自噬基因Atg5的缺失对精子成熟的影响

目的探讨自噬蛋白Atg5在维持附睾生理功能及精子的成熟过程中发挥的作用。方法通过基因敲除技术构建附睾头4-5段主细胞Atg5特异性敲除小鼠模型,根据基因型分为三组:对照组(野生型)、杂合敲除组以及纯合敲除组。8周龄时,做合笼实验并记录致孕率和产仔数,14周龄时通过精子动力分析仪检测小鼠精子活力和数量的变化,并通过病理组织染色观察小鼠附睾组织的形态学变化,以及通过Western blot和免疫荧光技术检测三组模型鼠中ATG5蛋白表达和自噬通路变化情况。结果与对照组、杂合敲除组相比,纯合敲除组小鼠的附睾头4-5段主细胞Atg5特异性敲除后,自噬标记蛋白LC3-Ⅰ以及p62得到积累,自噬过程成功阻断;但纯合敲除组小鼠的附睾组织形态、精子活力、精子数目、产仔率和致孕率均无变化。结论在正常条件下(无外界刺激),Atg5特异性敲除导致附睾头4-5段的自噬过程阻断后,对小鼠附睾的生理功能和精子成熟过程没有影响。

Impact of autophagy gene Atg5 deficiency on sperm maturation

Aim To investigate the role of autophagy core protein Atg5 in maintaining epididymal physiological functions and sperm maturation.Methods The Atg5 conditional knockout mouse model of principle cells in the 4-5 segments of the epididymal caput was constructed by Cre/LoxP system.The mice were divided into three groups according to genotype:Atg5f/f,Atg5f/-and Atg5-/-.The pregnancy rate and litter sizes were recorded by mating experiments at the age of 8 weeks-old.Sperm motility,sperm counts were evaluated with computer-aided sperm analysis(CASA)after 14 weeks of feeding.HE staining was conducted to observe the morphology of the epididymal caput.Western blot and immunofluorescence technologies were applied to verify the expression level of Atg5 and the impediment of autophagy after Atg5 conditional knock out.Results After tissue specific knockout of Atg5 in the 4-5 epididymal principle cells,the autophagy marker proteins,LC3-Ⅰand p62,were accumulated and autophagy was successfully blocked.There was no significant difference in the morphology of epididymal tissues of the three genotypes of mice,nor any statistically significant difference in sperm motility,sperm count,litter size and pregnancy rate.Conclusions Under normal conditions without external challenge,Atg5 conditional knockout led to autophagy blocking of epididymal caput,but had no effect on mouse epididymal function and sperm maturation process.