【作者】 杨明刚；

【导师】 蒋先镇；

【作者基本信息】 中南大学 ， 泌尿外科， 2011， 博士

【摘要】 在男性不育疾病中,生精障碍及其相关疾病是重要的一个组成部分。许多基因参与了精子生成,成熟,运动等方面的调控。有研究表明,大约2000多个不同的基因参与了男性生殖的各个方面[1,2]：睾丸发育,生殖细胞分化,减数分裂以及精子发生等阶段。其中不少生精相关基因的功能已经被充分研究,但是很大一部分基因的功能如何?与其他的基因怎样相互调控来影响人类生殖的不同阶段尚不完全清楚。因此对人类生精相关基因的研究具有重要意义。SUN家族是目前研究比较多的一个基因家族。SUN中文全称Sad-1/Unc-84家族。早期在探讨真核细胞的核移动、核重定位、核膜重构等过程中发现,内核膜蛋白Unc-84起到了关键作用[1]。后来又陆续发现部分SUN家族成员参予精子发生过程[3.4]。SUN家族成员的重要结构特征是肽链C端有一个高度保守的SUN domain氨基酸序列,靠近N端有跨膜区(TM),跨膜区和SUN domain之间有coiled-coil区。在人类蛋白中,SUN家族目前已发现的成员有五位：SUN1、SUN2、SUN3、SPAG4(又称SUN 4)和SPAG4L(又称SUN 5或者TSARG4)。SUN家族成员参予了精子发生过程：在小鼠精子尾部形成过程中,Xueping Shao等发现,SPAG4蛋白通过亮氨酸拉链结构,与Odfl(小鼠精子尾部纤维蛋白)相互作用,在精子尾部发生中起重要作用[3]；在哺乳动物精子头部形成过程中,Sun3与外核膜蛋白Nesprin1作用,Sunl与外核膜蛋白Nesprin3作用,将细胞核和细胞质骨架结构联系起来,在确保精子头部形成方面起重要作用[4]。我院中心实验室邢晓为老师在早期研究中,从SPAG4出发,克隆了人类睾丸新基因SPAG4L,并于2001年7月率先向GenBanK提交了该序列(GenBanK登录号：AF401350),随后克隆了SPAG4L的小鼠同源基因spag4l(又名SRG4) (GenBanK登录号：AY307077)。在早期的动物模型实验中发现,多组织的RT-PCR和Northern blot结果显示人SPAG4L的小鼠同源基因spag4l在小鼠睾丸组织中特异性表达,在其它组织中无明显表达；在研究小鼠同源基因spag4l表达水平变化时发现,RT-PCR结果显示小鼠出生2周内,spag4l表达量极低；3周开始,spag4l开始大量表达；到4-5周时表达量到达最高,说明小鼠同源基因spag4l可能在小鼠精子发育中起重要作用[6]。组织原位杂交结果显示,spag4l在正常小鼠睾丸中大量表达,主要表达于精母细胞和圆形精子细胞中；而在手术隐睾模型中,生殖细胞大量凋亡,精曲小管空泡化,仅在精曲小管管壁残存的生殖细胞中可见个别spag4l棕黄色阳性杂交信号。正常睾丸和隐睾阴性对照中均未见spag4l阳性杂交信号。表明spag4l可能对小鼠精子的生长发育起调控作用。以上动物实验结果均提示小鼠同源基因spag4l在小鼠的精子发育过程中起重要作用。而对于人类基因SPAG4L基因的功能和特点,目前所知甚少。在本研究中,我们对SPAG4L的蛋白家族归属、时间和空间表达特性；SPAG4L蛋白的原核表达与纯化；亚细胞定位以及SPAG4L蛋白在精子减数分裂发生中的变化等方面进行了初步探讨。第一章SPAG4L生物学信息学分析和表达特点目的：研究人睾丸生精新基因SPAG4L与已知的SUN蛋白家族成员的同源性分析,了解其蛋白家族归属；了解SPAG4L的组织表达特异性和时空表达特异性。方法：将SPAG4L基因全长序列与其他SUN家族成员基因序列比对,进行同源性分析。用Northern blot, RT-PCR,原位杂交等方法对SPAG4L的表达特性进行研究。结果：SPAG4L是SUN蛋白家族的一个新成员,并且与SUN3,SPAG4来源于同一个祖先。SPAG4L在睾丸组织中特异性表达,而在其余组织中无明显表达,说明其是一个睾丸特异性基因。SPAG4L主要是在成年男性睾丸中高表达,在胎儿睾丸中无表达；SPAG4L蛋白主要表达在精原细胞和精母细胞,在成熟精子中不表达。结论：SPAG4L是SUN家族的一个新成员；在睾丸组织中特异性表达且与睾丸成熟度有关；SPAG4L蛋白主要在精子发生减数分裂过程早期阶段出现,在成熟精子和长形精子中无表达。为我们进一步深入研究SPAG4L在精子发生机制,在减数分裂中的作用打下一定基础。第二章SPAG4L的原核表达和纯化目的：通过构建原核表达质粒PQE-30-SPAG4L,对SPAG4L基因在大肠杆菌中诱导表达并进行纯化,获得纯化SPAG4L蛋白并进行验证。方法：取人新鲜睾丸组织,提取总RNA,采用逆转录-聚合酶链反应(RT-PCR)扩增SPAG4L基因编码序列；将该基因克隆到表达载体PQE30中,构建原核表达载体PQE-30-SPAG4L,用限制性内切酶酶切分析及DNA序列分析鉴定重组质粒；将测序验证的重组质粒转化大肠杆菌JM15后诱导原核表达,并用NI-NTA磁性琼脂糖珠对表达出的融合蛋白进行纯化。并用Western Blot法对目的蛋白进行验证。结果：RT-PCR扩增出SPAG4L的(TSARG4)基因编码序列,目的插入片段长约253bp；产物经限制性内切酶酶切后,成功连接到表达质粒PQE-30。重组质粒PQE-30-SPAG4L经酶切鉴定构建成功；重组质粒转化大肠杆菌JM15后进行诱导并原核表达,获得带有6个组氨酸标签的融合蛋白6×His-SPAG4L。用NI-NTA磁性琼脂糖磁珠纯化6×His-SPAG4L蛋白,并Western Blot鉴定纯化蛋白成功。结论：成功构建了人睾丸基因SPAG4L (TSARG4)的原核表达载体PQE-30-SPAG4L并体外表达成功；所获得纯化6×His-SPAG4L蛋白可以用于下个阶段,对于SPAG4L蛋白功能学和蛋白相互作用等方面的研究。第三章SPAG4L的亚细胞定位及其在减数分裂中的变化目的：研究人SPAG4L蛋白的亚细胞空间定位,并明确对SPAG4L蛋白的精细亚细胞定位起关键作用的结构域。并初步观察人SPAG4L蛋白的小鼠同源基因spag41在小鼠精子减数分裂中的变化。方法：构建包含不同结构域的EGFP-N1-SPAG4L突变体的表达质粒,转染HeLa细胞,通过激光共聚焦观察绿色荧光蛋白发光情况,了解不同SPAG4L突变体的亚细胞定位情况,观察不同结构域对不同SPAG4L突变体其亚细胞定位变化的影响；用免疫荧光双标的方法,观察其小鼠同源蛋白spag4l和内质网标志蛋白PDI、核膜标志蛋白Lamin B1共定位情况；通过免疫荧光观察其小鼠同源蛋白spag41在小鼠精子减数分裂中的变化并初步分析其意义。结果：SPAG4L蛋白主要定位于核膜和内质网。其跨膜区(TM)和coiled-coil区对SPAG4L蛋白精确亚细胞定位是必需的,C端的SUN区对SPAG4L蛋白亚细胞定位无影响；在减数分裂中,其小鼠同源蛋白spag4l可能参与了细胞核迁移、定位和核膜重构。结论：SPAG4L是一个核膜和内质网蛋白；其亚细胞定位依赖于其跨膜区和coiled-coil区,C端的SUN区对其亚细胞定位无影响。其小鼠同源蛋白spag4l参与了减数分裂中核膜的迁移,折叠和重构。提示人SPAG4L蛋白也可能参予人精子减数中核膜的迁移,折叠和重构。更多还原

