Background Correct achievement of early ovarian folliculogenesis is a crucial phase for further ovarian function. This process is closely regulated by cell-cell interactions and coordinated expression of genes from oocyte and granulosa cells. But, despite of the large number of studies, little is known about the precise gene expression patterns driving early folliculogenesis. The experimental limitations concerned the very small size of these follicles and the mixture of the different developmental stages within an ovary that make the study of isolated follicular components much more difficult. The recently developed laser capture microdissection (LCM) technique coupled with microarrays experiments is promising in addressing the molecular specificity of each follicular compartment. Nevertheless, the isolation of unique cells or group of cells is still challenging to maintain RNA quality during this process and to obtain sufficient amount of RNA. In this study, we described a method allowing the analysis of oocyte and granulosa cells gene expression during the first stages of sheep early folliculogenesis. Results First we developed a new fixation protocol using a frizzed 70% ethanol fixation solution that ensures correct single cell capture and RNA integrity during microdissection time. After LCM capture of the compartments and follicular stages, RNA extraction and amplification, the expression of 6 oocyte-specific genes (SOHLH2, MAEL, MATER, VASA, GDF9, BMP15) and 3 granulosa cell-specific genes (KITLG, GATA4, AMH) confirmed the purity of the samples and documented their ovine expression profiles. Then, using bovine Affymetrix chip, we identified for the first time, a global gene expression for each follicular compartment during early developmental stages. Particularly the granulosa cell data set is quite unique. 1050 granulosa cell specific transcripts compared to oocyte and 759 oocyte specific transcripts were detected. The analysis of the expression of 2 genes (SIRT7, FST) confirmed this specificity of expression. Finally, the integration of the data stated the 3 main physiological events involved in early folliculogenesis and provided descriptive elements that confirmed the relevance and the potential of the LCM-derived RNAs. Conclusions This method should contribute through an additional genome wide expression profiling to give insights on molecular mechanisms involved in stage transitions and cell type interplays.
Transcriptome profiling of sheep granulosa cells and oocytes during early follicular development obtained by laser capture microdissection.
Age, Specimen part
View SamplesTesticular gene expression changes with loss of Topaz1
TOPAZ1, a germ cell specific factor, is essential for male meiotic progression.
Specimen part
View SamplesBasal cells represent a specific cell type in the epididymis, with specific functions. We performed gene expression analysis to detect differentially regulated genes in basal cells versus other, non-basal, cells in rat epididymis, in order to understand basal cell functions.
Isolated Rat Epididymal Basal Cells Share Common Properties with Adult Stem Cells.
Age, Specimen part
View SamplesMutations in leucine-rich repeat kinase 2 (LRRK2) are the most frequent cause of familial and sporadic Parkinsons disease (PD). Here, we investigated in parallel gene and microRNA transcriptome profiles of three different LRRK2 mouse models. Striatal tissue was isolated from adult LRRK2 knockout mice, as well as mice expressinghuman LRRK2 wildtype (hLRRK2-WT) or PD-associated R1441G mutation (hLRRK2-R1441G).
Gene and MicroRNA transcriptome analysis of Parkinson's related LRRK2 mouse models.
Age, Specimen part
View SamplesWe use mice containing a gene trap in the first intron of the Rest gene, which effectively eliminates transcription from all coding exons, to prematurely remove REST from neural progenitors. We find catastrophic DNA damage that occurs during S-phase of the cell cycle and concominant with activation of p53 pro-apoptotic sgnalling, with consequences including abnormal chromosome separation, apoptosis, and smaller brains.
The REST remodeling complex protects genomic integrity during embryonic neurogenesis.
Specimen part
View SamplesWe use mice containing a gene trap in the first intron of the Rest gene, which effectively eliminates transcription from all coding exons, to prematurely remove REST from neural progenitors. We find catastrophic DNA damage that occurs during S-phase of the cell cycle, with consequences including abnormal chromosome separation, apoptosis, and smaller brains. Further support for persistent effects is the latent appearance of proneural glioblastomas in adult mice also lacking the tumor suppressor, p53. A Rest deficient mouse line generated previously, using a conventional gene targeting approach, does not exhibit these phenotypes, likely due to a remaining C terminal peptide that still binds chromatin and recruits REST chromatin modifiers.Our results indicate that REST-mediated chromatin remodeling is required for proper S-phase dynamics, prior to its well-established role in relieving repression of neuronal genes at terminal differentiation.
