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Role of retinoblastoma protein (Rb) in mouse Sertoli cells function

ABSTRACT: Gene expression profiling study of Sertoli cells (Sc)-specific Retinoblastoma (Rb) knockout (KO) mice. We generated a Sc-specific Rb knockout (Sc-RbKO) line and characterised its testicular phenotype. Sc-RbKO showed a progressive germ cell depletion leading to infertility and Sc dysfunction. We observed that up to 10 days old, Sc-RbKO mice present a phenotype similar to that of their WT siblings. This age was choosen to study the gene expression changes that would eventually lead to the described phenotypical changes. Sc-RbKO testis gene expression was compared to WT and to Sc-Rb+/- samples. Our Microarray results show changes of many genes involved in cell cycle control, DNA repair, apoptosis and misregulation of genes that may be important for proper Sertoli cell function such as tight junction formation, cell membrane and tissue remodeling.

ORGANISM(S): Mus musculus

PROVIDER: GSE22234 | GEO | 2011/01/01

SECONDARY ACCESSION(S): PRJNA128847

REPOSITORIES: GEO

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Project description:Immune privileged Sertoli cells (SC) survive when transplanted across immunological barriers and prolong the survival of co-transplanted allogeneic and xenogeneic cells in rodent models. However, the mechanism for this survival and protection remains unresolved. We have recently identified a mouse Sertoli cell line (MSC-1) that lacks some of the immunoprotective abilities associated with primary SC. The objective of this study was to compare the survival and gene expression profiles of primary SC and MSC-1 cells to identify factors or immune-related pathways potentially important for SC immune privilege. Primary SC or MSC-1 cells were transplanted as allografts to the renal subcapsular area of naïve BALB/c mice and cell survival was analyzed by immunohistochemistry. Additionally, transcriptome differences were investigated by microarray and pathway analyses. While primary SC were detected within the grafts with 100% graft survival throughout the 20-day study, MSC-1 cells w ere rejected between 11 and 14 days with 0% graft survival at 20 days post-transplantation. Microarray analysis identified 3198 genes that were differentially expressed with a ± 4-fold or higher level in primary SC. Cluster and pathway analyses indicate that the mechanism of SC immune privilege is likely complex with multiple immune modulators being involved such as immunosuppressive cytokines and complement inhibitors, lipid mediators for controlling inflammation, and junctional molecules that control leukocyte movement in and out of the immune privileged space. Further study of these immune modulators will increase our understanding of SC immune privilege and in the long-term lead to improvements in transplantation success. We conducted microarray and pathway analyses to identify genes and immune-related pathways differentially regulated by aggregated primary Sertoli cells and MSC-1 cell line. Aggregated 19 to 20-day mice primary Sertoli cells and MSC-1 cell line were used to determine the global transcriptome differences important for the survival and protection of transplanted cells.

Project description:Abstract:The pRb tumor suppressor protein associates with chromatin and regulates gene expression. Numerous studies have identified Rb-dependent RNA signatures, but the proteomic effects of Rb-loss are largely unexplored. We have acutely ablated Rb in adult mice and conducted quantitative analysis of RNA and proteomic changes in colon and lung, where Rb-loss was sufficient or insufficient to induce ectopic proliferation respectively. As expected, RbKO caused similar increases in classic pRb/E2F-regulated transcripts in both tissues, but unexpectedly their protein products increased only in the colon, consistent with its increased proliferative index. Thus, these protein changes induced by Rb-loss are coupled with proliferation, but uncoupled from transcription. The proteomic changes in common between RbKO tissues showed a striking decrease in proteins with mitochondrial functions. Accordingly, RB1 inactivation in human cells decreased both mitochondrial mass and OXPHOS function. RBKO cells showed decreased mitochondrial respiratory capacity and the accumulation of hypopolarized mitochondria. Additionally, RB/Rb-loss altered mitochondrial pyruvate oxidation from 13C-Glucose through the TCA cycle in both cells and mouse tissues. Consequently, RBKO cells have an enhanced sensitivity to mitochondrial stress conditions. In summary, proteomic analyses provide a new perspective on Rb/RB1 mutation, highlighting the importance of pRb for mitochondrial function and suggesting vulnerabilities for treatment. Design: Two tissues (lung and colon) from adult control (Rb1 wild type) or variable (Rb1 acute knockout). We examined the consequences of pRb loss in an in vivo model. Mice were bred to introduce a tamoxifen inducible form of Cre recombinase (ROSA26Sortm1(cre/Esr1)Tyj ) into the genetic backround of either Rb wild type (Rb+/+) or Rb floxed (Rbf/f) alleles. These animals were then injected with tamoxifen, ablating Rb in Rbf/f mice and generating RbKO tissues. Lung and colon tissues were harvested 96 hrs after the tamoxifen injections and analyzed. Each cohort used 3 biological replicates. Methods: RNA extraction: Ninety six hours following the last injection of tamoxifen mice were processed were sacrificed and dissected tissues flash frozen in liquid nitrogen for RNA extraction. RNA was extracted using the TRIzol-choloroform method and then DNase treated. RNA quality was validated using a bioanalyzer. Library construction and RNA-squencing was performed at the MGH NextGeneration Sequencing facility. Three mice were used per genotype, taking matched tissues from each mouse. Methods: Alignment: Sequenced libraries were mapped to the mouse genome (mm10) using TOPHAT (Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks, Trapnell C et al, Nature protocols 2012, 7, 562-578, doi:10.1038/nprot.2012.016). A RefSeq GTF file was used to annotate the mapped transcripts. FPKM matrixes and differentially regulated gene lists were generated using the Cufflinks-Cuffmerge-Cuffdiff pipeline (Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks, Trapnell C et al, Nature protocols 2012, 7, 562-578, doi:10.1038/nprot.2012.016).

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Role of retinoblastoma protein (Rb) in mouse Sertoli cells function

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