Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Two-cell developmental block is due to the lack of cytoplasmic lattices in mouse antral oocytes

ABSTRACT: Mammalian oogenesis is a complex mechanism entailing the maturation of primordial follicles into preovulatory follicles followed by the release of antral oocytes. In the ovary, the antral compartment consists of two populations of oocytes whose main difference regards the ability to resume meiosis and to develop, after the egg-sperm fusion, until the blastocyst stage. Still inexplicably, the antral oocytes distinguishable as Surrounded Nucleolus (SN; 70% of the antral oocytes population) are able to develop to the blastocyst stage, while those showing a Not Surrounded Nucleolus (NSN) arrest at the two-cell stage. RNA labeling and hybridization on microarray was done independently for SN and NSN oocytes with 2 replications. Two sets of 50 antral oocytes were collected in M2 medium and then were labelled with Cy3-CTP. Fluorescently labelled microarray targets were prepared using a Low RNA Input Fluorescent Linear Amplification Kit (Agilent) with 2-round amplification. Cy5-CTP-labeled reference target was produced from mixture of Stratagene Universal Mouse Reference RNA and MC1 cell RNA. Targets were purified using an RNeasy Mini Kit (Qiagen), and then quantified on a NanoDrop scanning spectrophotometer (NanoDrop Technologies). Target cRNA was hybridized to the NIA Mouse 44K Microarray v3.0 (whole genome 60-mer oligo arrays, Agilent Technology, design ID 015087) according to manufacturers protocol (Two-Color Microarray-Based Gene Expression Analysis Protocol, Product # G4140-90050, Version 5.0.1). Slides were scanned with an Agilent DNA Microarray Scanner (model G2505-64120) at 100% and 10% PMT in both channels (except for the two oocyte Samples which were only scanned once each), with a scan resolution of 5um. All hybridizations compared one Cy3-CTP-labeled experimental target to the single Cy5-CTP-labeled reference target which was used for normalization. In the final analysis, we made a cutoff of 2.0 for the processed data (i.e., if value<2.0, it is set as 2.0) to minimize the noises from 2-round linear amplifications.

ORGANISM(S): Mus musculus

SUBMITTER: Minoru Ko

PROVIDER: E-GEOD-34671 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Developmental arrest and mouse antral not-surrounded nucleolus oocytes.

Monti Manuela M Zanoni Mario M Calligaro Alberto A Ko Minoru S H MS Mauri Pierluigi P Redi Carlo Alberto CA

Biology of reproduction 20130103 1

The antral compartment in the ovary consists of two populations of oocytes that differ by their ability to resume meiosis and to develop to the blastocyst stage. For reasons still not entirely clear, antral oocytes termed surrounded nucleolus (SN; 70% of the population of antral oocytes) develop to the blastocyst stage, whereas those called not-surrounded nucleolus (NSN) arrest at two cells. We profiled transcriptomic, proteomic, and morphological characteristics of antral oocytes and observed t ...[more]

PMID: 23136301

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Project description:Oct4 is an essential regulator of embryonic stem (ES) cell pluripotency in vivo and in vitro, as well as a key mediator of the reprogramming of somatic cells to form induced pluriopotent stem (iPS) cells. It is not known whether activation and/or repression of specific genes by Oct4 is relevant to these functions. Here we show that fusion proteins containing the coding sequence of Oct4 or Xlpou91 (the Xenopus homologue of Oct4) fused to activating regions, but not those fused to repressing regions, behave as Oct4, suppressing differentiation and promoting maintenance of undifferentiated phenotypes in vivo and in vitro. An Oct4 activation domain fusion supported ES cell self-renewal in vitro at lower concentrations than required for Oct4 while alleviating the ordinary requirement for the cytokine LIF. At still lower levels of the fusion, LIF-dependence was restored. We conclude that the necessary and sufficient function of Oct4 in promoting pluripotency is to activate specific target genes. Two independent clones from each cell line were used as biological replications. Cy3-CTP-labelled sample targets were prepared from 2.5 ?g of total RNA using the Low RNA Input Fluorescent Linear Amplification Kit (Agilent). Cy5-CTP-labelled reference target was produced from 2.5 ?g of Stratagene Universal Mouse Reference RNA. Purified target RNA were hybridized to the NIA Mouse 44K Microarray v3.0 (whole genome 60-mer oligo arrays, Agilent Technology, design ID 015087) (Carter et al., 2005) according to the manufacturer's protocol (Two-Colour Microarray-Based Gene Expression Analysis Protocol, Version 5.0.1). Slides were scanned with an Agilent DNA Microarray Scanner (model G2505-64120) at 100% PMT for the Cy3 channel and 10% PMT for the Cy5 channel with a scan resolution of 5µm. Data was analyzed using NIA Array Analysis (Sharov et al., 2005) using standard statistical conditions (FDR<0.05, 2-fold expression levels change) to unveil genes with changes in expression levels between the samples and controls.

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Dataset Information

Two-cell developmental block is due to the lack of cytoplasmic lattices in mouse antral oocytes

Publications

Developmental arrest and mouse antral not-surrounded nucleolus oocytes.

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