The REST remodeling complex protects genomic integrity during embryonic neurogenesis.
Specimen part
View SamplesMicroglia are key regulators of inflammatory response after stroke and brain injury. Here we profiled the microglia transcriptome isolated from a spontaneously hypertensive rat model of focal cerebral ischemia.
Transcriptomic characterization of microglia activation in a rat model of ischemic stroke.
Sex, Age
View SamplesOcular immune privilege (IP) limits immune surveillance of intraocular tumors as certain immunogenic tumor cell lines (P815, E.G7-OVA) that are rejected when transplanted in the skin grow progressively when placed in the anterior chamber (a.c.) of the eye. As splenectomy (SPLNX) is known to terminate ocular IP, we characterized immune mechanisms responsible for spontaneous rejection of intraocular tumors in SPLNX mice as a first step toward identifying how to restore tumoricidal activity within the eye. Microarray data showed a 3-fold increase in interferon (IFN)- and a 2.7-fold increase in Fas ligand (FasL). There was a robust increase in transcripts (127 of 408 surveyed) from interferon (IFN)-stimulated genes and a marked decrease (in 40 of 192 surveyed) in the expression of cell-cycle-associated genes. Non-microarray data confirmed that IFN, FasL and CD8+ T cells but not perforin or TNF were required for elimination of intraocular E.G7-OVA tumors that culminated in destruction of the eye (ocular phthsis). IFN and FasL did not target tumor cells directly as the majority of SPLNX IFNR1-/- mice and Fas-defective lpr mice failed to eliminate ocular E.G7-OVA tumors that expressed Fas and IFNR1. Bone marrow chimeras showed that immune cell expression of IFNR1 and Fas was critical and that SPLNX increased the frequency of activated macrophages within ocular tumors in an IFN- and Fas/FasL-dependent manner. Rejection of intraocular tumors was associated with increased ocular mRNA expression of several inflammatory genes including FasL, NOS2, CXCL2 and T-bet. Our data support a model in which IFN- and Fas/FasL-dependent activation of intratumoral macrophage by CD8+ T cells promotes severe intraocular inflammation that indirectly eliminates intraocular tumors by inducing phthisis. The immunosuppressive mechanisms which maintain ocular IP likely interfere with the interaction between CD8+ T cells and macrophage to limit immunosurveillance of intraocular tumors.
Splenectomy promotes indirect elimination of intraocular tumors by CD8+ T cells that is associated with IFNγ- and Fas/FasL-dependent activation of intratumoral macrophages.
Specimen part, Treatment
View SamplesNGS was used in order to discover novel downstream targets of the miR-17-92/106b clusters. Overall design: Comperasion of gene expression from miR-17-92/106b KO and control
miR-17-92 and miR-106b-25 clusters regulate beta cell mitotic checkpoint and insulin secretion in mice.
Specimen part, Cell line, Subject
View Samplesp63 mutations have been associated with several human hereditary disorders characterized by ectodermal dysplasia such as EEC syndrome, ADULT syndrome and AEC syndrome . The location and functional effects of the mutations that underlie these syndromes reveal a striking genotype-phenotype correlation. Unlike EEC and ADULT that result from missense mutations in the DNA-binding domain of p63, AEC is solely caused by missense mutations in the SAM domain of p63. We report a study on the TAp63a isoform, the first to be expressed during development of the embryonic epithelia, and on its naturally occurring Q540L mutant derived from an AEC patient. To assess the effects of the Q540L mutation, we generated stable cell lines expressing TAp63a wt, DeltaNp63 alpha or the TAp63 alpha-Q540L mutant protein and used them to systematically compare the cell growth regulatory activity of the mutant and wt p63 proteins and to generate, by microarray analysis, a comprehensive profile of differential gene expression. We found that the Q540L substitution impairs the transcriptional activity of TAp63a and causes misregulation of genes involved in the control of cell growth and epidermal differentiation.
The Hay Wells syndrome-derived TAp63alphaQ540L mutant has impaired transcriptional and cell growth regulatory activity.
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