精子β-actin下调对小鼠怀孕率影响的实验研究

Effects of gene silencing of sperm β-actin on the pregnant rate of rats

摘要:

目的：通过构建精子β-actin下调的小鼠模型探讨β-actin下调对小鼠精子活力及怀孕率的影响。方法：首先构建携带下调ACTB基因表达的shRNA序列的慢病毒载体。然后将昆明小鼠随机分为ACTB-RNAi-LV组（实验组）、5X-1-RNAi-LV组（空病毒对照组）、生理盐水组（空白对照组），每组雄性、雌性小鼠各20只，于雄鼠4周大时睾丸局部分别注射慢病毒载体、空病毒颗粒、生理盐水。饲养至性成熟后，将各组小鼠进行组内交配，观察各组雌鼠怀孕率并提取雄性小鼠精子进行real-time PCR、Western blot及精子活力的分析。结果：成功构建β-actin下调精子的小鼠模型；实验组与空病毒对照组、空白对照组相比，精子中β-actin的mRNA表达量、β-actin表达量、精子的活力均有所降低且有统计学意义（P均<0.05），空病毒对照组与空白对照组之间无统计学差异（P >0.05）。结论：β-actin下调精子活力明显下降，同时导致雌鼠受孕率降低。

Abstract:

Objective：To discuss the effects of gene silencing of sperm β-actin on the vitality of sperm and pregnant rate of rats. Methods：Firstly，lentivirus carrying shRNA sequence was constructed to down regulate the expression of ACTB gene. Secondly，the 60 male and 60 female rats were randomly divided into three groups：blank control group（normal saline group），empty lentivirus group（5X-1- RNAi-LV group） and experimental group（ACTB- RNAi-LV group）. Male rats were administrated with saline or lentivirus solution by tissue injection at testis at the fourth week. Rats were mated when they reached sexual maturity. Finally，the pregnancy rate of female rats and the motility of sperm were observed. The expression of ACTB gene and β-actin in sperm were detected by reverse transcription PCR and Western blot. Results：The model of sperm expressing low level of β-actin was successfully construed. The expression of β-actin and mRNA of ACTB gene as well as pregnant rate in experimental group were significantly reduced compared with those in blank control group and empty lentivirus group（P<0.05）.There was no difference in the expression of β-actin and pregnant rate in blank control group and empty lentivirus group（P >0.05）. Conclusion：The motility of sperm expressing the low level of β-actin could decline，which leads to the fall of pregnant rate.