Transgenerational impact of grand-paternal lifetime exposures to both folic acid deficiency and supplementation on genome-wide DNA methylation in male germ cells

Background: DNA methylation (DNAme) erasure and reacquisition occurs during prenatal male germ cell development; some further remodelling takes place after birth during spermatogenesis. Environmental insults during germline epigenetic reprogramming may affect DNAme, presenting a potential mechanism for transmission of environmental exposures across multiple generations. Objectives: We investigated how germ cell DNAme is impacted by lifetime exposures to diets containing either low or high, clinically relevant, levels of the methyl donor folic acid and whether resulting DNAme alterations were inherited in germ cells of male offspring of subsequent generations. Materials and Methods: Female mice were placed on a 7-fold folic acid deficient (7FD) and 10- or 20-fold supplemented (10FS and 20FS) diets before and during pregnancy. Resulting F1 litters were weaned on the respective diets. F2 and F3 males received control diets. Genome-wide DNAme was assessed in F1 spermatogonia, and in F1, F2 and F3 sperm. Results: In F1 germ cells, a greater number of differentially methylated cytosines (DMCs) was observed in spermatogonia as compared with F1 sperm for all folic acid diets. DMCs were lower in number in F2 versus F1 sperm, while an unexpected increase was found in F3 sperm. DMCs were predominantly hypomethylated, with genes in neurodevelopmental pathways commonly affected in F1, F2 and F3 male germ cells. While no DMCs were found to be inherited inter- or transgenerationally, we observed over-representation of repetitive elements, particularly young LINEs. Discussion and Conclusion: These results suggest that the prenatal window is the time most susceptible to folate-induced alterations in sperm DNAme in male germ cells. Altered methylation of specific sites in F1 sperm was not present in later generations. However, the finding of hypomethylated young LINE1 elements in sperm from F1 to F3 males provides insight into potential mechanisms of epigenetic inheritance of the effects of folate deficiency and supplementation. Overall design: In order to examine the effects of pre-natal exposures of folic acid supplementation and deficiency, F0 BALB/s females were placed on the different diets for 4 weeks prior to mating with Oct4-GFP +/- BALB/c males fed rodent chow. Females were maintained on diets throughout mating, gestation and lactation. F1 Oct4-GFP +/- males were sacrificed at PND 6 for spermatogonia isolation via fluorescent activated cell sorting. In order to study the effect of inter- and transgenerational effects of folic acid supplementation and deficiency, F0 BALB/s females were placed on the different diets for 4 weeks prior to mating with rodent chow fed males. The females were maintained on their folic acid defined diets throughout mating, gestation and lactation. Resultant F1 males were weaned onto their respective folic acid defined diets. Upon sexual maturity, F1 males were mated with rodent chow fed females. These females remained on the rodent chow diet throughout gestation and lactation. Subsequent F2 male pups were weaned and maintained on regular rodent chow until 18 weeks of age, then were mated with 10-week old female BALB/c fed with regular rodent chow to produce unexposed F3 litters.