生殖细胞条件性敲除Usp16基因小鼠的建立、鉴定及表型分析  

Establishment,identification and phenotyping of a germ cell-specific deubiquitinating enzyme 16 conditional knockout mouse

作　　者：李卫杰 梁冬丽 刘永忠[3] 王朝霞[2] 乔中东[1] 徐汪节[2] LI Weijie;LIANG Dongli;LIU Yongzhong;WANG Zhaoxia;QIAO Zhongdong;XU Wangjie(School of Life Sciences and Biotechnology,Shanghai Jiao Tong University,Shanghai 200240,China;Laboratory Animal Center,Shanghai Jiao Tong University,Shanghai 200240;State Key Laboratory of Oncogenes and Related Genes,Shanghai Cancer Institute,Renji Hospital,Shanghai Jiao Tong University School of Medicine,Shanghai 200240)

机构地区：[1]上海交通大学生命科学技术学院,上海200240 [2]上海交通大学实验动物中心,上海200240 [3]上海交通大学医学院仁济医院上海肿瘤研究所癌基因与相关基因国家重点实验室,上海200240

出　　处：《中国实验动物学报》2022年第3期376-383,共8页Acta Laboratorium Animalis Scientia Sinica

基　　金：上海市自然科学基金(20ZR1427400);上海交通大学决策咨询课题(JCZXSJA-02)。

摘　　要：目的建立生殖细胞特异性敲除去泛素化酶Usp16小鼠,并分析其生殖表型,探究Usp16是否在精子发生过程发挥功能。方法首先,将Vasa-cre转基因小鼠与Usp16条件性小鼠(Usp16flox/flox)杂交获得生殖细胞特异性敲除Usp16小鼠;其次,利用qPCR、Western Blot和免疫荧光检测睾丸中Usp16表达;并通过合笼交配、计算机辅助精子分析和HE染色法进行生殖表型分析。结果成功获得了生殖细胞特异性敲除Usp16纯合子(Usp16flox/flox/Vasa-cre+),及杂合子小鼠(Usp16flox/wt/Vasa-cre+)。与野生型相比,Usp16flox/wt/Vasa-cre+雄鼠生殖表型没有显著性变化,但与纯合子雄鼠交配的雌鼠受孕率仅为7%,精子数量减少约79%,活力下降约87%,精子畸形率高达83%,睾丸曲细精管中出现多核细胞,附睾中早熟精子数增多了约3倍。结论小鼠睾丸内生殖细胞Usp16纯合敲除会严重降低精子质量,从而降低雄性生育能力。Objective To study the role of deubiquitinating enzyme 16(Usp16)in spermatogenesis by establishing a mouse model of conditional Usp16 knockout in germ cells.Methods Vasa-cre transgenic mice were crossed with Usp16 transgenic mice(Usp16^(flox/flox))to obtain germ cell-specific Usp16 knockout mice.To confirm that Usp16 had been conditionally inactivated in germ cells,genotyping by PCR and western blotting was performed to evaluate Usp16 expression.Initial characterization of the phenotype of conditional knockout(CKO)mice was then conducted by co-cage mating,computer-assisted sperm analysis,and HE staining.Results We confirmed that Usp16 expression was appropriate in the conditional knockout mouse.Compared with the wild type,the conception rate of female mice mated with CKO mice had decreased to only 7%,while no significant difference was observed in heterozygous knockout mice male mice.The sperm count of CKO mice was decreased by approximately 79%,motility was decreased by approximately 87%,and the rate of sperm deformity was as high as 83%.Multinucleated cells appeared in the seminiferous tubules of the testes and the number of precocious sperm in the epididymis had increased by approximately 76%.Conclusions Homozygous mutation of Usp16 in germ cells severely reduces sperm quality,thereby reducing male fertility.

关 键 词：精子发生 Usp16 生殖力 CRE-LOXP 

分 类 号：Q95-33[生物学—动物学]