Folate, as the initial substrate in one-carbon metabolism, is involved in the synthesis of important substances such as DNA, RNA, and protein. Folate deficiency (FD) is associated with male subfertility and impaired spermatogenesis, yet the underlying mechanisms are poorly understood. In the present study, we established an animal model of FD to investigate the effect of FD on spermatogenesis. GC-1 spermatogonia were used as a model to investigate the effect of FD on proliferation, viability, and chromosomal instability (CIN). Furthermore, we explored the expression of core genes and proteins of spindle assembly checkpoint (SAC), a signaling cascade ensuring accurate chromosome segregation and preventing CIN during mitosis. Cells were maintained in medium containing 0, 20, 200, or 2000 nM folate for 14 days. CIN was measured by using a cytokinesis-blocked micronucleus cytome assay. We found that sperm counts decreased significantly (p < 0.001) and the rate of sperm with defects in the head increased significantly (p < 0.05) in FD diet mice. We also found, relative to the folate-sufficient conditions (2000 nM), cells cultured with 0, 20, or 200 nM folate exhibited delayed growth and increased apoptosis in an inverse dose-dependent manner. FD (0, 20, or 200 nM) significantly induced CIN (p < 0.001, p < 0.001, and p < 0.05, respectively). Moreover, FD significantly and inverse dose dependently increased the mRNA and protein expression of several key SAC-related genes. The results indicate that FD impairs SAC activity, which contributes to mitotic aberrations and CIN. These findings establish a novel association between FD and SAC dysfunction. Thus, FD-impaired spermatogenesis may be partly due to genomic instability and proliferation inhibition of spermatogonia.

中文翻译：

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低膳食叶酸对小鼠精子发生和纺锤体组装检查点功能障碍的影响可能导致叶酸缺乏引起的培养小鼠精原细胞染色体不稳定。

叶酸作为一碳代谢的起始底物，参与DNA、RNA、蛋白质等重要物质的合成。叶酸缺乏（FD）与男性生育力低下和精子发生受损有关，但其潜在机制尚不清楚。在本研究中，我们建立了FD动物模型来研究FD对精子发生的影响。使用 GC-1 精原细胞作为模型来研究 FD 对增殖、活力和染色体不稳定性 (CIN) 的影响。此外，我们还探索了纺锤体组装检查点（SAC）核心基因和蛋白质的表达，这是一种信号级联，确保准确的染色体分离并防止有丝分裂期间的 CIN。将细胞在含有 0、20、200 或 2000 nM 叶酸的培养基中维持 14 天。CIN 通过使用胞质分裂阻断的微核细胞分析来测量。我们发现，FD 饮食小鼠的精子数量显着下降 (p < 0.001)，而头部有缺陷的精子比例显着增加 (p < 0.05)。我们还发现，相对于叶酸充足的条件（2000 nM），用 0、20 或 200 nM 叶酸培养的细胞以反剂量依赖性方式表现出生长延迟和细胞凋亡增加。FD（0、20 或 200 nM）显着诱导 CIN（分别为 p < 0.001、p < 0.001 和 p < 0.05）。此外，FD 显着且呈反剂量依赖性地增加了几个关键 SAC 相关基因的 mRNA 和蛋白质表达。结果表明，FD 会损害 SAC 活性，从而导致有丝分裂畸变和 CIN。这些发现建立了 FD 和 SAC 功能障碍之间的新关联。因此，FD 损害的精子发生可能部分是由于基因组不稳定和精原细胞增殖抑制所致。