Genetic Regulation Of Spermatogenesis

精子发生的遗传调控

• 批准号: 6664182
• 负责人: Owen M Rennert
• 金额: --
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6664182
• 关键词: developmental genetics; genetic regulation; genetic transcription; laboratory mouse; microarray technology; molecular biology information system; serial analysis of gene expression; sperm; spermatogenesis

Spermatogenesis is a tightly regulated process characterized by spermatogonial stem cells undergoing mitotic expansion and differentiation. Distinct morphological and biological characteristics of germ cells at different stage of spermatogenesis allow preparation of these cells in relatively pure form. Animal models are available which permit study of arrest and restart of spermatogonial differentiation. This makes spermatogenesis a unique model for studying stem cells and the genetic factors that regulate cellular proliferation and differentiation in general. One of the goals of this research is to delineate the network of genes that regulate spermatogenesis. To achieve this goal, we study the global changes in gene expression in type A spermatogonia (Sg), pachytene spermatocytes (Sc) and round spermatids (Sd) of the mouse using cDNA microarrays. Two types of microarrays were employed, namely the Nylon-membrane based Mouse GeneFilters that contain 5,184 mouse genes (ResGen) and the glass-slide microarrays that were printed with the NIA 15K mouse cDNA clone set. In the experiment with GeneFilters, 79 differentially expressed genes and ESTs were identified among the three types of germ cells. Quantitative Real-Time PCR was used to confirm the differential expression of a number of these genes. In the experiment using glass-slide microarrays, we focused on studying the changes in gene expression in the transition of Sc to Sd. A total of 161 differentially expressed genes were identified. More than one-fourth (43/161) of these were uncharacterized genes. A larger number of genes (110/161) were found to be preferentially expressed in Sd. Functional categorization indicated that genes responsible for signal transduction, energy metabolism, biosynthesis and cellular transport were preferentially expressed in Sd, while genes for chromatin remodeling were expressed only in Sc. Several testis-specific genes (feminization 1b homolog; phosphatidylcholine transfer protein-like; sperm specific antigen 1; cDNA moderately similar to casein kinase) were found to be expressed preferentially in Sd. Confirmation of differential expression of these genes was achieved by both Quantitative Real-Time PCR and Serial Analysis of Gene Expression (SAGE). SAGE was performed on Sc and Sd using the I-SAGE kit (Invitrogen Corp). 101,068 and 106,212 tags of the Sc and Sd library respectively were sequenced. Excluding singletons, these represented 10,717 and 10,135 genes respectively. In the Sc library, 4 tags were present at more than 0.5%. These tags matched a mitochondrial sequence (0.65%), t-complex-associated testis expressed 3 (0.57%), and Y box protein 2 (0.50%). The third abundant tag (0.56%) had multiple hits in the SAGEmap database. In the Sd library, 4 tags were present at more than 0.5%. The most abundant tag matched protamine 2 (1.31%), followed by that matching FK506 binding protein (1.18%), and a tag that matched a mitochondrial sequence (0.64%). The fourth abundant tag (0.56%) had multiple hits. Virtual subtraction of the two libraries yielded 4,344 Sc-specific tags and 4,155 Sd-specific tags. The majority of these cell stage specific tags were present at less than 5 copies. 353 Sc-specific tags were present at more than 5 copies and only 38 of these were present at more than 10 copies. The corresponding figures for Sd-specific tags were 266 and 27. The most abundant Sc-specific tag matched Janus kinase 3 (43 tags, 0.040% of library), followed by that matching WW domain binding protein 4 (37 tags, 0.034%) and dynein (28 tags, 0.026%). Two of the 3 most abundant Sd-specific tags, CAGAAGGCGG and TATTAAAGCT, both at 18 copies (0.017%) were novel with no hit in the SAGEmap database. The other tag also present at 18 copies matched a RIKEN cDNA. Comparison of the results obtained by cDNA microarray hybridization and SAGE indicated a high degree of concordance (80%) between the two methods. Discordance was limited only to genes of low expression level. Our work succeeded in identifying a large number of genes previously unknown to be expressed in germ cells (characterized genes + ESTs more than 12,270) as well as novel genes. It is also the first in its kind to compare in detail the gene expression pattern in mouse germ cells. Results obtained provide the foundation for investigation of genetic regulation of spermatogenesis as well as abnormalities of such process in pathological conditions.

