小鼠精子激活力减弱模型制备技术的初步研究

A Primary Study for Establishment of Mouse Sperm Model with Decreased Oocyte Activation Capability

【目的】:人类辅助生殖技术中,低受精率的发生机理及辅助激活提高受精率的安全性尚不清楚,缺乏激活力减弱的精子模型是其中一个主要研究障碍。本研究拟探索减弱小鼠精子激活力的新技术。【方法】首先,分别对精子进行化学破膜、机械脱尾及其联合操作于37℃孵育12 h,比较各操作对显微授精后的受精率的影响;然后进一步结合时间因素,观察处理时间与精子激活力减弱程度的联系。同时,对操作后的胚胎早期发育的影响也进行了相应观察。【结果】①化学破膜法与机械脱尾法对精子破膜并温育后,均能有效降低精子受精率;②相对于正常的精子,对小鼠精子进行吹打机械脱尾,37℃下温育2、4、8、12 h后,受精率和卵裂率均出现程度逐步增加的显著降低(P<0.01);③与对照相比,以吹打机械脱尾并37℃下温育后的精子进行显微授精后,其受精卵的囊胚率和孵出率也显著降低(P<0.001)。【结论】将小鼠精子机械脱尾并在37℃下温育,精子激活力随温育时间增加而显著降低,同时其胚胎发育能力下降。本研究初步探索了程度可控的降低精子受精激活力的新技术,其安全性及导致精子激活力下降的机制尚需进一步研究。

[ Objective ] Due to absence of sperm model with decreased oocyte activation capability, the mechanism of low rate of fertilization and the safety of artificial activation techniques applied in intracytoplasmic sperm injection are unclear. The current research focuses on a novel technique of decreasing oocyte activation capability of mouse sperms in controlled manner. [ Methods ] First, sperm membrane was damaged with chemical interference, mechanical sperm tail-cutting off, or combing both manipulations respectively, and then incubated for 12 h at 37 ~C followed by intracytoplasmic sperm injection. The fertilization rates were analyzed to figure out the most effective method of decreasing the capability of sperm＇ s activation. Second, following the most effective treatment, the membrane-damaged sperms were incubated for different time to find out the association between the time and sperm＇s activation capability decreasing. Embryos＇ early development was also observed after intracytoplasmic sperm injection with activation capability decreased sperms. [Results] 1） Both of chemical interference and the mechanical tail-cutting off decreased fertilization rate significantly. 2） When sperm tails were cut off mechanically and then incubated at 37 ℃ for 2 h, 4 h, 8 h or 12 h, the rates of fertilization and cleavage were decreased significantly with the increase of incubation time （P 〈 0.01 ） accordingly. 3） Compared with control group, the percentage of blastocyst population and hatching （P 〈 0.001） also decreased significantly while intracytoplasmic sperm injection were applied using tail-cut sperms after incubation. [ Conclusion] When sperm tails were cut off and incubated at 37 ℃, the capability of sperms to activate oocytes had significantly decreased with the increase of incubation time, and the embryo development was also disrupted. In this study, a novel technique of decreasing the sperm activation capability in controlled manner has been build up primarily. However the safety and the underlying mechanism need further explored.