Increased human AP endonuclease 1 level confers protection against the paternal age effect in mice

Abstract

Increased paternal age is associated with a greater risk of producing children with genetic disorders originating from de novo germline mutations. Mice mimic the human condition by displaying an age-associated increase in spontaneous mutant frequency in spermatogenic cells. The observed increase in mutant frequency appears to be associated with a decrease in the DNA repair protein, AP endonuclease 1 (APEX1) and Apex1 heterozygous mice display an accelerated paternal age effect as young adults. In this study, we directly tested if APEX1 over-expression in cell lines and transgenic mice could prevent increases in mutagenesis. Cell lines with ectopic expression of APEX1 had increased APEX1 activity and lower spontaneous and induced mutations in the lacI reporter gene relative to the control. Spermatogenic cells obtained from mice transgenic for human APEX1 displayed increased APEX1 activity, were protected from the age-dependent increase in spontaneous germline mutagenesis, and exhibited increased apoptosis in the spermatogonial cell population. These results directly indicate that increases in APEX1 level confer protection against the murine paternal age effect, thus highlighting the role of APEX1 in preserving reproductive health with increasing age and in protection against genotoxin-induced mutagenesis in somatic cells.

Keywords: APEX1; DNA repair; LacI; Mutagenesis; Paternal Age; Spermatogenesis.

Fig. 1

mApex1 expression vector. The murine Apex1 (mApex1) cDNA was subcloned into pBluescript(r) II SK+ (Stratagene). The murine phosphoglycerol kinase (Pgk-1) promoter directs expression of Apex1. Rabbit β-globin exons II and III and intron II were added to potentially enhance expression of Apex1.

Fig. 2

Southern blot identification of stable Apex1 transfectants. Puromycin resistant clones were subjected to Southern blot analysis to identify clones carrying the mApex1 expession vector, using radiolabeled mApex1 cDNA probe. Top panel: a 1kpb mApex1-hybridizing band indicates the presence of the expression vector. Clone Pur 1, not transfected with mApex1 expression vector does not have a 1kbp hybridizing Apex1 band and serves as a puromycin transfected control cell line. Bottom panel: Gapdh hybridization was used to correct for loading and transfer differences among samples. The representative negative control sample is taken from the same gel, but was separated by clones that did not carry the Apex1 expression vector. These lanes were removed digitally and the negative control lane placed adjacent to the first sample that displayed hybridization to the probe.

Fig. 3

Confocal immunofluorescence detection of APEX1 subcellular localization. Confocal fluorescence images (left) show the distribution of APEX1 (XY) in a single optical section selected from a series of images collected throughout the cell volume. YZ1 and YZ2 depict APEX1 distribution through the depth of the cells at positions XY1 and XY2 respectively. Differential interference contrast (DIC) images (right) show the cell morphology. (A) Detection of endogenous APEX1 in Pur 1 cells transfected with the puromycin resistance plasmid only. (B) APEX1 localization in Pap2a (C) APEX1 localization in clone Pap 6a.

Fig. 4

APEX1 activity assays. Decreased activity was observed in extracts prepared from Apex1 heterozygous primary fibroblasts compared to all other tested cell lines. A modest increase in activity, P < 0.05, was observed in extracts prepared from clones Pap 2a and Pap 6a compared to the transfected control cell line, Pur 1. Data are presented as means ± SEM and represent 3 independent preparations of whole cell extracts with 2 repetitions of the assay for each preparation. WT = wild-type primary fibroblasts obtained from a C57BL/6J mouse. Het = primary fibroblasts obtained from Apex1 heterozygous mice. BBR = primary fibroblasts obtained from Big Blue Rat^®. a = significantly different from Het, b = significantly different from Pur 1. Analysis of variance (ANOVA) was used to analyze APEX1 activity data.

Fig. 5

Clones display varying levels of Apex1 RNA over-expression. A greater abundance of Apex1 RNA was observed in multiple clones. Pap 2a and Pap 6a represent low and moderate over-expression of Apex1 RNA, respectively.

Fig. 6

A greater mutant frequency was observed in all MMS treated cell lines compared to untreated and IR-treated conditions. A greater mutant frequency was observed in all IR treated cell lines compared to untreated spontaneous controls except for Pur 1, which displayed no significant difference between spontaneous and IR treatments. A minimum of 350,000 PFUs were screened for each cell line and treatment. Error bars represent standard errors. a = significantly different from spontaneous, same cell line; b = significantly different from MMS, same cell line; c =nsignificantly different from IR, same cell line. Cross-hatched bars = Pur 1, white bars = Pap 2a, diagonal bars = Pap 6a.

Fig. 7

Changes in APEX1 activity with age in mixed germ cells. APEX1 activity is increased in GCs isolated from hAPEX1 transgenic mice. GC extracts were prepared from four transgenic 6-months-old, three non-transgenic 6-months-old wild-type, two transgenic 28-months-old and three non-transgenic 28-months-old wild-type mice. APEX1 activity is plotted as percent incision of the oligonucleotide containing the synthetic AP site relative to wild-type activity for each age. Black bars represent wild-type; white bars represent transgenic. Error bars represent standard error of the mean. a = significantly different from WT, same age, P ≤ 0.01.

Fig. 8

hAPEX1 overexpression in mice does not alter the overall subcellular distribution pattern of the APEX1 protein. APEX1 immunohistochemistry was performed on testes sections prepared from four transgenic 6-months-old and three non-transgenic 6-months-old wild-type mice. Predominantly nuclear staining for APEX1 is detected in transgenic and wild-type mice. Upon greater magnification, APEX1 is also detected largely in nuclei (insets).

Fig. 9

(A) The percent of seminiferous tubule cross sections containing apoptotic cells was determined for each genotype. The fraction of tubule cross sections containing apoptotic cells is increased in transgenic and wild-type animals with increasing age. a = significantly different from 6-months-old, same genotype; b = significantly different from 16-months-old, same genotype. Wild-type are black bars, hAPEX1 transgenic are white bars. (B) The number of TUNEL positive spermatogonial cells increased with increasing age in transgenic and wild-type mice. There are significantly more TUNEL positive spermatogonial cells in the 26-months-old hAPEX1 testes compared to the 26-months-old wild-type revealing a greater level of apoptosis in this cell type at old age. Wild-type are black bars, hAPEX1 transgenic are white bars. (C) Both non-transgenic wild-type testes and hAPEX1 transgenic testes demonstrated an age-dependent increased in TUNEL positive pachytene spermatocytes. Error bars represent standard errors. Wild-type are black bars, hAPEX1 transgenic are white bars. a = significantly different from 6-months-old, same genotype; b = significantly different from 16-months-old, same genotype. c = significantly different from WT, same age.

Fig. 10

Spontaneous mutant frequencies for GCs obtained from control lacI and hAPEX1 transgenic mice. Mutant frequencies were determined for the lacI gene carried in mixed germ cells isolated from control lacI and hAPEX1 transgenic mice, at 6, 16, and 26 months of age. The mutant frequency is significantly increased in cells obtained from 26-month-old control lacI transgenic mice, but remains low in cells obtained from old hAPEX1 transgenic mice. Control lacI transgenic are black bars, hAPEX1 transgenic are white bars. a = significantly different from control lacI transgenic at 26-months-old. b = significantly different from 16-months-old of the same genotype.