Paternal MTHFR deficiency leads to reproductive decline across generations in association with hypomethylation of young retrotransposons

5, 10-Methylenetetrahydrofolate reductase (MTHFR) is a crucial enzyme in the folate metabolic pathway with a key role in generating methyl groups. As MTHFR deficiency impacts male fertility and sperm DNA methylation, there is the potential for epimutations to be passed to the next generation. Here, we assessed whether the impact of MTHFR deficiency on testis morphology and sperm DNA methylation is exacerbated across generations. While MTHFR deficiency in F1 fathers has only minor effects on sperm counts and testis weights and histology, F2 generation sons show further deterioration in reproductive parameters. Extensive loss of DNA methylation is observed in both F1 and F2 sperm, with >80% of sites shared between generations, suggestive of regions consistently susceptible to MTHFR deficiency. These regions are generally methylated during late prenatal germ cell development and are enriched in young retrotransposons. As retrotransposons are resistant to reprogramming of DNA methylation in prenatal germ cells, their hypomethylated state in the sperm of F1 males could contribute to the worsening reproductive phenotype observed in F2 MTHFR- deficient males, findings compatible with the intergenerational passage of epimutations. In order to study the reproductive health of Mthfr mice across generations, Mthfr +/- mice were mated in order to obtain an F1 generation of both Mthfr +/+ and Mthfr -/- male mice. Upon sexual maturity, male mice of the F1 generation were mated with Mthfr +/- mice to obtain the F2 generation. An external cohort of mice (Mat_Def_colony) was also examined, where maternal- and paternal-deficient Mthfr mice were mated to obtain Mthfr -/- male mice.

5,10-亚甲基四氢叶酸还原酶（5, 10-Methylenetetrahydrofolate reductase, MTHFR）是叶酸代谢通路中的关键酶，在甲基基团生成过程中发挥核心作用。由于MTHFR缺乏会影响男性生育力与精子DNA甲基化，因此存在表观突变（epimutation）传递至下一代的可能性。本研究旨在探究MTHFR缺乏对睾丸形态与精子DNA甲基化的影响是否会在跨代传递过程中加剧。尽管F1代雄性小鼠的MTHFR缺乏仅对精子计数、睾丸重量及组织学结构产生轻微影响，但F2代雄性小鼠的生殖参数却出现了进一步恶化。F1与F2代小鼠的精子中均观察到大范围DNA甲基化丢失，且两代间共有超过80%的甲基化丢失位点，这提示存在一类对MTHFR缺乏持续敏感的基因组区域。此类区域通常在产前生殖细胞发育晚期发生甲基化修饰，且富集于年轻反转录转座子（retrotransposon）。由于反转录转座子在产前生殖细胞中对DNA甲基化重编程具有抗性，因此F1代雄性小鼠精子中此类区域的低甲基化状态，可能是导致F2代MTHFR缺陷雄性小鼠生殖表型进一步恶化的原因，该发现与表观突变的跨代传递现象相符。为跨代研究Mthfr基因缺陷小鼠的生殖健康状况，研究人员通过Mthfr+/-小鼠交配，获得了同时携带Mthfr+/+与Mthfr-/-基因型的F1代雄性小鼠。待F1代雄性小鼠性成熟后，将其与Mthfr+/-小鼠交配以获得F2代小鼠。此外本研究还对一个外部小鼠队列（Mat_Def_colony）进行了检测：该队列通过母源与父源Mthfr缺陷小鼠交配，获得Mthfr-/-雄性小鼠。