体内生精系统重塑模型的建立及生精过程相关基因RSD-9功能研究

【作者】 李树春；

【导师】 王琳芳； 缪时英；

【作者基本信息】 中国协和医科大学 ， 生物化学与分子生物学， 2008， 博士

【摘要】 精子发生是由精原细胞增殖分化形成精子的一系列复杂而有序的过程。精原细胞经过有丝分裂、减数分裂,依次分化为初级精母细胞、次级精母细胞、精子细胞,最终成为成熟的精子。在这一分化过程中的每一特定阶段均受到细胞特异性和发育阶段特异性的基因的调节,这些生精过程相关基因的转录和表达也就成为精子发生的基础。因此,揭示生精细胞特异表达基因的功能,对阐明精子发生的分子机制具有十分重要的意义。由于精子发生的高度复杂性和对体内微环境的依赖性,目前各个生精阶段相应细胞系的缺乏是研究生精特异基因功能的瓶颈。从而,探索研究精子发生的新方法是摆在我们面前亟待解决的课题。在本研究中,我们首先对体内生精系统重塑模型进行了完善,发现GC1细胞移植入小鼠睾丸曲细精管后可以继续发育成成熟精子。在此基础上,我们建立了一种在体研究精子发生相关基因功能的新方法:建立稳定表达目的基因RNAi及对照质粒的GC1细胞株,将稳定表达目的基因RNAi的GC1细胞株移植入小鼠一侧睾丸曲细精管,将稳定表达对照质粒的GC1细胞株移植入另一侧睾丸的曲细精管。经过两至三个生精周期后,检测精子发生缺陷,从而确定目的基因的功能。并应用该方法对精子发生相关基因RSD-9的功能进行了探讨。首先在本实验室以往工作基础上,对体内生精系统重塑模型进行了完善。以Busulfan 40mg/kg腹腔注射后3～5周雄性小鼠为受体小鼠,将稳定表达GFP的GC1细胞移植入受体小鼠睾丸曲细精管中,供体细胞移植后在受体小鼠曲细精管成活、增殖、分化,并产生了成熟精子。本实验成功证明了以SV40 large Tantigen永生化的精原细胞系GC1 spg,不但能够在受体小鼠曲细精管中生存,而且能够定位于相当于精原干细胞所处的niche中进行增殖和分化,并产生成熟精子。此发现为建立在体研究生精相关基因功能技术平台提供了基础。在此基础上,针对我们实验室所筛选出的一个新的精子发生相关基因RSD-9,对这种体内生精系统重塑模型进行了应用研究。RSD-9是本组以rtSH3p13的N端保守区+可变区(C+V区)为诱饵,应用酵母双杂交的方法从小鼠睾丸cDNA文库中筛选获得的编码结合蛋白的新基因。该基因全长为575bp,开放读码框534bp,编码一个含177个氨基酸的蛋白质,获得GenBank接受号为AY278322。我们建立了稳定表达RSD-9-RNAi及对照质粒的GC1细胞株,将稳定表达RSD-9-RNAi的GC1细胞株移植入小鼠一侧睾丸曲细精管,将稳定表达对照质粒的GC1细胞株移植入另一侧睾丸的曲细精管。经过两个生精周期后,检测精子发生情况。我们发现,缺少RSD-9蛋白表达的精子细胞呈幼稚状态,并且没有顶体。为了确证RSD-9对精子顶体形成的重要作用,我们通过曲细精管内抗体注射确定了RSD-9蛋白影响精子顶体发生的功能。在得到RSD-9在体功能后,我们对其发挥功能的分子机制进行了探讨。我们首先通过GST-Pull down和Co-IP验证了该蛋白与rtSH3P13的相互作用。然后通过Northern blot发现RSD-9为睾丸特异性基因,免疫组化研究表明RSD-9从圆形精子开始表达,直至成熟精子,说明RSD-9基因是一个新的生精过程相关基因。为获得进一步的功能提示,利用PROSITE网上软件分析发现RSD-9羧基端含有一个P-loop区,提示RSD-9可能参与内吞中的耗能步骤。又根据PropSearch网上资源,发现RSD-9与GrpE功能相关,后者参与内吞中clathrin的释放。以此为思路,我们围绕RSD-9参与受体介导的内吞过程开展了初步的功能研究。利用ATP和GTP结合、水解实验证明RSD-9可以与ATP和GTP结合并水解它们,但其点突变体ARSD-9不能结合ATP和GTP,而且水解能力也比野生型蛋白低。然后通过转铁蛋白内吞实验证明RSD-9蛋白抑制网格蛋白介导的内吞。因为网格蛋白介导的内吞在精子顶体发生过程中具有重要作用,故RSD-9蛋白在精子顶体发生过程中具有重要作用。更多还原

