Genetic Regulation Of Spermatogenesis

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的扩张和分化。生殖细胞在精子发生的不同阶段具有不同的形态和生物学特性，因此可以制备相对纯净的生殖细胞。已有动物模型可用于研究精原细胞分化的停止和重新启动。这使得精子发生成为研究干细胞和总体上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控精子发生的基因网络。为了实现这一目标，我们利用基因芯片技术研究了小鼠A型精原细胞(Sg)、粗线期精母细胞(SC)和圆形精细胞(SD)中基因表达的整体变化。使用了两种类型的微阵列，即含有5,184个小鼠基因(ResGen)的尼龙膜小鼠基因过滤器和印有NIA 15K小鼠克隆集的玻片微阵列。在GeneFilters的实验中，在三种类型的生殖细胞中鉴定出79个差异表达基因和EST。用实时定量聚合酶链式反应(Real-time PCR)证实了其中一些基因的差异表达。在使用玻片芯片的实验中，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)是未确定特征的基因。有较多基因(110/161)在SD中优先表达。功能分类表明，参与信号转导、能量代谢、生物合成和细胞转运的基因在SD中优先表达，而染色质重塑基因仅在SC中表达。一些睾丸特异性基因(女性化1b同源基因、类磷脂酰胆碱转移蛋白基因、精子特异性抗原1基因、与酪蛋白激酶基因相似的基因)在SD中优先表达。通过实时定量聚合酶链式反应和基因表达系列分析(SAGE)验证这些基因的差异表达。 使用I-SAGE试剂盒(Invitgen公司)对SC和SD进行SAGE。分别对SC和SD文库的101,068和106,212个标签进行了测序。不包括独生子女，这些基因分别代表10,717和10,135个基因。在SC文库中，有4个标签的含量超过0.5%。这些标签与线粒体序列匹配(0.65%)，T-复合体相关睾丸表达3(0.57%)，Y盒蛋白2(0.50%)。第三个丰富的标签(0.56%)在SAGE图谱数据库中有多次匹配。在SD文库中，有4个标签出现在0.5%以上。最丰富的标签与鱼精蛋白2匹配(1.31%)，其次是匹配的FK506结合蛋白(1.18%)，以及与线粒体序列匹配的标签(0.64%)。第四个丰富标签(0.56%)有多次命中。两个文库的虚拟减法得到4,344个SC特有标签和4,155个SD特有标签。大多数这些细胞阶段特异性标签的拷贝数少于5个。有353个SC特异性标签存在于5个拷贝以上，其中只有38个存在于10个拷贝以上。特定于SD的标签的相应数字为266和27。最丰富的SC特异性标签匹配Janus kinas3(43个标签，占文库的0.040%)，其次是匹配的WW结构域结合蛋白4(37个标签，0.034%)和dynein(28个标签，0.026%)。在3个最丰富的SD特异标签中，CAGAAGGCGG和TATTAAAGCT都是新的，均为18个拷贝(0.017%)，在SAGE图谱数据库中没有命中。另一个标签也有18个拷贝，与RIKEN的cDNA匹配。 将基因芯片杂交结果与SAGE结果进行比较，结果显示两种方法具有较高的一致性(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。这也是第一次详细比较小鼠生殖细胞中的基因表达模式。这些结果为研究精子发生的遗传调控以及病理条件下精子发生过程的异常奠定了基础。

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.