Paternal aging over a natural lifetime is directly associated with genetic and epigenetic alterations in the male gamete

Abstract

There is evidence that a combination of genetic mutations and epigenetic alterations may support an association between paternal age at human conception and neurodevelopmental disorders in offspring, including autism and schizophrenia. The aim of this study was to investigate the exome and methylome of individual male gametes over the course of a natural lifetime. Longitudinal animal study. This study examined sperm of individual male mice over their natural lifetimes from youth (5 months) to old age (15 months). Young males (5 months) with proven virility had 1 testicle surgically removed for mature sperm collection. Fecundity was then assessed monthly, utilizing young virgin females (6-8 weeks) to control for any female factor. The remaining testicle was excised for mature sperm collection when the male reached old age. Individual sperm samples were subjected to somatic cell lysis prior to sperm DNA isolation for exome sequencing (ION PI v2 chip; Thermo Scientific) and methylome analysis (Mouse Genome Microarray 2x105K; Agilent Technologies). DNA sequence reads were aligned to mouse reference genome UCSC mm10 and compared to UCSC dbSNP 137 with Avadis NGS platform. Methylome statistics utilized a BATMAN algorithm (Bayesian Tool) to calculate methylated or unmethylated probes, significance at P<0.05. All males experienced significantly reduced fecundity with old age (15 months) and no viable offspring were produced after 12 months. Sperm exome sequencing generated 75 million reads, high coverage and 99.5% accuracy in single base nucleotide calls. A total of 625 de novo point mutations resulting in direct amino acid changes were detected only in older age sperm (12-15 months) and never observed in sperm from these same males during their youth (P<0.05). The amino acid changes are predicted to impact protein/enzyme function, and pathway analysis of the 625 de novo point mutations revealed enrichment of genes involved in neurological system pathways. Significant methylation alterations were also observed with paternal aging across the genome and on every chromosome. Specifically, 308 probes lost methylation (85 promoter regions) and 102 probes gained methylation (35 promoter regions), in older age sperm when compared to the sperm collected in their youth (P<0.05). Methylation validation using targeted bisulfite mutagenesis confirmed alterations with paternal aging in Pex7, a gene associated with autism (P<0.05). This novel study examined sperm DNA over the natural lifetime of an individual male revealing an accumulation of de novo point mutations in the spermatozoa coding sequence, as well as significant methylation alterations. Combined, these sperm DNA changes associated with paternal aging are predicted to have severe consequences to downstream gene transcription and cellular signaling in the offspring, thereby impacting developmental pathways including early stages of neurodevelopment.