Genetic Regulation Of Spermatogenesis

精子发生的遗传调控

• 批准号: 6813935
• 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/6813935
• 关键词: cell differentiation; cell growth regulation; cell proliferation; cytogenetics; developmental genetics; genetic library; genetic regulation; laboratory mouse; microarray technology; serial analysis of gene expression; sperm; spermatogenesis

Spermatogenesis is a tightly regulated process characterized by spermatogonial stem cells undergoing mitotic expansion, meiosis and postmeiotic differentiation. Distinct morphological and biological characteristics of germ cells at different stages 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. The transcriptome of mouse type A spermatogonia (Spga), pachytene spermatocytes (Spcy), and round spermatids (Sptd) purified by the STAPUT technique were determined by sequencing of the respective SAGE (Serial Analysis of Gene Expression) libraries. A total of 444,015 SAGE tags derived from one Spga, two Spcy, and one Sptd libraries were analyzed. A total of 34,619 different species of transcripts were identified among these SAGE tags. Among these transcripts, 42.6% were derived from known genes, 42.1% were uncharacterized cDNAs and 15.3% were not described previously. Analysis of the spermatocyte transcriptome indicates the size of germ cell transcriptome could comprise of more than 30,000 transcripts, different from what was observed for other cells (Zhang et al., Science, 276[1997]1268). For each type of germ cells analyzed, 8-14 % of the transcriptomes are novel transcripts, 44-46% are uncharacterized cDNAs and 42-46% are transcripts of known genes. 12-19.5% of the transcriptomes consist of cell-specific transcripts while approximately 6,000 transcripts are shared by the three types of cells. It is worth noting that the most abundant tag matched with three UniGene clusters, the second most abundant tag represented an uncharacterized cDNA, while the third most abundant tag had no match in the SAGEmap database. Differential expression between the SAGE libraries was validated by quantitative realtime polymerase chain reaction (QPCR) of randomly selected transcripts. Components of the protein biosynthetic machinery are highly expressed in Spga. In Spcy, transcription factors are abundantly expressed while chromosome remodeling genes and testis-specific genes are prominent in Sptd. Comparative analysis of SAGE and QPCR data were used to reveal the presence of alternatively spliced variants. The present study represents the first global study of gene expression in mouse germ cells. Based on this study it is evident that a core set of genes may be required for basic biological activities in all germ cells. However, cell-specific stages of spermatogenesis (mitotic, meiotic, and post-meiotic) require the concerted action of distinct sets of genes. It has been reported that aberrant expression of some X-linked genes would cause failure in spermatogenesis as well as improper sexual development. A recent report also indicated that spermatogonia harbor a substantial number of X-linked genes related to spermatogenesis. We have identified a number of X-linked genes that escape transcription inactivation at meiosis and in the post-meiotic stage in the SAGE study. In order to have a better understanding of the regulation of expression of X-linked genes, we clustered the expression of all X-linked genes. Two of such genes, the Testis expressed gene 13 (Tex13) and Sex comb on midleg-like 2 (Scml2), were found to express predominantly in type A spermatogonia. In silico studies indicated the existence of a potential antisense transcript of Tex13, which is complementary to the 3!| end of the Tex13 sense transcript. Reverse transcription-polymerase chain reaction (RT-PCR) studies revealed that the trend of change in the expression of the sense and antisense transcripts were similar, but the relative expression levels of the transcripts in different stages of germ cells were dissimilar. The regulation of expression, biological activities, as well as the relationship between the sense and antisense transcripts of Tex13 are currently under study. Scml2 is a member of the Polycomb group genes, which encode transcriptional repressors essential to appropriate development in the fly and in mammals. At least 3 different Scml2 transcripts were found in mouse testes, with each of them having a different 5!| untranslated region. Alternative use of exons in the coding region was also observed. By RT-PCR analyses, we found that the various forms of Scml2 transcripts were expressed at different levels during spermatogenesis, implying preferential use of the transcripts during germ cell development. In vivo as well as in vitro gene knockdown experiments are planned to study the functional roles of these X-linked genes during spermatogenesis. A number of novel as well as uncharacterized cDNAs identified to be differentially expressed in germ cells by SAGE as well cDNA microarray experiements performed previously were cloned and characterized. A novel ~1.1kb transcript shown to be preferentially expressed in round spermatids was cloned by cDNA library screening and 5!| RACE. Another spermatid-specific EST identified by cDNA microarray experiment was also cloned. SAGE analyses of germ cell transcriptomes identified a number of novel transcripts. The more abundant novel transcripts are being cloned using RT-PCR with extended primers. Characterization and developmental studies of these cDNAs are expected to yield information of their roles in spermatogenesis.

