摘要
目的用精子DNA完整性评价NaOH溶液对小鼠精子进行头尾分离的最优条件。方法取2～3月龄雄鼠附睾尾精子，稀释到合适浓度（5×10^6～10×10^6／ml），0～37℃下，加入不同浓度NaOH溶液孵育，显微镜下观察精子的头尾分离率，流式细胞仪检测精子的质膜完整性及DNA完整性。结果NaOH对精子头尾分离的效果，与碱浓度、作用时间和作用温度呈正相关，作用温度越高，时间越长，头尾分离效果越好，但同时对精子DNA的损伤程度也就越大。本研究证实精子在10mmol／LNaOH溶液，0℃下孵育2h，精子头尾分离的效率可达55％，而精子DNA损伤与空白对照相比，则无显著性差异。结论在本研究条件下低浓度NaOH处理小鼠精子，方法简单易行，可有效去除小鼠尾巴，而又不使其DNA受损。
Objective To optimize the method of separation of mouse sperm head and tail by treating with different concentration of NaOH so as to improve the efficiency of ICSI. Methods The sperm were collected from the epididymis of male mice at the age of 2-3 months and diluted to an appropriate density. The sperm head and tail separation rate was determined under the microscope after incubating with different concentration of NaOH at the range of 0-37℃. Flow cytometry was utilized to evaluate both of membrane and DNA integrity of the sperm cells. Results The sperm head and tail separation rate was positively correlated with NaOH concentration, incubation time and temperature. However, DNA integrity of the sperm cells was negatively correlated to NaOH concentration and incubation temperature and time. The lower NaOH concentration and temperature as well as the shorter incubation time led to less serious DNA damage. The results indicated that the condition of 10 mmol/L NaOH for 2 h at 0℃ was the optimal treatment to achieve 55% of sperm head and tail separation rate without injury of their DNA. Conclusion The low concentration NaOH treatment of sperm with the optimized condition in our study is a simple and convenient methodwhich can effectively separated the tail from sperm while remain the high DNA integrity.
出处
《实验动物与比较医学》
CAS
2010年第2期82-86,共5页
Laboratory Animal and Comparative Medicine
基金
【基金项目】国家自然科学基金（30872765）和上海市科技发展基金（071409004）
