Base excision repair is limited by different proteins in male germ cell nuclear extracts prepared from young and old mice

Abstract

The combined observations of elevated DNA repair gene expression, high uracil-DNA glycosylase-initiated base excision repair, and a low spontaneous mutant frequency for a lacI transgene in spermatogenic cells from young mice suggest that base excision repair activity is high in spermatogenic cell types. Notably, the spontaneous mutant frequency of the lacI transgene is greater in spermatogenic cells obtained from old mice, suggesting that germ line DNA repair activity may decline with age. A paternal age effect in spermatogenic cells is recognized for the human population as well. To determine if male germ cell base excision repair activity changes with age, uracil-DNA glycosylase-initiated base excision repair activity was measured in mixed germ cell (i.e., all spermatogenic cell types in adult testis) nuclear extracts prepared from young, middle-aged, and old mice. Base excision repair activity was also assessed in nuclear extracts from premeiotic, meiotic, and postmeiotic spermatogenic cell types obtained from young mice. Mixed germ cell nuclear extracts exhibited an age-related decrease in base excision repair activity that was restored by addition of apurinic/apyrimidinic (AP) endonuclease. Uracil-DNA glycosylase and DNA ligase were determined to be limiting in mixed germ cell nuclear extracts prepared from young animals. Base excision repair activity was only modestly elevated in pachytene spermatocytes and round spermatids relative to other spermatogenic cells. Thus, germ line short-patch base excision repair activity appears to be relatively constant throughout spermatogenesis in young animals, limited by uracil-DNA glycosylase and DNA ligase in young animals, and limited by AP endonuclease in old animals.

FIG. 1.

UDG-BER activities for nuclear extracts prepared from enriched populations of spermatogenic cell types obtained from CD1 mice. Results are presented as mean ± standard error of the mean (SEM) of three replicate assays for each of three independent nuclear extract preparations. Cell types exhibiting similar UDG-BER activity are indicated by having the same letter. Prim A, primitive type A spermatogonia; Type A, type A spermatogonia; Type B, type B spermatogonia; Pach., pachytene spermatocytes; R-Tids, round spermatids.

FIG. 2.

UDG-BER activities for MGC nuclear extracts prepared from 6-day-old (6-d/o), 8-day-old, 3-month-old (3-m/o), 16-month-old, and 28-month-old male B6D2F₁ mice. Results are presented as means ± SEM of three replicate assays for each of three independent nuclear extract preparations. MGC nuclear extracts from different-aged animals with similar UDG-BER activity are indicated by having the same letter.

FIG. 3.

Long-patch BER activities on reduced AP site cccDNA for MGC nuclear extracts prepared from 3-month-old, 16-month-old, and 28-month-old male B6D2F₁ mice. Results are presented as means ± SEM of assays for each of three independent nuclear extract preparations. Solid circles, 3-month-old mice; open circles, 16-month-old mice; solid squares, 28-month-old mice.

FIG. 4.

Western blot analysis of BER proteins in nuclear extracts prepared from enriched populations of primitive type A spermatogonia (PA), type A spermatogonia (A), type B spermatogonia (B), pachytene spermatocytes (P), and round spermatids (R). Bands corresponding to DNA ligase I (130 kDa), DNA ligase III (93 kDa), Xrcc-1 (69 kDa), β-pol (39 kDa), and Ape/Ref-1 (37 kDa) proteins were visualized. Triplicate assays for each of three independent nuclear extract preparations were performed. Some variation between replicates was noted. A summary of all data is shown in Table 1. Molecular mass protein standards and purified DNA ligases I and III, β-pol, and Ape/Ref-1 (lane STD) are shown for comparison.

FIG. 5.

Western blot analysis of BER proteins in MGC nuclear extracts prepared from 3-month-old, 16-month-old, and 28-month-old mice. Bands corresponding to DNA ligase I (130 kDa), DNA ligase III (93 kDa), Xrcc-1 (69 kDa), β-pol (39 kDa), and Ape/Ref-1 (37 kDa) proteins were visualized. Triplicate assays were performed for each of three independent nuclear extract preparations. Some variation in signal intensities was observed among Western blots. A summary of all data is shown in Table 2. Molecular mass protein standards and purified DNA ligases I and III, β-pol, and APE/REF-1 (lane STD) are shown for comparison.

FIG. 6.

UDG-BER activities for MGC nuclear extracts prepared from 3-month-old B6D2F₁ (open), 28-month-old B6D2F₁ (solid), and 3-month-old Apex heterozygous knockout mice (striped). Results are presented as means ± SEM of three replicate assays for each of three independent nuclear extract preparations. ∗, significantly (P < 0.05) different from addition of 0 ng of protein within a specific group. †, amount of purified APE/REF-1 required to restore activity to that of 3-month-old mice with 0 ng of protein added.

FIG. 7.

UDG-BER activities for MGC nuclear extracts prepared from 3-month-old (open) and 28-month-old (solid) male B6D2F₁ mice. Results are presented as means ± SEM of three replicate assays for each of three independent nuclear extract preparations. Results are for UDG-BER assays in which purified uracil-DNA glycosylase (a), DNA ligase III (b), β-pol (c), and DNA ligase I (d) were added to MGC nuclear extracts. ∗, significantly (P < 0.05) different from addition of 0 ng of protein within a specific age group.