Gene expression profile in juvenile Spo11 knockout testes

Spo11, a meiosis-specific protein, introduces double strand breaks on chromosomes and initiates meiotic recombination in a wide variety of organisms. Mouse null Spo11 spermatocytes fail to synapse chromosomes and to progress beyond zygotene stage of meiosis. We analyzed gene expression profiles in Spo11-/- adult and juvenile wild type testis to determine genes expressed in different stages of meiosis and spermatogenesis. These genes were characterized using Gene Ontology. To focus on genes involved in meiosis we performed comparative gene expression analysis of Spo11 -/- and wild type testes from 12 and 15 day mice. We found that the knockout of Spo11 gene causes dramatic changes in the level of expression of genes that participate in meiotic recombination (Hop2, Brca2, Mnd1, FancG) and in the meiotic checkpoint (cyclin B2, Cks2) at 15 day, but does not affect genes encoding protein components of the synaptonemal complex. Finally, we uncovered unknown genes that are affected by disruption of the Spo11gene and therefore, may be specifically involved in meiosis and spermatogenesis. Keywords: repeat sample Microarray experiments were performed in quadruplicate with different mouse pairs from the same mouse litter. The chips were scanned using the GenePix 4000A scanner (Axon Instruments) and primary data were analyzed using the Genepix 3.0 software. Primary data were flagged using four default parameters set in the Genepix 3.0 program. For further analysis, the data were imported into Excel (Microsoft) and normalized by the Median Centering Method. We performed the statistical analysis using a modified t-test implemented in SAM software (Tusher et al., 2001). We defined differentially expressed genes at a 1% false discovery rate confidence level and a cutoff for differential expression equal to 1.5 for juvenile Spo11-/- microarray experimentsSpo11作为一种减数分裂特异性蛋白，可在多条染色体上引入双链断裂，并在众多生物体内启动减数分裂重组。敲除Spo11基因的小鼠精母细胞无法完成染色体联会，且无法越过减数分裂的合子期继续发育。本研究通过分析Spo11基因敲除（Spo11-/-）成年与幼年野生型小鼠的睾丸基因表达谱，以鉴定在减数分裂与精子发生不同阶段特异性表达的基因。我们通过基因本体论（Gene Ontology）对这些基因进行了功能注释。为聚焦于减数分裂相关基因，我们对12日龄与15日龄的Spo11-/-及野生型小鼠睾丸开展了对比基因表达分析。研究发现，在15日龄小鼠中，Spo11基因敲除会显著改变参与减数分裂重组的基因（Hop2、Brca2、Mnd1、FancG）以及减数分裂检验点相关基因（cyclin B2、Cks2）的表达水平，但不会影响联会复合体蛋白编码基因的表达。最后，我们还发现了一批受Spo11基因敲除影响的未知基因，这类基因可能特异性参与减数分裂与精子发生过程。关键词：重复样本 本微阵列实验采用同窝不同小鼠配对样本，共设置四次生物学重复。芯片扫描使用GenePix 4000A扫描仪（Axon Instruments公司）完成，原始数据通过Genepix 3.0软件进行分析。原始数据通过Genepix 3.0软件内置的四项默认参数进行标记筛选。后续分析阶段，我们将数据导入Excel（Microsoft公司），并通过中位数中心化法完成数据标准化。统计分析采用SAM软件中实现的修正t检验（Tusher等人，2001年）。针对幼年Spo11-/-小鼠的微阵列实验，我们以1%的错误发现率置信水平以及1.5倍表达差异阈值作为筛选标准，定义差异表达基因。