Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of mouse ovaries lacking nobox from newborns

ABSTRACT: 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. Experiment Overall Design: 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 deficiency

ORGANISM(S): Mus musculus

SUBMITTER: Aleksandar Rajkovic

PROVIDER: E-GEOD-7775 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Transcription profiling of mouse ovaries lacking nobox from newborns

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