摘要
目的通过对睾丸精子超微结构的观察和实验室前期数据的生物信息学再分析,探讨睾丸Clock基因下调使小鼠精子前向运动百分率降低的机制。方法选取SPF级ICR雄性小鼠(8周龄)14只,随机分为实验组和对照组,每组6只,另2只小鼠作为免疫共沉淀实验样本。获取小鼠Clock基因下调的睾丸组织,透射电镜观察其中精子的超微结构。结合对实验室前期数据再分析的结果,ELISA检测睾丸组织中α-KGDH的活性,并以RT-qPCR检测睾丸中二氢硫辛酸脱氢酶(DLD)的转录组水平。最后在野生型小鼠睾丸组织中验证CLOCK蛋白与DLD蛋白的相互作用关系。结果与对照组相比,实验组Clock基因的表达显著降低(P<0.001);实验组睾丸中精子细胞线粒体形态和排列出现异常;差异蛋白的GO和KEGG富集分析提示,实验组精子线粒体中三羧酸循环限速酶α-KGDH的亚基构成蛋白之一的DLD明显下调(P<0.05);与对照组相比,实验组睾丸α-酮戊二酸脱氢酶复合体(α-KGDH)酶活性明显降低(P<0.05);与对照组相比,实验组睾丸Dld基因mRNA水平也明显下调(P<0.05);而野生型小鼠睾丸中CLOCK蛋白与DLD蛋白能够相互作用。结论睾丸Clock基因的下调可导致小鼠精子线粒体结构和功能蛋白发生改变,从而限制了线粒体能量的产出,最终导致精子前向运动百分率降低。
Objective Through the observation of the ultrastructure of testicular sperm and the bioinformatics re-analysis of preliminary laboratory data,to study the mechanism by which the down-regulation of the testicular Clock gene reduces the percentage of forward sperm movement in mouse.Methods Twenty male SPF ICR mice(8 weeks old)were randomly divided into experimental group,the control group,with 6 mice in each group,and the other 2 mice were used as the samples of co-immunoprecipitation experiment.Get Clock genes in mice testicular tissue,tem observation of the ultrastructure of sperm.The activity ofα-KGDH in the testicular tissue was detected with ELISA,and the transcriptome level of dihydrolipoic acid dehydrogenase(DLD)in the testicular tissue was detected with RT-qPCR.Finally,the interaction between CLOCK protein and DLD protein was verified in testicular tissue of wild-type mice.Results The morphology and arrangement of sperm cell mitochondria in the testis of the experimental group were abnormal.The GO and KEGG enrichment analysis of differential proteins indicated that DLD,one of the subunit constituent proteins of the TCA-cycle’s rate-limiting enzymeα-KGDH,was significantly down-regulated in the sperm mitochondria of the experimental group.Theα-KGDH enzyme activity in the testis of the experimental group was significantly reduced.The mRNA level of Dld gene in the testis was also significantly down-regulated in the experimental group.And the testis CLOCK protein and DLD protein can interact with each other in the wild type mouse.Conclusion Down-regulation of the Clock gene in the testis can lead to changes in the structure and functional protein of mouse sperm mitochondria,which limits the energy output of mitochondria and ultimately leads to a decrease in the percentage of forward sperm movement.
作者
王禹蒙
成姝婷
后望
骆芷寒
代泽咏
江舟
肖静
汪宇辉
郭慧玲
刘延友
WANG Yumeng;CHENG Shuting;HOU Wang;LUO Zhihan;DAI Zeyong;JIANG Zhou;XIAO Jing;WANG Yuhui;GUO Huiling;LIU Yanyou(Health Ministry Key Laboratory of Chronobiology,West China School of Basic Medical Sciences,Forensic Medicine,Sichuan University,Chengdu 610041,China)
出处
《西部医学》
2022年第10期1409-1413,1419,共6页
Medical Journal of West China
