【作者】 周作民；

【导师】 周开亚； 王义权；

【作者基本信息】 南京师范大学 ， 动物学， 2003， 博士

【摘要】 人睾丸发育／精于发生相关基因的研究 人睾丸是一个特殊的器官，其功能是产生精子和分泌雄激素。精子产生是一个复杂的过程，涉及到由精原细胞转化为初级精母细胞的有丝分裂过程，又经历了从初级精母细胞形成精子细胞的减数分裂过程，最后精子细胞经过变态形成精子。由于减数分裂是生殖细胞特有的一种细胞分裂方式，睾丸特异表达的基因中相当一部分与减数分裂有关，因此睾丸的基因表达必然不同于体内的其它脏器。此外，睾丸的功能状态有明显的阶段特异性，因此睾丸在不同发育和功能阶段的基因表达也必然不同，筛选并研究这些差异表达的基因有望发现与睾丸发育／精子发生相关的新基因，或发现已知基因的新功能，研究结果对于揭示人睾丸发育／精子发生的分子机制、建立男性不育在分子水平的诊断方法、发现精子发生障碍的基因治疗手段和寻找男性避孕的新方法均具有重要意义。关于差异表达基因的筛选，有多种方法，本实验室应用基因表达芯片方法，比较胚胎和成年人睾丸组织的基因表达差异，获得了1522个差异表达克隆，其中独立基因499个，在此基础上，本研究选择其中的三个基因(Rtn-T、NYD-TSP1和NYD-SP6)进行了功能研究。 第一章 Rtn-T基因的研究 Reticulon(Rtn)是一个基因家族，分布于细胞的内质网上，该家族成员的共同特征是表达产物在C末端均含有一个200个氨基酸组成的保守序列，在人、牛、大鼠、小鼠、果蝇和蠕虫等物种均已发现了Rtn的家族成员，研究发现Rtn蛋白与轴突生长和凋亡等功能有关，但Rtn蛋白与睾丸功能的关系尚未见报道。本研究在筛选获得差异表达克隆的基础上，对差异克隆进行测序和同源序列的比较，发现了一个新基因，为Rtn家族成员，由于其在睾丸特异表达，故命名为Rtn-T(Reticulon-Testis),Rtn-T在成年和胚胎睾丸的杂交强度分别为180.64和50.15。序列测定显示Rtn-T cDNA全长为3419bp，编码蛋白为108KDa，含986个氨基酸，该基因已被Genbank收录，收录号为AF333336。基因结构分析显示，Rtn-T由9个内元组成，定位于2p16，软件分析显示在N-末端含有一个由26个氨基酸组成的信号肽，在C-末端含有两个疏水区，分别由37和 南京师范大学博士研究生毕业论文35个氨基酸组成。在Genb加中进行Blast搜索，发现Ru1与6个基因高度同源，这些基因均属于Ru家族成员。脏器表达特异性研究显示，Rtn下仅在睾丸内特异表达，其它重要脏器均未见表达。研究结果提示Rtn下可能与睾丸发育，特别是精子发生有关，但Rtn1是否通过抑制生精细胞的凋亡来促进精子的发生尚有待进一步的研究。第二章NYD－TSPI基因的研究 精子发生是一个多基因表达的复杂过程，这一过程受到多种转录调控因子的影响，在一些转录调控基因的表达产物中常带有特殊的功能域，basic helix－loophelix（bHLH）就是其中的一个。本研究用基因芯片方法比较人胚胎睾丸和成年睾丸的基因表达差异，也发现了一个在成年睾丸高表达的带有 bHLH功能域的蛋白，命名为 NYD丁（NYD4estis specificprotein）；NY’D1 为成年高表达，胚胎低表达，信号强度分别为 134．27和 22．53。N’YD丁 CDNA含 1848个核旮酸，编码蛋白含430个氨基酸，编码蛋白的分子量为49．skDa。该基因已被Genbank收录，登录号为AF333098，并已被GenBank确定为Unigene，号码为Hs．98266。查阅人类基因组序列，显示NYD丁PI定位于5…3．3。软件分析NYD1PI的氨基酸序列，显示NYD1PI编码蛋白含有两个功能域，一个是位于付末端的 basic helix－loonhelix（bHLH），另一个是位于 C一末端的 Zip。同源序列比较显示，TSPI与小鼠的SpZI同源，同源性为58％，SpZI同样带有bHLH和Zip功能域，两个基因在这两个功能域的同源性分别为59．9％和97．3％，SPZI也为小鼠睾丸表达的基因，提示两个基因有相同的功能。PCR分析脏器表达特异性显示，TSP 仅在人睾丸呈高表达。由于bHLH呕ip蛋白常为转录调控因子，具有促进细胞增殖和分化的作用，有些还是 Oncogene，提示 NYD1SP也可能是一个转录调控因子，由于N－YD1S＊在成年睾丸呈特异性高表达，因此 NYD1SN很可能与睾丸的功能，特别是精子发生有关。第三章 NYD－SP6基因的研究 睾丸内的Sertoli细胞对精子的发生具有重要的调控作用，但是Sertoli细胞调控精子发生的分子机制尚不完全清楚。本研究用人睾丸CDNA芯片杂交比较胚胎和成年人睾丸基因表达差异，获得了一个在支持细胞内特异表达的基因，命名为N’YDSP6基因，该基因在成年和胚胎睾 2 南京师范大学博士研究生毕业论文丸的杂交信号强度分别为2126．37和58．74。本研究同时提取胚胎和成年小鼠睾丸组织的RNA，分别与人睾丸CDNA芯片杂交，结果显示NYDsP6基因在胚胎小鼠和成年小鼠睾丸的杂交信号强度与用胚胎和成年人睾丸杂交的结果基本一致。序歹’1测定显示，NYDEP6 cDNA全长为 1858 hp，编码更多还原

