Age-dependent increases in L1 in the mouse hippocampus genome. (A) Age-related variation in the copy numbers in the hippocampus (left panel) and frontal cortex (right panel). Six groups (3-, 8-, 15-, 18-, 20-, and 55-week-old) of C57BL/6J inbred mice were subjected to quantitative polymerase chain reaction (qPCR) analysis. The copy numbers of L1-ORF2 were measured using the 5S ribosomal gene as a reference. We analyzed a total of 42 mice (21 female, 21 male) at 3 (3 female, 3 male), 8 (6 female, 6 male), 15 (3 female, 3 male), 18 (3 female, 3 male), 20 (3 female, 3 male) and 55 (3 female, 3 male) weeks of age. The error bars show the standard error of the mean (SEM) values. The copy numbers in the hippocampus were significantly higher in 8-week-old inbred mice (Steel–Dwass test, 3-week-old vs 8-week-old, P = 0.008; 8-week-old vs 15-week-old, P = 0.4; 8-week-old vs 18-week-old, P = 0.08; 8-week-old vs 20-week-old, P = 0.0001). (B) The copy numbers of L1-5′UTR and -ORF1. The measurement analysis was done using the 5S ribosomal gene as a reference. A total of 18 mice (9 female, 9 male) were analyzed at 3 (3 female, 3 male), 8 (3 female, 3 male), and 20 (3 female, 3 male) weeks of age. Bars show the SEM. The copy numbers in the hippocampus were significantly higher in 8-week-old inbred mice (Tukey's test, 5′UTR; P = 0.0001, ORF1; P = 0.00003). (C) Stavudine (d4T)-induced inhibition of the age-dependent L1 increase in the hippocampus genome. Mice were administered d4T in drinking water beginning 3 weeks after birth and continued for 5 weeks, and then the L1 copy numbers in the hippocampus genome were analyzed. Bars show the SEM. The analysis included 47 control mice (3-week-old; 24 female, 8 male, 8-week-old; 9 female, 6 male) and 12 d4T-treated mice (3 female, 9 male). Of note, d4T treatment inhibited the L1 increase in 8-week-old mice (Steel–Dwass test, P = 0.0000009). (D) Representative results of the immunohistochemical analysis of d4T-treated mice. Ki67 (red; arrowheads), calretinin (green), 5-ethynyl-2-deoxyuridine (EdU) (red) and calretinin-EdU double-positive cells (yellow; arrowheads, inset) were visualised in the dentate gyrus of d4T-treated mice. Nuclei are depicted in blue. Scale bar, 50 µm. (E) Comparison of Ki67-positive cells and calretinin-EdU double-staining cells in mice with or without d4T treatment. Under graph shows calretinine-EdU double-staining cells (blue column) and EdU-positive cells (red column). The analysis included a total of 16 mice (8 female, 8 male); 8 control mice (4 female, 4 male) and 8 d4T-treated mice (4 female, 4 male). Ki67-positive cells and calretinin-EdU double-staining cells were counted in 3 randomly selected sections that were prepared from each mouse.