精子发生是一个严格调节的过程，其特征是精子干细胞经历有丝分裂膨胀和分化。生物发生不同阶段生殖细胞的独特形态和生物学特征允许以相对纯净的形式制备这些细胞。可以使用动物模型，可以研究逮捕并重新开始精子分化。这使得精子发生成为研究干细胞以及调节细胞增殖和一般分化的遗传因素的独特模型。 这项研究的目标之一是描述调节精子​​发生的基因网络。为了实现这一目标，我们使用cDNA微阵列研究了A型精子（SG），Pachytene精子细胞（SC）和圆形精子（SD）中基因表达的全局变化。采用了两种类型的微阵列，即包含5,184个小鼠基因（RESGEN）的基于尼龙膜的小鼠基因词，以及用NIA 15k小鼠cDNA克隆集打印的玻璃丝滑层微阵列。在与基因词的实验中，在三种类型的生殖细胞中鉴定了79个差异表达的基因和EST。定量实时PCR用于确认许多这些基因的差异表达。在使用玻璃扫描微阵列的实验中，我们专注于研究SC向SD过渡中基因表达的变化。总共确定了161个差异表达的基因。其中超过四分之一（43/161）是未表征的基因。发现更多基因（110/161）在SD中优先表达。功能分类表明，负责信号转导，能量代谢，生物合成和细胞转运的基因优先在SD中表达，而染色质重塑的基因仅在SC中表达。发现几种睾丸特异性基因（女性化1B同源物；磷脂酰胆碱转移蛋白样；特异性抗原1； cDNA中适度相似的酪蛋白激酶）在SD中优先表达。通过定量实时PCR和基因表达（SAGE）的序列分析来确认这些基因的差异表达。 使用I-SAGE试剂盒（Invitrogen Corp）在SC和SD上进行SAGE。 SC和SD库的101,068和106,212标签被测序。除了单例外，这些分别代表10,717和10,135个基因。在SC库中，有4个标签以0.5％以上。这些标签匹配线粒体序列（0.65％），T-复合物相关的睾丸表达3（0.57％）和Y盒蛋白2（0.50％）。第三个丰富的标签（0.56％）在SAGEMAP数据库中具有多个命中。在SD库中，有4个标签以0.5％以上。最丰富的标签匹配了精蛋白2（1.31％），其次是匹配的FK506结合蛋白（1.18％），而与线粒体序列（0.64％）相匹配的标签。第四大标签（0.56％）具有多次命中。两个库的虚拟减法产生了4,344个SC特异性标签和4,155个SD特异性标签。这些细胞阶段特异性标签中的大多数都以少于5份的形式存在。 353个SC特异性标签以5个以上的副本存在，其中只有38个以上的副本存在。 SD特异性标签的相应数字为266和27。最丰富的SC特异性标签匹配Janus激酶3（43个标签，库的0.040％），其次是匹配的WW域结合蛋白4（37个标签，0.034％）和Dynein（28 tags，0.026％）。在3个最丰富的SD特异性标签中，有两个Cagaaggcgg和Tattaaagct，均为18份（0.017％），在SageMap数据库中没有命中。另一个标签也存在于18份，与Riken cDNA相匹配。 通过cDNA微阵列杂交和鼠尾草获得的结果比较表明两种方法之间具有高度的一致性（80％）。不一致仅限于低表达水平的基因。我们的工作成功地鉴定了许多以前未知的基因（在生殖细胞中表达） 12,270）以及新基因。它也是第一个详细比较小鼠生殖细胞中基因表达模式的人。获得的结果为研究精子发生的遗传调节以及在病理条件下这种过程的异常提供了基础。