Page 7 - 南京医科大学自然版

第44卷第12期 南京医科大学学报（自然科学版）
2024年12月 Journal of Nanjing Medical University（Natural Sciences） ·1621 ·

·基础研究·

环磷酰胺对卵母细胞发育潜能影响的研究

程 丽，倪 曼，王 洁，郭 爽，覃莲菊 ，马 翔 *
*
南京医科大学第一附属医院生殖医学中心，江苏 南京 210029

［摘 要］ 目的：探究环磷酰胺活性代谢物 4⁃羟基环磷酰胺（4⁃hydroxycyclophosphamide，4⁃HC）对卵母细胞质量的影响及机
制。方法：将小鼠生发泡（germinal vesicle，GV）期卵丘卵母细胞复合体（cumulus⁃oocyte complex，COC）随机分为8组，空白对照
组不做处理，溶剂对照组中添加与实验组相同浓度溶剂DMSO，实验组中分别加入终浓度为0.3、1.0、3.0、10.0、30.0、100.0 μmol/L
的4⁃HC，观察各组COC 在体外培养后的第一极体排出率、二细胞率、囊胚率，最终确定实验组最佳浓度。检测1 μmol/L 浓度
下卵母细胞内活性氧水平、线粒体膜电位水平、还原型谷胱甘肽水平等以评估卵母细胞质量。RT⁃qPCR、免疫荧光等检测
4⁃HC对卵母细胞DNMT3A表达的影响。结果：随着4⁃HC浓度的增加，各组卵母细胞2⁃细胞率相当或略有下降，实验组卵母细
胞受精后囊胚率随4⁃HC浓度增加而降低（P < 0.05）。1 μmol/L 4⁃HC下，小鼠卵母细胞线粒体膜电位下降、细胞内超氧化物阴
离子含量上升、还原型谷胱甘肽含量下降（P均< 0.05）且囊胚形成率下降（0.809 ±0.087 vs. 0.566±0.175，P < 0.05），RT⁃qPCR及免
疫荧光结果显示卵母细胞DNMT3A表达增加（P < 0.05）。结论：4⁃HC会引发卵母细胞发生氧化应激，线粒体损伤，导致其发育
潜能降低，并对卵母细胞表观遗传产生影响。
［关键词］ 氧化应激；表观遗传；环磷酰胺；谷胱甘肽；卵母细胞
［中图分类号］ R321.1 ［文献标志码］ A ［文章编号］ 1007⁃4368（2024）12⁃1621⁃09
doi：10.7655/NYDXBNSN240530

Effect of cyclophosphamide on developmental potential of oocytes

* *
CHENG Li，NI Man，WANG Jie，GUO Shuang，QIN Lianju ，MA Xiang
Reproductive Medicine Center，the First Affiliated Hospital of Nanjing Medical University，Nanjing 210029，China

［Abstract］ Objective：This study investigated the effect and mechanism of 4⁃hydroxycyclophosphamide（4⁃HC），the active metabolite
of cyclophosphamide（CTX），on oocyte quality. Methods：Mouse cumulus⁃oocyte complexes（COCs）in the germinal vesicle（GV）stage
were randomly divided into eight groups. The blank control group received no treatment，while the solvent control group was treated
with DMSO at the same concentration as the experimental group. The experimental groups were exposed to 4⁃HC at concentrations of
0.3，1.0，3.0，10.0，30.0，and 100.0 μmol/L. The in vitro matured oocytes were observed for the first polar body discharge rate，2⁃cell
rate，and blastocyst rate. The 1 μmol/L concentration group was further evaluated for reactive oxygen species（ROS）levels，

mitochondrial membrane potential（MMP），and reduced glutathione（GSH）content to assess oocyte quality and explore the mechanism.
RT⁃qPCR and immunofluorescence were used to detect the effect of 4⁃HC on DNMT3A expression in oocytes. Results：With increasing
4⁃HC concentration，the 2⁃cell rate of oocytes remained similar or slightly decreased，while the blastocyst rate decreased significantly
（P < 0.05）. Treatment with 1 μmol/L 4 ⁃ HC reduced mitochondrial membrane potential，increased intracellular superoxide anion
content，and decreased reduced glutathione content（all P < 0.05）. Additionally，the blastocyst formation rate（0.809±0.087 vs. 0.566±
0.175，P < 0.05）was significantly reduced. PCR and immunofluorescence results showed increased DNMT3A expression in oocytes
（P < 0.05）. Conclusion：4⁃HC induces oxidative stress and mitochondrial damage in oocytes，impairing their developmental potential
and affecting oocyte epigenetics.
［Key words］ oxidative stress；epigenetics；cyclophosphamide；glutathione；oocytes
［J Nanjing Med Univ，2024，44（12）：1621⁃1628，1648］

［基金项目］ 国家重点研发计划（2021YFC2700402）；江苏省科技厅社会发展面上项目（BE2021743）
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通信作者（Corresponding author），E⁃mail：sxmaxiang@126.com；ljqin@njmu.edu.cn