【作者】 李婷；

【导师】 罗孟成；

【作者基本信息】 武汉大学 ， 人体解剖与组织胚胎学（专业学位）， 2019， 硕士

【摘要】 精子发生是二倍体生殖细胞通过减数分裂最终形成有功能的单倍体精子的过程,主要包括减数分裂的启动,精母细胞的减数分裂和精细胞变形,精子的发生过程对雄性生殖具有决定性作用,其中任何阶段出现异常都极可能导致精子发生异常,引发雄性不育。研究表明在精子发生过程中涉及多种特异蛋白的表达,参与调控精子发生这一精细而复杂的过程。本研究中,通过一种优化的染色质纯化方法,将不同时期小鼠睾丸的染色质纯化后进行质谱分析,筛选调控精子发生过程的可能蛋白。在质谱结果中,泛素特异性激酶26(Ubiquitin-specific protease26,USP26)引发了关注。有文献报道,去泛素化酶作为蛋白质泛素化的负性调控因子,在泛素化调控的过程中发挥着重要作用,USP26即为该家族的成员之一。有研究表明,在非梗阻性无精子症的男性病人中发现USP26基因突变,会引发男性精子发生障碍,但是具体的作用机制仍不清楚。因此本课题以小鼠为实验模型,开展研究,探究USP26在精子发生过程中的作用及可能机制。首先,本课题通过构建Pet-42b-USP26的重组质粒,然后进行原核蛋白表达以及纯化,制备了USP26的兔多克隆抗体;其次,通过Western blot对小鼠不同组织进行USP26蛋白表达分析,结果显示USP26是一个睾丸特异性表达的蛋白;通过冷冻切片及免疫荧光实验确定USP26定位在精子顶体。而后本课题采用基于CRISPR-CAS9的基因编辑技术对小鼠进行USP26基因的敲除,探究USP26在小鼠精子发生过程中的作用机制。通过Western blot验证USP26蛋白的敲除,然后通过分析敲除小鼠表型发现,敲除USP26后对小鼠的睾丸重量没有影响,但是精子数量减少,同时体外受精分析发现小鼠胚胎的体外发育未受到明显阻滞,敲除鼠的产仔量也没有明显差异。综上结果显示,虽然在人类男性中有报道USP26基因的突变会导致男性不育,但是在小鼠中,USP26的敲除对于雄鼠的生育能力没有显著影响。因此在小鼠中,虽然USP26的敲除可能导致某些精子形成的相关蛋白无法去泛素化,使精子的发生过程受到部分阻滞,但该调控并不是精子发生所必须的。更多还原

【Abstract】 Spermatogenesis is the process by which diploid germ cells eventually form functional haploid sperm through meiosis.It mainly includes the initiation of meiosis,spermatocytes meiosis and spermatids formation.The process of sperm production plays a decisive role in male reproduction,and abnormalities at any stage are likely to cause defects in spermatogenesis and lead to male infertility.Previous studies have shown that the expression of various of specific proteins during spermatogenesis is involved in the regulation of spermatogenesis,a delicate and complex process.In this study,an optimized chromatin purification method was used to purify the chromatin of mouse testes at different stages and mass spectrometry was performed to screen potential proteins that regulate spermatogenesis.In the mass spectrometry results,Ubiquitin-specific protease 26(USP26)has drawn our attention.It has been reported that deubiquitinating enzymes,as negative regulators of protein ubiquitination,play an important role in the regulation of ubiquitination,and USP26 is a member of this family.Studies has shown that USP26 gene mutations in male patients with non-obstructive azoospermia can cause male spermatogenesis disorders,however,the molecular mechanism remains unclear.Therefore,this subject was conducted to explore the function and possible mechanism of USP26 in mouse spermatogenesis.Firstly,we made the rabbit polyclonal antibody of USP26 through constructing the recombinant plasmid of Pet-42b-USP26,performing prokaryotic expression and purification and immunizing the rabbit with the purified protein.Secondly,the expression and distribution of USP26 was examined by western blot and immunofluorescent assay.The results showed that USP26 is testis-specific and mainly located in sperm acrosome.Afterwards,the USP26 gene was knocked out through the CRISPR-CAS9-based gene editing technology,with the gene edited mouse the mechanism of USP26 in mouse spermatogenesis was studied.The knockout efficiency of USP26 was confirmed by Western blot,and then the knockout mouse phenotypes were analyzed.It was found that knocking out USP26 had no effect on testicular weight of mice,however,the number of sperm decreased,and in vitro fertilization analysis revealed there was no obvious difference in the development of mouse embryos between wild type and USP26 knockout mouse.Meanwhile,there was no significant difference in the littersize of the knockout mice compared with wild-type mice.The above results showed that knockout of USP26 has no significant effect on male fertility in mice,though the mutation in men cause male infertility.Therefore,although the knockout of USP26 in mice may cause abnormal ubiquitination to some sperm-related proteins,and affect the spermatogenesis partially,but this regulation is not necessary for spermatogenesis.更多还原