Cfap65基因敲除对小鼠精子发生的影响  

Effect of Cfap65 deficiency on mouse spermatogenesis

作　　者：余怡 王家雄[1] 刘彩钊 郑邦旭 王改改 杨慎敏[1] Yu Yi;Wang Jiaxiong;Liu Caizhao;Zheng Bangxu;Wang Gaigai;Yang Shenmin(Reproductive and Genetic Center,the Affiliated Suzhou Hospital of Nanjing Medical University,Suzhou 215002,China;Reproductive Medicine Center,Ningbo Women and Children's Hospital,Ningbo 315012,China)

机构地区：[1]南京医科大学附属苏州医院生殖与遗传中心,苏州215002 [2]宁波市妇女儿童医院生殖医学中心,宁波315012

出　　处：《中华生殖与避孕杂志》2022年第8期829-836,共8页Chinese Journal of Reproduction and Contraception

基　　金：中华医学会临床医学研究专项(18010270756);江苏省卫生健康委资助项目(F201866,H2018050);浙江省医药卫生科技计划(2019KY622);姑苏卫生人才项目(GSWS2019053)。

摘　　要：目的探讨Cfap65基因敲除对小鼠精子发生的影响。方法利用CRISPR/Cas9技术构建Cfap65基因敲除小鼠;使用PCR和Sanger测序方法进行小鼠基因型鉴定;依据小鼠基因型将小鼠分为Cfap65-/-组(n=3)与野生型组(n=3);生育力试验评估小鼠生育力;使用HE染色、免疫荧光、透射电子显微镜观察小鼠附睾精子与睾丸精子形态;使用定量实时聚合酶链锁反应检测Cfap65 mRNA在小鼠心、肝、脾、肺、肾和睾丸组织中的表达。结果Cfap65-/-组小鼠表现为完全不育,附睾精子数量减少和活动率低下,形态观察可见精子出现短尾、卷尾和尾部缺失,头部畸形率也显著高于野生型小鼠(1.67%±0.44%比33.00%±1.53%),差异有统计学意义(P<0.001)。Cfap65基因敲除导致小鼠精子领结构异常。Cfap65高表达于成年小鼠的睾丸、肺中;Cfap65在小鼠睾丸中的表达从4周龄到6周龄有一个急剧的增加(901.90±33.19比2144.00±22.92),差异有统计学意义(P<0.001)。结论Cfap65表达具有组织特异性,缺失后导致雄性小鼠精子发生障碍,这可能与精子领运输障碍有关。Objective To explore the effect of Cfap65 deficiency on mouse spermatogenesis.Methods CRISPR/Cas9 technology was utilized to construct Cfap65 deficient mice.PCR and Sanger sequencing were adopted to identify mouse genotypes.Mice were divided into Cfap65-/-group(n=3)and wild-type(WT)group(n=3)based on genotypes of mice.Fertility test was applied to evaluate the fertility of mice.Sperm morphology of Cfap65 deficient mice was observed by hematoxylin-eosin(HE)staining,immunofluorescence and transmission electron microscope.Real-time fluorescent quantitative PCR was used to detect the expression of Cfap65 mRNA in heart,liver,spleen,lung,kidney and testis tissues of mice.Results Cfap65 deficient male mice were completely infertile.Compared with wild-type male mice,Cfap65 deficient mice had fewer and less motile epididymal spermatozoa,whose flagellums tend to be short,curled,bent and even absent,and heads tend to be deformed(1.67%±0.44%vs.33.00%±1.53%),and the differences were statistically significant(P<0.001).Besides,Cfap65 deficiency led to anomalous structure of manchette of mice.Cfap65 was highly expressed in the testes and lung of adult mice,and the expression of Cfap65 in testes embodied a sharp increase trend from mice aged 4 to 6 weeks(901.90±33.19 vs.2144.00±22.92),and the differences were statistically significant(P<0.001).Conclusion The expression of Cfap65 is tissue-specific,and the deletion of Cfap65 leads to spermatogenesis failure in male mice,which might be related to the dysfunction of intra-manchette transport.

关 键 词：精子发生 精子变态 精子鞭毛 Cfap65 

分 类 号：R698.2[医药卫生—泌尿科学] R-332[医药卫生—外科学]