Environmental exposure of the mouse germ line: DNA adducts in spermatozoa and formation of de novo mutations during spermatogenesis

Abstract

Background: Spermatozoal DNA damage is associated with poor sperm quality, disturbed embryonic development and early embryonic loss, and some genetic diseases originate from paternal de novo mutations. We previously reported poor repair of bulky DNA-lesions in rodent testicular cells.

Methodology/principal findings: We studied the fate of DNA lesions in the male germ line. B[a]PDE-N(2)-dG adducts were determined by liquid chromatography-tandem mass spectrometry, and de novo mutations were measured in the cII-transgene, in Big Blue mice exposed to benzo[a]pyrene (B[a]P; 3 x 50 mg/kg bw, i.p.). Spermatozoa were harvested at various time-points following exposure, to study the consequences of exposure during the different stages of spermatogenesis. B[a]PDE-N(2)-dG adducts induced by exposure of spermatocytes or later stages of spermatogenesis persisted at high levels in the resulting spermatozoa. Spermatozoa originating from exposed spermatogonia did not contain DNA adducts; however de novo mutations had been induced (p = 0.029), specifically GC-TA transversions, characteristic of B[a]P mutagenesis. Moreover, a specific spectrum of spontaneous mutations was consistently observed in spermatozoa.

Conclusions/significance: A temporal pattern of genotoxic consequences following exposure was identified, with an initial increase in DNA adduct levels in spermatozoa, believed to influence fertility, followed by induction of germ line de novo mutations with possible consequences for the offspring.

Figure 1. B[a]PDE-N²-dG adducts in caput spermatozoa from untreated and exposed male germ cells at different stages of spermatogenesis.

The male germ cell stage is indicated along with day of sacrifice. Untreated cauda spermatozoa = CASZ (number of animals n = 4); epididymal spermatozoa = ESZ (n = 4); spermatids = ST (n = 4); spermatocytes = SC (n = 6); differentiating spermatogonia = Diff. SG (n = 4); stem cell spermatogonia = Stem cell SG (n = 5). B[a]PDE-N²-dG adduct levels are given as means±SE. For untreated CASZ and exposed Diff. SG and Stem cell SG the values were below detection. Statistically significant differences and p-values are indicated in the figure (ANOVA and post hoc LSD).

Figure 2. B[a]PDE-N²-dG adducts in liver from untreated and exposed mice.

The day of sacrifice is indicated for untreated and exposed mice. B[a]PDE-N²-dG adduct levels from 4–8 mice per group are given as means±SE. Reduction in B[a]PDE-N²-dG adduct levels were tested statistically (Mann-Whitney) between groups and are indicated with p-values in the figure.

Figure 3. Mutation frequencies in cauda spermatozoa from B[a]P-exposed male germ cells at different stages of spermatogenesis.

The male germ cell stage is indicated along with day of sacrifice. Cauda spermatozoa = CASZ (untreated, n = 3); epididymal spermatozoa = ESZ (n = 4); spermatids = ST (n = 6); spermatocytes = SC (n = 5); differentiating spermatogonia = Diff. SG (n = 6); stem cell spermatogonia = Stem cell SG (n = 6). Mf values are given as means (per 10⁶ pfu) ±SE. Differences in mean Mf were tested statistically (Mann-Whitney); The B[a]P-exposed were compared with untreated control, and B[a]P-exposed at 119 days were also compared with its time-matched corn oil treated control. Statistically significant differences are indicated (p = 0.029).

Figure 4. Mutation frequencies in the liver of Big Blue® mice exposed to B[a]P.

The Mf values are given as means (per 10⁶ pfu) ±SE, and the days of sacrifice are indicated on the x-axis. Mice sacrificed at day 0 are untreated controls. Treatments groups and number of mice (n) are given: 0 days untreated (n = 3); 4 days B[a]P (n = 4); 16 days B[a]P (n = 6); 30 days B[a]P (n = 5); 44 days B[a]P (n = 6); 120 days B[a]P (n = 6) and 120 days corn oil (n = 6). Statistically significant differences (Mann-Whitney) between the Mf of the groups and p-values are indicated.

Figure 5. Mutation spectra in cauda spermatozoa and liver.

The figure shows the distributions of mutation types from cauda spermatozoa of untreated mice (A), mice treated with corn oil (B), B[a]P (C) 119 days before sacrifice, or from livers of mice treated with corn oil (D) or B[a]P (E) 119 days before sacrifice. The distributions are given as percentages (%) of the total number of mutations.