【Abstract】 Spermatogenesis is a complex developmental process that includes the mitotic proliferation of spermatogonial stem cells,meiotic prophase,division of spermatocytes, and morphological changes of haploid spermatids to highly specialized spermatozoa.The developmental program of spermatogenesis is dependent upon precise,developmental stage and germ cell type-specific gene expression.Therefore,the cloning and identification of these differentially expressed genes is of great value to delineate the mechanism of spermatogenesis.In the present study,we’ve developed a novel in vivo system to further elucidate the mechanism of spermatogenesis based on the technique of microinjection into seminiferous tubules of mouse.Then,we determined the functions of the RSD-9 gene that is a germ cell type-specific gene.In the past,we established the technique of microinjection into seminiferous tubules of mouse and the germ cell transplantation system.Based on it,The GC1 cells stably expressed GFP were transplanted into mouse seminiferous tubules.After three spermatogenic cycles,the frozen sections from testis tissues were observed by microscopy.In the seminiferous tubules of mouse testis of the experimental group, donor-derived cells developed into elongated spermatids identified by the staining of the acrosomes with TRITC-PNA.The green fluorescent-labeled cells stained with TRITC-PNA showed that donor GC1 spg cells did undergo differentiation into elongated spermatids.However,no green fluorescence from the spermatogenic clone was observed within the control side of the testicular seminiferous tubules,or within the epididymis.Based on the above findings,with each recipient mouse,one testis was used to transplant GC1 spg cells that stably expressed pRNAT/H1-RSD-9-RNAi-negative and the other one was transplanted GC1 spg cells that stably expressed pRNAT/H1-RSD-9-RNAi.After two to three spermatogenic cycles,the transplanted GC1 spg cells that stably expressed pRNAT/H1-RSD-9-RNAi-negative can differentiate and form donor-derived spermatogenic cell colonies and sperms in the recipient testes.But, the transplanted GC1 spg cells that stably expressed pRNAT/H1-RSD-9-RNAi can’t differentiate and form donor-derived spermatogenic cell colonies in the recipient testes. Knock-down of the RSD-9 gene led to the stagnation of the process of spermatogenesis and the absence of the acrosome. RSD-9 was isolated from the mouse testis cDNA library using the fragment coding the coiled-coil region and the variable region of rtSH3P13 as bait in yeast two-hybrid system.RSD-9 protein has a structure similarity with GrpE,which serves as an ADP/ATP exchange factor in the Hsp70 chaperone machinery.Northern blot results revealed that RSD-9 gene was specifically expressed in brain and testis.Furthermove, immunohistochemistry has been performed to detect the localization of RSD-9 protein in testis tissues a significant staining pattern with anti-RSD-9 was detected from round and elongated spermatids to mature sperms,whereas no staining was detected in spermatogonia,spermatocytes closing to basal membrane as well as in Sertoli cells and Leydig cells.Then,we demonstrated that the RSD-9 protein interacted with rtSH3P13 in vitro and in vivo by GST-pulldown and Co-IP.Based on this background,we began our functional research of RSD-9 gene.By Blot overlay assay,we found RSD-9 protein bound with both ATP and GTP through its P-loop motif.Because rtSH3P13 is known to be involved in clathrin-dependent endocytosis,we examined the effect of overexpressing RSD-9 on this process.And we found that expression of RSD-9 and△RSD-9 could regulate the endocytosis of transferrin.The clathrin-mediated endocytosis is very crucial for the process of architecture of acrosome,Knock-down of endogenous RSD-9 interfered the architecture of the acrosome,which confirms that RSD-9 is needed for this acrosome biogenesis process.更多还原