny. To evaluate mutagenic responses in spermatozoa derived from germ cells exposed to a mutagen at different stages of spermatogenesis, we compared cII mutant frequencies (MFs) in spermatozoa collected from male lambda transgenic medaka exposed to ethylnitrosourea (ENU) as either post-meiotic or pre-meiotic germ cells. cII MFs in spermatozoa exposed to ENU as spermatogonial stem cells were induced significantly, 9-fold, compared to controls, whereas, cII MFs in spermatozoa exposed as spermatozoa/late spermatids were not elevated. To directly compare responses in spermatozoa with those in progeny, we analyzed cII MFs directly in spermatozoa and in the offspring produced from identical sperm samples of ENU-exposed males. cII MFs in isolated spermatozoa exposed to ENU as post-meiotic germ cells were not significantly elevated, whereas 11-30% of the progeny derived from the identically exposed germ cells exhibited significantly elevated cII MFs, approximately 2-fold to >130-fold, compared to controls. The contradictory responses between spermatozoa and progeny analyses can be attributed to induced pre-mutational lesions that remain intact in spermatozoa but were not detected as mutations. Progeny analyses, by contrast, revealed mutant individuals with elevated cII mutant frequencies because persistent DNA damage in the spermatozoa was fixed as mutations in cells of the early stage embryo. Spermatozoa exposed to a mutagen as spermatogonial stem cells can provide an efficient means to detect the portion of transmissible mutations that were fixed as mutations in spermatozoa. The caveat is that direct analyses of mutations in spermatozoa excludes the contribution of mutations that arise from post-fertilization processes in cells of early stage embryos, and therefore may underestimate the actual frequency of mutant offspring.
Norris
Michelle B
MB
Aquatic Biotechnology and Environmental Laboratory, Warnell School of Forestry and Natural Resources, University of Georgia, Athens, GA 30602, United States.
Winn
Richard N
RN
D000818:Animals; D030801:Animals, Genetically Modified; D004252:DNA Mutational Analysis / Q000379:methods*; D005038:Ethylnitrosourea; D005240:Feasibility Studies; D018095:Germ-Line Mutation; D008297:Male; D009153:Mutagens; D009990:Oryzias; D013091:Spermatogenesis; D013094:Spermatozoa / Q000648:ultrastructure*; D014157:Transcription Factors / Q000235:genetics*; D014764:Viral Proteins / Q000235:genetics*
D009153:Mutagens; D014157:Transcription Factors; D014764:Viral Proteins; C039000:cII protein, bacteriophage lambda; D005038:Ethylnitrosourea