Mutagenesis is elevated in male germ cells obtained from DNA polymerase-beta heterozygous mice

Abstract

Gametes carry the DNA that will direct the development of the next generation. By compromising genetic integrity, DNA damage and mutagenesis threaten the ability of gametes to fulfill their biological function. DNA repair pathways function in germ cells and serve to ameliorate much DNA damage and prevent mutagenesis. High base excision repair (BER) activity is documented for spermatogenic cells. DNA polymerase-beta (POLB) is required for the short-patch BER pathway. Because mice homozygous null for the Polb gene die soon after birth, mice heterozygous for Polb were used to examine the extent to which POLB contributes to maintaining spermatogenic genomic integrity in vivo. POLB protein levels were reduced only in mixed spermatogenic cells. In vitro short-patch BER activity assays revealed that spermatogenic cell nuclear extracts obtained from Polb heterozygous mice had one third the BER activity of age-matched control mice. Polb heterozygosity had no effect on the BER activities of somatic tissues tested. The Polb heterozygous mouse line was crossed with the lacI transgenic Big Blue mouse line to assess mutant frequency. The spontaneous mutant frequency for mixed spermatogenic cells prepared from Polb heterozygous mice was 2-fold greater than that of wild-type controls, but no significant effect was found among the somatic tissues tested. These results demonstrate that normal POLB abundance is necessary for normal BER activity, which is critical in maintaining a low germline mutant frequency. Notably, spermatogenic cells respond differently than somatic cells to Polb haploinsufficiency.

FIG. 1.

Western blot analysis. Western blot analysis of nuclear extracts prepared from mixed germ cells (MGC) (A), liver (B), and brain (C) obtained from wild-type control and Polb+/− animals. Each tissue was analyzed separately from other tissues. Alongside samples, lanes were also loaded with purified APEX1 and POLB as standards. APEX1 protein is distinct as two bands on MGC Western blots at 37 kDa and at 34 kDa. POLB is at 39 kDa, XRCC1 is at 69 kDa, and DNA ligase IIIα is at 93 kDa. D) Summary of Western blot quantification using nuclear extracts prepared from Polb+/− and wild-type mice. Data are given as mean ± SEM after normalization to values for wild-type controls. At least four animals were used per genotype. Bands were quantified as IDV per microgram of protein. Solid black bars, MGC; solid gray bars, liver; hatched bars, brain; and a, significantly different from wild-type control in same tissue.

FIG. 2.

BER activity assay results. A) Linearity determinations for mixed germ cells (MGC), liver, and brain BER activities relative to protein concentration. The best-fit line is shown for wild-type control MGC (solid line), liver (long dashed line), and brain (short dashed line). a, significantly different from liver and brain; b, significantly different from brain. B–D) BER activities for nuclear extracts prepared from MGC (B), liver (C), and brain (D) obtained from wild-type (WT) control and Polb+/− animals. Forty micrograms of MGC nuclear extracts and 160 μg of liver and brain nuclear extracts were used. Results are given as mean ± SEM (E). Four animals were used per genotype for MGC and liver and three for brain. Results are from at least two replicate assays for each sample. Solid bars, control; hatched bars, Polb+/−; a, significantly different from wild-type control in same tissue; b, significantly different from MGC and brain same of genotype; and c, significantly different from MGC and liver of same genotype.

FIG. 3.

Spontaneous mutant frequencies for MGC and somatic tissues obtained from wild-type control (solid bars) and Polb(+/−),lacI+ (hatched bars) animals. Results are given as mean ± SEM. Five animals per genotype were used minimally, and at least two replicate assays were performed on DNA obtained from each animal. a, significantly different from wild-type control in same tissue; b, significantly different from liver of same genotype; and c, significantly different from brain of same genotype.

FIG. 4.

TUNEL staining of testes cross-sections obtained from 10-day-old and young adult wild-type control mice. A) TUNEL-stained cross-section from a 10-day-old mouse. B) An H&E-stained testis from a 10-day-old mouse. C) TUNEL-stained young adult testis. D) An H&E-stained young adult testis. Brown precipitate on TUNEL-stained slides indicates TUNEL-positive nuclei, indicative of cells scored positive for apoptosis. Prevalence of apoptosis among the testis decreases approximately by half as the animal ages from 10 days to adulthood in wild-type controls and in Polb+/− mice. Examples of apoptotic nuclei are designated by black arrows. Original magnification A and B ×16; C and D ×6.3.