Aging results in hypermethylation of ribosomal DNA in sperm and liver of male rats

Abstract

There is a concern that increased paternal age may be associated with altered fertility and an increased incidence of birth defects in man. In previous studies of aged male rats, we have found abnormalities in the fertility and in the embryos sired by older males. Aging in mammals is associated with alterations in the content and patterns of DNA methylation in somatic cells; however, little is known in regard to germ cells. A systematic search for global and gene-specific alterations of DNA methylation in germ cells and liver of male rats was done. Restriction landmark genomic scanning, a method used to determine specific methylation patterns of CpG island sequences, has revealed a region of the ribosomal DNA locus that is preferentially hypermethylated with age in both spermatozoa and liver. In contrast, all single copy CpG island sequences in spermatozoa and in liver remain unaltered with age. We further demonstrate that a large proportion of rat ribosomal DNA is normally methylated and that regional and site-specific differences exist in the patterns of methylation between spermatozoa and liver. We conclude that patterns of ribosomal DNA methylation in spermatozoa are vulnerable to the same age-dependent alterations that we observe in normal aging liver. Failure to maintain normal DNA methylation patterns in male germ cells could be one of the mechanisms underlying age-related abnormalities in fertility and progeny outcome.

Figure 1

Global genomic DNA determination by TLC. (a) Phase-contrast photographs of purified pachytene spermatocytes (Left), round spermatids (Center), and cauda sperm (Right) (×400). (b) Representative TLC plates of HpaII- and MspI-digested genomic DNA in germ cells and liver of young and old animals. The MspI-digested DNA reveals the relative content of cytosine (C) to m⁵C. (c) Graphical representation of quantified TLC results of germ cells and liver of young (black bars) and old (gray bars) animals. *, P < 0.05. Error bars represent ± SEM. Two-way ANOVA (n = 4–6 per age per group) revealed no significant effect of age, but methylation was significantly higher in liver when compared to pachytene spermatocytes (P = 0.019), round spermatids (P < 0.001), caput sperm (P = 0.005), and cauda sperm (P < 0.001).

Figure 2

RLGS of cauda spermatozoa and liver of young and old animals. (a) Representative RLGS scans of young and old cauda spermatozoa. Numbers across the top of the scans represent first dimension fragment sizes (kb), and numbers down the left side of the scans represent second dimension fragment sizes (bp). Small spots represent single-copy genomic fragments, and large, dense spots represent repeat copy fragments. Arrows indicate fragments A–D. (b) Enlargements of fragments A–D in both cauda spermatozoa and liver of young and old animals.

Figure 3

RLGS restriction sites within the rat rDNA locus and specific PCR amplification of RLGS fragments A–D. (a) Rat rDNA consists of tandem repeats containing the 45S preRNA-transcribed region (rectangle) and the nontranscribed spacer (NTS) region (line). The 5′-ETS region is indicated as well as regions coding for the 18S, 5.8S, and the 28S ribosomal RNAs. The recognition sites and the RLGS fragments (A/B, C, and D) generated by the restriction enzymes used in the first dimension RLGS separation, NotI (N) and EcoRV (RV), are indicated. (b) Enlargement of the NotI sites within the 5′-ETS and 28S regions. The positions of HinfI (H) recognition sites adjacent to NotI sites are illustrated. The NotI–HinfI fragments that generate second dimension RLGS fragments A–D are indicated. The positions of PCR primer pairs 1–4 are indicated. (c) PCR primer pairs 1–4 are used to specifically amplify DNA from excised RLGS fragments A–D by using H₂O and genomic DNA (G) as a negative and positive amplification control, respectively. The PCR product sizes are listed.

Figure 4

Southern blot analysis of NotI-containing regions of the 5′-ETS and 28S. (a) Fragment sizes generated by digestion of genomic DNA with BamHI (B) and NotI (N) in the 5′-ETS region are illustrated above. Genomic DNA from spermatozoa and liver was digested with BamHI only in lanes 1 and 4, respectively. Both BamHI and NotI were used to digest spermatozoa DNA from young and old animals in lanes 2 and 3, respectively, and liver DNA from young and old animals in lanes 5 and 6, respectively. (b) Fragment sizes generated by digestion of genomic DNA with EcoRI (RI) and NotI (N) in the 28S region are illustrated. The identical arrangement of enzyme digestions, tissues, and ages used in a were applied to the 28S region except for the replacement of BamHI with EcoRI.

Figure 5

Graphical representation of the densitiometric analysis of Southern blots. The four possible methylation states of the two NotI sites present in the 5′-ETS region (a) and the 28S region (b) are illustrated as restriction fragments with the corresponding fragment sizes (Left). The relative contribution of each band to the total lane intensity in spermatozoa (Center) and liver (Right) of young (black bars) and old (gray bars) animals is illustrated (n = 4 per age per group). Statistical analysis was performed by the Mann–Whitney rank sum test (*, P < 0.05). Error bars represent ± SEM.