Microarray Analyses of Newborn Mouse Ovaries Lacking Nobox

Nobox is a homeobox gene expressed in oocytes and critical in oogenesis. Nobox deficiency leads to rapid loss of postnatal oocytes. Early oocyte differentiation is poorly understood. We hypothesized that lack of Nobox perturbs global expression of genes preferentially expressed in oocytes as well as microRNAs. We compared Nobox knockout and wild type ovaries using Affymetrix 430 2.0 microarray platform. We discovered that 28 out of 38 (74%) of the genes down-regulated more than five fold in the absence of Nobox were preferentially expressed in oocytes, while only 5 out of 33 (15%) of genes up-regulated more than five fold in the absence of Nobox, were preferentially expressed in oocytes. Protein binding microarray helped identify nucleotide motifs that NOBOX binds, and that several down-regulated genes contain within putative promoter regions. MicroRNA population in newborn ovaries deficient of Nobox, was largely unaffected. Genes whose proteins are predicted to be secreted, but previously unknown to be significantly expressed in early oogenesis, were down regulated in Nobox knockouts and included astacin-like metalloendopeptidase (Astl), Jagged 1 (Jag1), oocyte secreted protein 1 (Oosp1), fetuin beta (Fetub) and R-spondin 2 (Rspo2). In addition, pluripotency associated genes, Pou5f1 and Sall4 are drastically down-regulated in Nobox deficient ovaries, while testes determining gene Dmrt1 is over-expressed. Our findings indicate that Nobox is likely an activator of oocyte-specific gene expression, and suggest that oocyte plays an important role in suppressing expression of male determining genes such as Dmrt1. Keywords: Genetic Modification Newborn ovaries were pooled separately from wild type and Nobox -/- animals and total RNA isolated using RNeasy mini kit (Qiagen, CA). Newborn ovaries were collected within 12 hours of delivery. Animal experimentation was approved by the Institutional Animal Care and Use Committee of Baylor College of Medicine. Three independently pooled RNA samples from wild type and Nobox -/- newborn ovaries were used to generate biotinylated cRNA. Biotinylated cRNA was hybridized to GeneChip Mouse Expression Set 430 2.0 (Affymetrix, Inc.). Since three independent experiments were performed from three independent pools of wild type and Nobox -/- RNA, signal intensities for particular genes were averaged between the three chips and ratio of the wild type over knockout signal calculated. Signal less than 100 was considered background. The raw data in the Affymetrix CEL files were normalized by the RMA method (robust multi-array analysis) [15, 16]. Then the null hypothesis was tested that there is no significant changes in gene expression between the treatment pairs. This was done by LIMMA [17] and the pooled local error (LPE) method [18]. The raw pvalues were adjusted by the Benjamini-Hochberg method for the false discovery rate of 5% [19]. We used DAVID database [20] to aid in functional annotation of genes affected by Nobox deficiencyNobox是一种在卵母细胞中表达的同源盒基因（homeobox gene），在卵子发生过程中发挥关键作用。Nobox缺陷会导致出生后卵母细胞快速丢失。早期卵母细胞分化的分子机制尚不明确。我们提出假说：Nobox的缺失会扰乱卵母细胞偏好表达基因以及微小核糖核酸（microRNAs）的全局表达谱。 我们使用Affymetrix 430 2.0微阵列平台，对Nobox敲除与野生型小鼠卵巢进行了比较转录组分析。结果发现，在Nobox缺失情况下下调幅度超过5倍的38个基因中，有28个（74%）在卵母细胞中偏好表达；而在Nobox缺失情况下上调幅度超过5倍的33个基因中，仅有5个（15%）在卵母细胞中偏好表达。 蛋白质结合微阵列（protein binding microarray）辅助鉴定了NOBOX蛋白结合的核苷酸基序，且多个下调基因的推定启动子区域内包含该基序。Nobox缺陷新生卵巢中的微小核糖核酸群体基本未受显著影响。 那些编码分泌型蛋白、但此前未被报道会在早期卵子发生中显著表达的基因，在Nobox敲除小鼠卵巢中出现显著下调，其中包括类阿斯塔辛金属内肽酶（astacin-like metalloendopeptidase, Astl）、Jagged1（Jag1）、卵母细胞分泌蛋白1（oocyte secreted protein 1, Oosp1）、胎球蛋白β（fetuin beta, Fetub）以及R-spondin 2（Rspo2）。此外，多能性相关基因Pou5f1和Sall4在Nobox缺陷卵巢中表达量显著下调，而睾丸决定基因Dmrt1则出现过表达。 我们的研究结果表明，Nobox可能是卵母细胞特异性基因表达的激活因子，同时提示卵母细胞在抑制男性决定基因（如Dmrt1）的表达中发挥重要作用。 关键词：遗传修饰（Genetic Modification） 新生卵巢分别从野生型与Nobox纯合敲除（Nobox -/-）动物中单独收集并混合，总RNA提取使用RNeasy迷你试剂盒（Qiagen公司，加利福尼亚州）。新生卵巢在分娩后12小时内完成采集。本动物实验经贝勒医学院（Baylor College of Medicine）机构动物护理与使用委员会批准。 我们使用三组独立的野生型与Nobox -/-新生卵巢混合RNA样本，制备了生物素标记的cRNA。将生物素标记的cRNA与GeneChip Mouse Expression Set 430 2.0（Affymetrix公司）进行杂交。由于我们针对野生型与Nobox -/- RNA的三个独立混合样本分别开展了三次独立实验，因此将三张芯片中特定基因的信号强度取平均值，并计算野生型与敲除组的信号比值。信号强度低于100的被视为背景信号。 Affymetrix CEL文件中的原始数据通过RMA法（稳健多阵列分析，robust multi-array analysis）[15,16]进行标准化。随后我们检验了无效假设：处理组间基因表达无显著差异。该检验通过LIMMA [17]与合并局部误差（pooled local error, LPE）法[18]完成。原始P值通过Benjamini-Hochberg法进行校正，以将错误发现率控制在5%[19]。我们使用DAVID数据库（DAVID database）[20]辅助对受Nobox缺陷影响的基因进行功能注释。