Iodoacetic Acid Exposure Alters the Transcriptome in Mouse Ovarian Antral Follicles. Iodoacetic Acid Exposure Alters the Transcriptome in Mouse Ovarian Antral Follicles

The process of drinking water disinfection forms compounds known as disinfection byproducts (DBPs). Studies have shown that DBPs can be harmful to human and animal health. Iodoacetic acid (IAA) is a non-regulated DBP that is cytotoxic and genotoxic to mammalian cells. In addition, IAA has been shown to be an ovarian toxicant in vitro and in vivo. However, the mechanisms of action underlying IAA toxicity on ovarian follicles in vivo remain unclear. In this study, we determined whether IAA exposure alters gene expression patterns in ovarian antral follicles in mice. Adult female CD-1 mice were dosed with water or IAA (10 or 500mg/L) in the drinking water for 35-40 days. Antral follicles were dissected from the ovaries based on size (220–400 μm). Sera were collected to measure estradiol levels. RNA-sequencing was applied to uncover the global gene expression of the antral follicles in response to IAA exposure. RNA-sequencing analysis identified 410 and 653 differentially expressed genes (DEGs) in the 10 and 500mg/L IAA treatment groups (FDR < 0.1), respectively, compared to controls. Gene Ontology Enrichment analysis showed that DEGs were involved with RNA processing and regulation of angiogenesis (10mg/L) and the cell cycle, cell division, and mitotic nuclear division (500mg/L). In addition, Pathway Enrichment analysis showed that DEGs were involved in the phosphatidylinositol 3-kinase and protein kinase B (PI3K-Akt) signaling pathway, gonadotropin-releasing hormone (GnRH) signaling pathway, estrogen signaling pathway, and insulin signaling pathway (10mg/L). In the 500mg/L group, Pathway Enrichment analysis showed that DEGs were involved in the oocyte meiosis signaling pathway, GnRH signaling pathway, and oxytocin signaling pathway. In addition, RNA-sequencing analysis identified 809 DEGs when comparing the 10 and 500mg/L IAA groups (FDR < 0.1). DEGs were related to ribosome, translation, mRNA processing, oxidative phosphorylation, chromosome, cell cycle, cell division, protein folding, platelet activation, and the oxytocin signaling pathway. Moreover, IAA exposure significantly decreased estradiol levels (500mg/L) in serum compared to control. This study identified key candidate genes and pathways involved in IAA toxicity that could help to further understand the molecular mechanisms of IAA toxicity in ovarian follicles. Overall design: Murine mRNA profile of ovarian antral follicles. Adult female mice (40 days old) were dosed with vehicle (deionized, reverse osmosis filtered water) or IAA (10 or 500 mg/L) dissolved in reverse osmosis water for 35 days (n=5 per treatment group) via the drinking water. Two experiments were performed a month apart. In experiment 1, animals were exposed to only water (control) or 500mg/L of IAA in drinking water. In experiment 2, animals were exposed to only water (control) or 10mg/L of IAA in drinking water. Antral follicles were mechanically isolated from 1 individual mouse and submitted for mRNA sequencing as 1 experimenal unit. Global analyses combined control groups from both experiments for comparisons with IAA treated groups.

饮水消毒过程会生成一类被称为消毒副产物（disinfection byproducts, DBPs）的化合物。现有研究证实，消毒副产物可对人类与动物健康产生危害。碘乙酸（iodoacetic acid, IAA）属于未受监管的消毒副产物，对哺乳动物细胞兼具细胞毒性与遗传毒性。此外，多项研究表明IAA在体外及体内均表现出卵巢毒性。然而，IAA对体内卵巢窦状卵泡（antral follicles）的毒性作用机制至今仍不明确。

本研究旨在探究IAA暴露是否会改变小鼠卵巢窦状卵泡的基因表达模式。实验选取成年雌性CD-1小鼠，通过饮用水分别给予去离子水（对照组）或浓度为10mg/L、500mg/L的IAA溶液，持续染毒35~40天。根据粒径（220~400μm）从卵巢中机械分离窦状卵泡。采集血清样本以检测雌二醇水平。采用RNA测序（RNA-sequencing）技术解析IAA暴露后窦状卵泡的全局基因表达谱。

RNA测序分析显示，与对照组相比，10mg/L和500mg/L IAA处理组分别鉴定出410个和653个差异表达基因（differentially expressed genes, DEGs）（错误发现率<0.1，false discovery rate, FDR）。基因本体（Gene Ontology, GO）富集分析结果表明，10mg/L处理组的DEGs参与RNA加工及血管生成调控过程，500mg/L处理组的DEGs则与细胞周期、细胞分裂及有丝分裂核分裂密切相关。

通路富集分析显示，10mg/L处理组的DEGs富集于磷脂酰肌醇3-激酶-蛋白激酶B（phosphatidylinositol 3-kinase and protein kinase B, PI3K-Akt）信号通路、促性腺激素释放激素（gonadotropin-releasing hormone, GnRH）信号通路、雌激素信号通路及胰岛素信号通路。500mg/L处理组的DEGs则富集于卵母细胞减数分裂信号通路、GnRH信号通路及催产素信号通路。

此外，对比10mg/L与500mg/L两个IAA处理组，共鉴定出809个DEGs（FDR<0.1），这些DEGs与核糖体功能、翻译过程、mRNA加工、氧化磷酸化、染色体结构、细胞周期、细胞分裂、蛋白质折叠、血小板活化及催产素信号通路相关。同时，与对照组相比，500mg/L IAA暴露可显著降低血清雌二醇水平。

本研究鉴定出参与IAA毒性作用的关键候选基因与通路，可为进一步解析IAA对卵巢窦状卵泡的毒性分子机制提供重要依据。

实验整体设计：本数据集为小鼠卵巢窦状卵泡的mRNA表达谱。实验选用40日龄成年雌性小鼠，通过饮用水给予赋形剂（去离子反渗透过滤水）或溶解于反渗透水的IAA（10或500mg/L），持续暴露35天（每组n=5）。实验分为两批开展，间隔1个月。第一批实验仅设置纯水对照组与500mg/L IAA暴露组；第二批实验仅设置纯水对照组与10mg/L IAA暴露组。从单只小鼠卵巢中机械分离窦状卵泡，作为1个实验单元进行mRNA测序。全局分析阶段将两次实验的对照组合并，与各IAA处理组进行对比分析。