【Abstract】 Dyszoospermia and other correlative diseases is an important part of male infertility. Many genes participate in the controls of spermatogenesis, sperm maturity, sperm movement and other processes. About 2000 different genes been reported have taken part in the every procese of male reproduction:testis growth, germ cells differentiation, meiosis, spermatogenesis and so on. Many genes have been studied absolutely, but to the others, there are still many genes which can not been studied throughly. What are the details of the function of them? How do they cooperate with each other to control the different stage of human procreation? So it is very important to do the research of human genes involved in the spermatogenesis.It is reported that some members of SUN gene family may cooperate with the human spermatogenesis. Teacher Xiao Wei Xing has coloned a new sperm gene located in the chromosome 20q 11.21 region, named SPAG4L (TSARG4), from the gene SPAG4 in early research. In july 2001 he submit the full sequence of SPAG4L to GenBanK (GeneBanK Access number:AF401350) and coloned another gene named spag41 (SRG4) (GeneBanK Access number:AY307077) which is a mouse homologous gene of SPAG4L from mouse ESTs. The result of multiple orgnizations revealed that spag41 expressed specifically in mouse testicle by the means of RT-PCR and Northern blot. The result of RT-PCR revealed that spag4l express very low in 2 weeks after mouse’s birth. From the third week Spag4l expressed more and more and to it’s peak in the period of 4-5 weeks, indicating that spag4l may play important role in the spermatogenesis progress. In situ hybridization, spag4l express obviously in the normal control sperm, mostly in spermatocyte and round spermatids. But in normal sperm and enorchia negative control, No obvious signals were detected, indicating that Spag41 may in charge of the development of mouse spermatogenesis.But to the human homologous gene SPAG4L, there are still many spaces needed to be explored. In this research, we study the expression specility of SPAG4L, the accurate subcellular localization and the key gene region which respond for the subcellular location、the changes of spag41 proteins in the process of mouse meiosis.Chapter One:The bioinformation analyzation of SPAG41 and the expression specialityObjective:We try to clear the expression specility of SPAG4L and to confirm which gene family it should belong to by means of homology analysis with the known SUN-domain proteins. Methods:Comparative homology analysis of full length of SPAG4L with the known SUN proteins, we kown which protein family it belong to and speculate the original function of it. By means of Northern bolt、RT-PCR and In situ hybridization, we clear the expression characteristic of SPAG4L.Result:SPAG4L is a new member of SUN family and may originate from the same ancestor with SUN3, SUN4 (SPAG4). SPAG4L only expressed in human testis, but in other tissues no expression of SPAG4L been detected. SPAG4L expressed in mature male testis, mostly been observed in spermatogonium and spermatocyte, but not in mature sperm, neither in fetus testisConclusion:SPAG4L is a new member of SUN family and expressed specially in human testis, correlating with the mature degree of testis. SPAG4L expressed in mature male testis, mostly been observed in spermatogonium and spermatocyte but not in mature sperm, neither in fetus testis Chapter Two:Prokaryotic expression and purification of SPAG4LObjective:Get the protein of SPAG4L through prokaryotic expression and purified it.Methods:We amplify the 126-379bp of SPAG4L by means of RT-PCR from freash human testis. Then we subclone the fragment to pUCm-T vector and PQE-30 expressing vector. After transforming to Bacterium coli JM109, we extract the protein and examine it by means of Western blot. At last we purify the protein through NI-NTA magnectic agarose beads.Result:We amplify the coding sequence of SPAG4L by the means of RT-PCR and the fragment inserted is about 253bp. The product dealed with restriction enzyme then subclone to expressing vector PQE30. The recominated vector PQE30-SPAG4L been transformed to Bacterium coli JM109 and then successfully expressed out the fusion protein. The fusion protein been purifyied by means of NI-NTA magnectic agarose beads and then confirmed by means of Western Blot.Conclusion:We successfully constructed the prokaryotic expressing vector of SPAG4L and get the fusion protein. Then the purified product which been confirmed by Western Blot can be used into next research. Chapter Three:The subcellular localization of SPAG4L and the expression features in meiosisObjective:Try to clear the accurate subcellular localization of SPAG4L and confirm the key region which decide the correct position. We observed the changing feature of spag4l, the mouse homologous of human SPAG4L, in the development of mouse meiosis and speculate the meaning of it’s function.Methods:The accurate subcellular localization of SPAG4L was confirmed by immunofluorescence. To clear the key region which decide the subcellar position we construct a few mutants and infected the constructed EGFP-N1-SPAG4L vectors to Hela cells. We observed the changing features of spag4l, the mouse homologous, in mouse sperm meiosis.RESULT:SPAG4L localized in nuclear envelope and endoplasmic reticulum. The transmember and coiled-coil region but not the SUN region in it’s C-terminal are the necessary region for it’s accurate position. SPAG4L may involved in the migration and position of nuclear envelope and endoplasmic reticulum in the process of meiosis, indicating that it’s importance in spermatogenesis.Conclusion:SPAG4L is a protein of nuclear envelope and endoplasmic reticulum. The transmember and coiled-coil region is the key region for the accurate position. SPAG4L may be a new breakout for us next research of male infernity.更多还原