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控的过程，其特征是精原干细胞经历有丝分裂扩张和分化。精子发生不同阶段的生殖细胞具有独特的形态和生物学特征，因此可以以相对纯净的形式制备这些细胞。动物模型可用于研究精原细胞分化的停滞和重启。这使得精子发生成为研究干细胞和调节细胞增殖和分化的遗传因素的独特模型。 这项研究的目标之一是描绘调节精子发生的基因网络。为了实现这一目标，我们使用 cDNA 微阵列研究了小鼠 A 型精原细胞 (Sg)、粗线期精母细胞 (Sc) 和圆形精子细胞 (Sd) 基因表达的整体变化。采用两种类型的微阵列，即包含 5,184 个小鼠基因 (ResGen) 的基于尼龙膜的小鼠基因过滤器和印有 NIA 15K 小鼠 cDNA 克隆集的载玻片微阵列。在GeneFilters的实验中，在三种类型的生殖细胞中鉴定出了79个差异表达基因和EST。使用定量实时 PCR 来确认其中许多基因的差异表达。在使用载玻片微阵列的实验中，我们重点研究了Sc向Sd转变过程中基因表达的变化。共鉴定出161个差异表达基因。其中超过四分之一 (43/161) 是未表征的基因。发现大量基因 (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、生物合成和细胞运输的基因优先在Sd中表达，而负责染色质重塑的基因仅在Sc中表达。发现几个睾丸特异性基因（女性化 1b 同源物；磷脂酰胆碱转移蛋白样；精子特异性抗原 1；与酪蛋白激酶适度相似的 cDNA）在 Sd 中优先表达。通过定量实时 PCR 和基因表达系列分析 (SAGE) 确认了这些基因的差异表达。 使用 I-SAGE 试剂盒（Invitrogen Corp）对 Sc 和 Sd 进行 SAGE。 Sc和Sd文库的101,068和106,212个标签分别被测序。排除单例基因，这些分别代表 10,717 和 10,135 个基因。在Sc文库中，有4个标签的存在率超过0.5%。这些标签与线粒体序列 (0.65%)、t 复合物相关睾丸表达 3 (0.57%) 和 Y 盒蛋白 2 (0.50%) 相匹配。第三个丰富的标签 (0.56%) 在 SAGEmap 数据库中有多次命中。在Sd文库中，有4个标签的存在量超过0.5%。最丰富的标签与鱼精蛋白 2 匹配 (1.31%)，其次是与 FK506 结合蛋白匹配的标签 (1.18%)，以及与线粒体序列匹配的标签 (0.64%)。第四个丰富的标签（0.56%）有多次点击。两个文库的虚拟扣除产生了 4,344 个 Sc 特异性标签和 4,155 个 Sd 特异性标签。大多数这些细胞阶段特异性标签的拷贝数少于 5 个。 353 个 Sc 特异性标签存在超过 5 个拷贝，其中只有 38 个存在超过 10 个拷贝。 Sd 特异性标签的相应数字为 266 和 27。最丰富的 Sc 特异性标签与 Janus 激酶 3 匹配（43 个标签，占文库的 0.040%），其次是与 WW 结构域结合蛋白 4 匹配的（37 个标签，0.034%）和动力蛋白（28 个标签，0.026%）。 3 个最丰富的 Sd 特异性标签中的两个 CAGAAGGCGG 和 TATTAAAGCT 均为 18 个拷贝 (0.017%)，都是新颖的，在 SAGEmap 数据库中没有命中。另一个标签也有 18 个拷贝，与 RIKEN cDNA 相匹配。 cDNA 微阵列杂交和 SAGE 获得的结果的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大量以前未知的在生殖细胞中表达的基因（特征基因+ESTs超过 12,270）以及新基因。这也是同类中第一个详细比较小鼠生殖细胞中基因表达模式的研究。获得的结果为研究精子发生的遗传调控以及病理条件下该过程的异常奠定了基础。