Sperm DNA methylation defects in a new mouse model of the 5,10-methylenetetrahydrofolate reductase 677C>T variant and correction with moderate dose folic acid supplementation.
5,10-Methylenetetrahydrofolate reductase (MTHFR) is an enzyme that plays a key role in providing methyl groups for DNA methylation, including during spermatogenesis. A common genetic variant in humans (MTHFR 677C>T), results in reduced enzyme activity and has been linked to various disorders, including male infertility. A new animal model has been created by reproducing the human equivalent of the polymorphism in mice using CRISPR/Cas9. Biochemical parameters in the Mthfr 677TT mice recapitulate alterations found in MTHFR 677TT men. Our aims were to: 1) characterize the sperm DNA methylome of the Mthfr 677CC and TT mice on a control diet (2mg folic acid/kg diet) and 2) assess the effects of folic acid deficiency (0.3mg/kg diet) and supplementation (10 mg/kg diet) on the sperm DNA methylome. Body and reproductive organ weights, testicular sperm counts, and histology were examined. DNA methylation in sperm was assessed using bisulfite pyrosequencing, Illumina Mouse Methylation Array and whole genome bisulfite sequencing. Reproductive parameters and imprinted gene methylation were unaffected by genotype or diets. The largest effect was due to genotype, with sperm from 677TT mice showing more hypo- than hypermethylation. Folate-deficient diets resulted in sperm hyper- and hypomethylation in CC and TT mice. Folic acid supplementation caused mostly hypermethylation in sperm of males of both genotypes and was found to partially correct the DNA methylation alterations in sperm associated with the TT genotype. The new mouse model will be useful in understanding the role of MTHFR deficiency in male fertility and in designing folate supplementation regimens for the clinic. Overall design: The experimental design consists of six groups of male C57Bl/6J mice: Mthfr TT mice and wild-type mice as control (Mthfr CC) on 3 different diets. The diets consisted of control (CD, 2mg folic acid/kg diet), folate-deficient (FD, 0.3mg folic acid/kg diet) and moderate dose folic acid-supplemented (FASD, 5-fold supplemented: 10 mg folic acid/kg diet) diets. Mice were placed on their respective diets starting at four weeks of age and maintained on them for four months.  At the end of diet treatments mice were sacrificed and tissues and organs were collected.