Zfy基因在精子尾部发育过程中的作用机制研究

doi:10.16431/j.cnki.1671-7236.2025.03.024

摘要/Abstract

摘要： 【目的】研究Y染色体锌指基因(Zfy)在成年雄性小鼠精子尾部发育过程中的作用机制。【方法】选取20只8周龄昆明品系雄性小鼠，分为试验组与对照组，每组随机分配10只小鼠。试验组采用睾丸网注射法注射Zfy基因干扰载体与Lipofectamine^TM LTX&PLUS^TM转染试剂混合液，建立小鼠睾丸Zfy基因干扰模型，对照组注射空载体与Lipofectamine^TM LTX&PLUS^TM转染试剂混合液；注射72 h后采集试验组与对照组小鼠睾丸组织，通过实时荧光定量PCR检测Zfy基因干扰载体对小鼠睾丸Zfy基因干扰效果，同时检测试验组与对照组小鼠精子尾部发育相关基因SUN结构域基因5(SUN5)、精子特异性钙离子通道基因1(CATSPER1)、外层致密纤维基因1(Odf1)的表达量变化，并利用Western blotting检测上述基因在蛋白质水平表达量；利用免疫共沉淀检测小鼠睾丸中Zfy蛋白与SUN5、CATSPER1、Odf1蛋白关联性；采集试验组与对照组小鼠附睾精子，利用伊红精子染色计数对比8项头部、尾部畸形率变化，并通过计算机辅助精子分析(CASA)系统检测精子运动参数变化。【结果】注射72 h后，与对照组相比，试验组Zfy基因mRNA及蛋白相对表达量均极显著降低(P＜0.01)；试验组小鼠睾丸中SUN5、CATSPER1、Odf1基因mRNA及蛋白相对表达量均极显著降低(P＜0.01)；Zfy蛋白与SUN5、CATSPER1蛋白可形成免疫沉淀复合物；与对照组相比，试验组小鼠精子无钩头部畸形率、胖头畸形率、不定形畸形率、尾部折叠畸形率、尾部卷曲畸形率和断尾畸形率均极显著或显著升高(P＜0.01；P＜0.05)；CASA结果显示，与对照组相比，试验组小鼠精液参数(精子密度、活力、前向运动精子)和精子运动参数(曲线速率、直线速率、平均轨道速度、侧摆幅度、直线性、摆动性、向前性、交打频率)均显著或极显著降低(P＜0.05；P＜0.01)。【结论】小鼠睾丸Zfy基因干扰模型中SUN5、CATSPER1、Odf1基因及其蛋白表达量降低，影响精子头尾连接、尾部运动供能及尾部鞭毛外层结构形成，造成精子尾部畸形及运动能力下降。

关键词: Zfy基因; 精子尾部发育; 基因干扰

Abstract: 【Objective】 The aim of this experiment was to explore the mechanism of Y chromosome (Zfy) gene during the development of sperm tails in adult male mice.【Method】 Twenty 8-week-old Kunming strain (KM) male mice were selected and divided into experimental and control groups,with 10 mice in each group.The mouse testis Zfy gene interference model was established by injecting the Zfy gene interference vector and the mixture of Lipofectamine^TM LTX & PLUS^TM transfection reagent into the rete testis,and injecting the empty vector and the mixture of Lipofectamine^TM LTX & PLUS^TM transfection reagent into the rete testis as the control group.After injection for 72 hours,testis tissues from both groups were collected for detecting the interference effect of Zfy gene interference vector on testis Zfy gene by Real-time quantitative PCR,and measuring the expression changes of SUN5,CATSPER1 and Odf1 genes related to the development of sperm tail of mice.The protein expression levels of the above genes were detected by Western blotting.The correlation between Zfy protein and SUN5,CATSPER1 and Odf1 proteins were detected by immunoprecipitation.The epididymal sperm of experimental and control groups were collected,for comparing the changes of 8 head and tail malformation rates by eosin sperm staining,and detecting the changes of sperm movement parameters by computer assisted sperm analysis (CASA) system.【Result】 After interference for 72 hours,compared with the control group,the mRNA and protein relative expression levels of Zfy gene in experimental group were extremely significant decreased (P＜0.01).The mRNA and protein relative expression levels of SUN5,CATSPER1 and Odf1 genes in testis of experimental group were extremely significantly decreased (P＜0.01).Zfy protein could form immunoprecipitation complex with SUN5 and CATSPER1 proteins.Compared with control group,the rates of head without hook deformity,abnormally large head deformity,amorphous deformity,tail folding deformity,tail curling deformity and tail breaking deformity of mouse sperm in the experimental group were extremely significant or significantly increased (P＜0.01 or P＜0.05).The results of CASA showed that compared with the control group,the semen parameters (sperm density,motility,progressive motility) and sperm locomotion parameters (curvilinear velocity,straight-line velocity,average path velocity,amplitude of lateral head displacement,linearity,wobble,straightness and beat-cross frequency) of mouse in experimental group were significantly or extremely significantly decreased (P＜0.05 or P＜0.01). 【Conclusion】 The expression levels of SUN5,CATSPER1 and Odf1 genes and their proteins decreased in the Zfy gene interference model of mouse testis,which affected the head-tail connection of sperm,the energy supply of tail motor and the formation of outer layer structure of tail flagella,resulting in the abnormality of sperm tail and the decrease of motor ability.

Key words: Zfy gene; sperm tail development; gene interference

中图分类号:

S813.1

李超程, 刘亚星, 李健, 李云蕾, 贾斌. Zfy基因在精子尾部发育过程中的作用机制研究[J]. 中国畜牧兽医, 2025, 52(3): 1222-1230.

LI Chaocheng, LIU Yaxing, LI Jian, LI Yunlei, JIA Bin. Study on the Mechanism of Zfy Gene in the Development of Sperm Tail[J]. China Animal Husbandry and Veterinary Medicine, 2025, 52(3): 1222-1230.

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