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. Overall design: 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。MTHFR缺乏可影响男性生育力与精子DNA甲基化，存在表观突变向下一代传递的潜在可能。本研究旨在评估MTHFR缺乏对睾丸形态及精子DNA甲基化的影响是否会在跨世代中加剧。F1代父本的MTHFR缺乏仅对精子计数、睾丸重量及组织学特征产生轻微影响，但F2代雄性小鼠的生殖参数出现进一步恶化。F1与F2代精子中均观察到广泛的DNA甲基化丢失，且两代间共享超过80%的异常甲基化位点，提示存在持续易受MTHFR缺乏影响的基因组区域。此类区域通常在产前晚期生殖细胞发育过程中完成甲基化，且富集于年轻反转录转座子。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，F1代雄性精子的低甲基化状态可能会加剧F2代MTHFR缺陷雄性小鼠的生殖表型恶化，该发现与表观突变的跨代传递现象相符。实验设计概述：为研究跨世代Mthfr缺陷小鼠的生殖健康状况，我们通过Mthfr+/-小鼠交配获得F1代，包含Mthfr+/+与Mthfr-/-雄性小鼠。待其性成熟后，将F1代雄性小鼠与Mthfr+/-小鼠交配以获得F2代。同时还设置了外部小鼠队列（Mat_Def_colony），该队列通过母本与父本均为Mthfr缺陷的小鼠交配获得Mthfr-/-雄性小鼠进行检测。