精子发生是一个严格调节的过程，其特征是经过有丝分裂扩张，减数分裂和症状后分化的精子干细胞。生物发生不同阶段生殖细胞的独特形态和生物学特征使这些细胞相对纯净的形式制备。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。 通过Staput技术纯化的小鼠A型精子（SPGA），Pachytene精子细胞（SPCY）和圆形精子（SPTD）的转录组，通过测序相应的SAGE（基因表达的串行分析）库来确定。分析了一个来自一个SPGA，两个SPCY和一个SPTD库的444,015个鼠尾草标签。在这些鼠尾草标签中，总共确定了34,619种不同种类的转录本。在这些转录本中，有42.6％来自已知的基因，42.1％是未表征的cDNA，而15.3％则没有描述。对精子细胞转录组的分析表明，生殖细胞转录组的大小可能包括30,000多个转录物，与其他细胞观察到的不同（Zhang等，Science，276 [1997] 1268）。对于分析的每种类型的生殖细胞，有8-14％的转录组是新的转录本，44-46％是未表征的CDNA，而42-46％是已知基因的转录本。 12-19.5％的转录组由细胞特异性转录本组成，而三种类型的细胞共享了大约6,000个转录本。值得注意的是，最丰富的标签与三个Unigene群集匹配，第二大最丰富的标签代表一个未表征的cDNA，而第三大最丰富的标签在SageMap数据库中没有匹配。鼠尾草文库之间的差异表达通过随机选择的转录本的定量实时聚合酶链反应（QPCR）验证。蛋白质生物合成机械的成分在SPGA中高度表达。在SPCY中，转录因子大量表达，而染色体重塑基因和睾丸特异性基因在SPTD中很明显。 SAGE和QPCR数据的比较分析用于揭示存在剪接变体的存在。本研究代表了小鼠生殖细胞中基因表达的首次全球研究。基于这项研究，很明显，在所有生殖细胞中的基本生物学活性可能需要一组核心基因。然而，精子发生的细胞特异性阶段（有丝分裂，减数分裂和脂点后）需要一组不同基因的协同作用。 据报道，某些X连锁基因的异常表达会导致精子发生以及性发育不当。最近的一份报告还表明，精子含有大量与精子发生有关的X连锁基因。我们已经确定了许多X连锁基因，这些基因在Sage研究中逃脱了减数分裂的转录灭活和杀伤力后阶段。为了更好地了解X连锁基因表达的调节，我们聚集了所有X连锁基因的表达。其中两个基因，即睾丸表达的基因13（TEX13）和类似Midleg样的2（SCML2）的性梳子，主要在A型精子中表达。在计算机研究中表明，存在Tex13的潜在反义转录本，这是3！|的补充！ Tex13 Sense成绩单的结尾。逆转录 - 聚合酶链反应（RT-PCR）研究表明，意义和反义转录本表达的变化趋势是相似的，但是在生殖细胞不同阶段，转录本的相对表达水平不同。目前正在研究Tex13的表达，生物学活性以及意义和反义转录本之间的关系。 SCML2是Polycomb基因的成员，该基因编码转录阻遏物对于果蝇和哺乳动物中适当发育所必需的转录阻遏物。在鼠标睾丸中发现了至少3个不同的SCML2转录本，每个睾丸具有不同的5！未翻译区域。还观察到在编码区域中外显子的替代用途。通过RT-PCR分析，我们发现在精子发生过程中以不同的水平表达了各种形式的SCML2转录本，这意味着在生殖细胞发育过程中优先使用转录本。计划在体内以及体外基因敲低实验中研究这些X连锁基因在精子发生过程中的功能作用。 并表征了许多新颖的和未表征的cDNA在生殖细胞中差异表达的cDNA以及先前执行的cDNA微阵列体验。 cDNA库筛选和5！种族。还克隆了另一个通过cDNA微阵列实验鉴定的精子特异性EST。生殖细胞转录组的鼠尾草分析确定了许多新的转录本。使用带有扩展引物的RT-PCR克隆了更丰富的新型转录本。这些CDNA的表征和发育研究有望产生其在精子发生中的作用信息。