【Abstract】 Human testis is a specific organ. There are two fundamental functions in human tetstis: androgen secretion and spermatogenesis. Spermatogenesis is a complex and very efficient process that begins with the division and differentiation of the spermatogenic stem cell within the seminiferous tubule of the testis. Because meiosis is different from mitosis, the expression of genes is different between testis and other organs. Also, spermatogenesis begins in puberty, so the expression of genes is different in different stages, for example in adult and fetal testis. Study of genes specifically expressed in the testis at different stages of development may reveal new genes related to the function of testis, especially spermatogenesis or find new function for reported genes. In our laboratory, human testis cDNA microarrays were used to identify the genes specifically expressed in adult or fetal testes and 1522 differentially expressed clones were found. Among these clones, 499 were unique genes. In this study, three of these genes (Rtn-T, NYD-TSP1 and NYD-SP6) were selected for functional study. Part 1. Study on Rtn-T geneReticulon (Rtn) is a gene family. All Rtn proteins show a 200-amino-acid residue region of sequence similarity at the C terminus of the protein. Related sequences have been recognized in human, bovine, mouse, rat, fly and worm genomes. It has been reported that Rtn is related to axon extention and apoptosis. The relationship between Rtn and testis function has not been reported. In this study, by using human testis cDNA microarray, a new Rtn like gene was found. It was named as Rtn-T for its specific expression in testis. The hybridization intensity of Rtn-T in adult and fetal testes was 180.64 and 50.15, respectively. The full length cDNA of Rtn-T was 3491 bp and encodes a 108 kDa protein of 986 amino acids. This Rtn-T has Genbank accession number AF333336. Blast search in the human genome datebase showed that Rtn-T consisted of 9 exons. Blast search of the contig map showed that Rtn-T was mapped to chromosome 2pl6. Analysis of the amino acid sequence revealed one signal sequence of 26 amino acids at theN-terminal and two long hydrophobic stretches of 37 and 35 amino acids at the C-terminal. Blast searches revealed that Rtn-T was highly homologous to six other genes. All of them belong to the Rtn family and come from humans. PCR showed that Rtn-T was particularly and strongly expressed in human testis. These results indicate that Rtn-T may be related to testis development, especially process of spermatogenesis. Whether Rtn-T influence apoptosis, and thus affects the process of spermatogenesis should be determined. Part 2. Study on NYD-TSP1Spermatogenensis involves the expression of many genes. The expression of these genes is promoted by testis-expressed transcription factors at the transcription level. One of the major classes of growth and development regulatory proteins consists of the basic helix-loop-helix (bHLH) motif-containing proteins. In this study, by using human testis cDNA microarray, a novel human testis specific gene, NYD-TSP1, was identified. NYD-TSP1 encodes a putative bHLH-Zip containing protein and is highly and specifically expressed in adult human testis. The level of hybridization intensity of NYD-TSP1 in adult and fetal testis was 134.27 and 22.53 respectively. The full length of NYD-TSP1 cDNA was 1848 bp and encoded a 49.4 KDa protein of 430 amino acids. The cDNA sequence of this clone was deposited with Genbank. The accession number was AF333098. NYD-TSP1 was also determined as Interim Gen Symbol (Unigene). The name is NYD-TSP1: testis-specific protein NYD-TSP1. UniGene number is Hs.98266. Predicted protein analysis showed that NYD-TSP1 contained two functional dormains: an N-terminal basic helix-loop-helix (bHLH) and a C-terminal Zip. Homologous analysis showed that the 430 amino acid sequences had sequence homology with Spz 1 protein of mouse. The positive rate of homology for full sequences was 58%. In bHLH and Zip motif the positive rate of homology was 59.更多还原