冻存后大鼠精子活力变化与其结构的关系

The relationship between the motility and structure of the cryopreserved spermatozoa from rat

目的 观察冷冻对大鼠精子活动能力的影响 ,并探讨其活动能力的变化与细胞膜完整性、DNA结构、线粒体鞘和顶体的关系。 方法 使用计算机辅助精子活动分析仪 (CASA)检测冻存前后大鼠精子活动力的变化 ;荧光素乙酰乙酸盐 (FDA)染色法检测大鼠精子细胞膜的完整性 ;双氢鲁丹明荧光反应观察精子线粒体鞘的变化 ;金霉素 (CTC)荧光检测法观察冻存对大鼠精子顶体的影响。 结果 复苏后精子的活动力下降 ,为冻存前的 39.7% ;FDA检测显示冻存后的大鼠精子细胞膜完整 ,未受到破坏 ;冻存后的大鼠精子线粒体鞘荧光强度下降 ,且荧光不连续、甚至消失 ;CTC荧光反应显示冻存后精子顶体反应 (AR)的类型发生改变 ,AR型精子比例明显下降 ,由冻存前的 6 8.6 %降至冻存后的 13.4 %。 结论 冻存后大鼠精子细胞膜的完整性未受到破坏 ;精子的线粒体鞘和顶体受到较明显的破坏。大鼠精子冻存复苏后活力下降与线粒体鞘和AR存在相关性。

Objective: To study the relationship among the motility,the membrane integrity,acrosomal reaction and the mitochondrial function of cryopreserved spermatozoa from rat.Methods:(1)The membrane integrity of frozen spermatozoa was determined with FDA fluorescence staining. (2)The structure of rat sperm mitochondrial sheath was checked with rhodamine fluorescence staining.(3)The effect of cryopreservation on the acrosomal reaction of spermatozoa was analyzed with chlortetracycline (CTC) staining. (4)The motibility of both the unfrozen and frozen spermatozoa was determined with computer aided sperm assay.Results: After being cryopreserved,the cytoplasmic membrane of rat spermatozoa was intact,but the rate of acrosomal reaction reduced significantly,and the structure of mitochondrial sheath damaged. The motibility of the frozen spermatozoa decreased significantly.Conclusion: Cryopreservation impaired the function of acrosome and the mitochondria of rat spermatozoa,while there was no impairment on the cytoplasmic membrane. The relationship among the mitochondrial function,acrosomal reaction and the motility of rat spermatozoa need to be further studied,which will be useful to improve the cryopreservation protocol and cryoprotectant components for rat spermatozoa.