联合ATAC-seq和RNA-seq探究无机砷诱导小鼠雄性生殖损伤的作用机制

Exploring the Mechanisms of Inorganic Arsenic-Induced Male Reproductive Damage in Mice via Integrated ATAC-seq and RNA-seq Analysis

    摘要
  • 摘要:
    目的 探讨无机砷（Inorganic arsenic， iAs）暴露对小鼠雄性生殖功能的影响，从染色质可及性及基因表达层面解析其表观遗传调控机制。
    方法 以6周龄ICR雄性小鼠为实验对象，采用饮水染毒方式建立亚慢性iAs暴露模型，设对照组（自来水）、低剂量组（5 mg·L-1 NaAsO₂）、中剂量组（25 mg·L-1 NaAsO₂）和高剂量组（125 mg·L-1NaAsO₂），分别于暴露12周和20周取样。检测睾丸组织病理、精子质量、血清激素水平及组织中砷含量；取暴露20周高剂量组和对照组睾丸进行ATAC-seq和RNA-seq测序，联合分析筛选关键调控基因；在TM3和TM4细胞中验证关键基因表达。
    结果 iAs暴露导致小鼠睾丸组织结构异常、精子活力降低、精子密度下降、精子畸形率升高及血清睾酮水平降低，且损伤随暴露时间的延长和暴露剂量增加而加重。ATAC-seq显示iAs暴露诱导全基因组染色质重塑。RNA-seq显示下调基因富集于精子发生、线粒体氧化磷酸化及血睾屏障通路，上调基因富集于组蛋白H3K4甲基化等表观遗传调控通路。联合分析筛选出组蛋白H3K4甲基转移酶基因（Kmt2a、Kmt2c、Kmt2d、Kmt2e、Ash1l、Setd1b）及精子发生相关基因（Sox5、Rarb、Diaph3、Tdrkh、Tssk6）。细胞实验证实，iAs暴露可诱导TM4支持细胞中组蛋白H3K4甲基转移酶及其下游染色质重塑因子表达上调。
    结论 亚慢性iAs暴露能够诱导雄性小鼠生殖功能损伤。组蛋白H3K4甲基转移酶及其下游染色质重塑因子异常上调是iAs介导雄性生殖损伤的关键分子机制之一。

     

    Abstract:
    OBJECTIVE To investigate the effects of inorganic arsenic (iAs) exposure on male reproductive function in mice and to elucidate the underlying epigenetic regulatory mechanisms at the levels of chromatin accessibility and gene expression.
    METHODS Six-week-old male ICR mice were used to establish a subchronic iAs exposure model via drinking water. The mice were divided into four groups: control (tap water), low dose (5 mg·L⁻¹ NaAsO₂), medium dose (25 mg·L⁻¹ NaAsO₂), and high dose (125 mg·L⁻¹ NaAsO₂). Samples were collected at 12 and 20 weeks of exposure. Testicular histopathology, sperm quality, serum hormone levels, and tissue arsenic content were measured. Testes from the 20-week high-dose group and the control group were subjected to ATAC-seq and RNA-seq; integrated analysis was performed to identify key regulatory genes. The expression of key genes was validated in TM3 and TM4 cells.
    RESULTS iAs exposure induced abnormal testicular architecture, reduced sperm motility, decreased sperm density, increased sperm deformity rate, and lowered serum testosterone levels in mice, with damage intensifying with prolonged exposure duration and increased dose. ATAC-seq revealed iAs exposure induced genome-wide chromatin remodeling. RNA-seq showed that downregulated genes were enriched in spermatogenesis, mitochondrial oxidative phosphorylation, and blood-testis barrier pathways, while upregulated genes were enriched in epigenetic regulatory pathways such as histone H3K4 methylation. Integrated analysis identified histone H3K4 methyltransferase genes (Kmt2a, Kmt2c, Kmt2d, Kmt2e, Ash1l, Setd1b) and spermatogenesis-related genes (Sox5, Rarb, Diaph3, Tdrkh, Tssk6). Cellular experiments confirmed that iAs exposure upregulated histone H3K4 methyltransferases and their downstream chromatin remodeling factors in TM4 Sertoli cells.
    CONCLUSION Subchronic iAs exposure can induce male reproductive impairment in mice. Aberrant upregulation of histone H3K4 methyltransferases and their downstream chromatin remodeling factors represents one of the key molecular mechanisms underlying iAs-mediated male reproductive damage.

     

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