Gene expression profile after GGPPS deletion in sertoli cell
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ABSTRACT: GGPPS was the key enzyme of mevalonate metabolic pathway which was used to synthesize the geranylgeranyl pyrophosphate (GGPP). When we deletion the GGPPS in the sertoli cell and the germ cell loss were found . We used microarrays to detail the global programme of gene expression after GGPPS deletion in sertoli cell and identified 1623 gene expression level change more than 2 times. Primary sertoli cells from 3 days old of control and KO mice for RNA extraction and hybridization on Affymetrix microarrays, every group primare sertoli cell were from more than 30 mice.
Project description:To elucidate the role(s) of WNT/CTNNB1 signaling in the testis, transgenic Ctnnb1 tm1Mmt/+;Amhr2 tm3(cre)Bhr/+ mice were generated to obtain sustained activation of the WNT/CTNNB1 pathway in Sertoli cells. Male Ctnnb1 tm1Mmt/+;Amhr2 tm3(cre)Bhr/+ mice were sterile because of testicular atrophy starting at 5 wk of age, associated with degeneration of seminiferous tubules and the progressive loss of germ cells. Sustained WNT/CTNNB1 pathway activation was obtained in Ctnnb1 tm1Mmt/+;Amhr2 tm3(cre)Bhr/+ Sertoli cells. Sertoli cells often exhibited morphological characteristics suggestive of incomplete differentiation that appeared in a manner coincident with germ cell loss, accompanied by an increase in the expression of the immature Sertoli cell marker AMH. Microarray analyses performed on cultured Sertoli cells showed that CTNNB1 induces the expression of genes associated with the female sex determination pathway as well as cell-cycle associated genes . Together, these data suggest that the WNT/CTNNB1 pathway regulates Sertoli cell functions critical to their capacity to support spermatogenesis and to proliferate in the postnatal testis. Sertoli cells were obtained from 3wks-old Ctnnb1(tm1Mmt/tm1Mmt) mice. Cells were infected with adenoviruses to express eGFP or Cre in serum-free medium, and subsequently harvested for RNA extraction. Preliminary experiments demonstrated that an infection efficiency of nearly 100% could be obtained at an MOI of 50 (as determined by analysis of fluorescent signal in Ad-eGFP-infected cells) and that the Ad-Cre virus produced complete recombination of the floxed Ctnnb1 allele after 12 hours. Microaaray analyses were done using triplicate RNA samples from each adenoviral treatment using MouseRef-6 v.2.0 expression BeadChips technology (Illumina, San Diego, CA).
Project description:Environmental toxicants have been shown to induce the epigenetic transgenerational inheritance of adult onset disease, including testis disease and male infertility. The exposure of a gestating female during the period of gonadal sex determination has been shown to promote sperm epimutations, differential DNA methylation regions (DMR), that transmit transgenerational disease to subsequent generations. The current study was designed to determine the impact of an altered sperm epigenome on the subsequent development of an adult somatic cell (Sertoli cell) that influences the onset of a specific disease (male infertility). A gestating female rat (F0 generation) was exposed to the agriculture fungicide vinclozolin during gonadal sex determination and then the subsequent F3 generation progeny used for the isolation of Sertoli cells and assessment of testis disease. As previously observed, a spermatogenic cell apoptosis was observed. The Sertoli cells that provide the physical and nutritional support for the spermatogenic cells were isolated and alterations in gene expression examined. Over 400 genes were differentially expressed in the F3 generation control versus vinclozolin lineage Sertoli cells. A number of specific signaling pathways and cellular processes were identified to be transgenerationally altered. One of the key metabolic processes affected was pyruvate/lactate production that is directly linked to spermatogenic cell viability. The Sertoli cell epigenome was also altered with over 100 promoter differential DNA methylation regions (DMR) modified in the vinclozolin F3 generation Sertoli cell. The genomic features and overlap with the sperm DMR were investigated. Observations demonstrate that the transgenerational sperm epigenetic alterations subsequently alters the development of a specific somatic cell (Sertoli cell) epigenome and transcriptome that then has a role in the adult onset disease (male infertility). The environmentally induced epigenetic transgenerational inheritance of testis disease appears to be a component of the molecular etiology of male infertility. Environmental toxicants have been shown to induce the epigenetic transgenerational inheritance of adult onset male infertility. The exposure of a gestating female during the period of gonadal sex determination has been shown to promote sperm epimutations, differential DNA methylation regions (DMR), that transmit transgenerational disease to subsequent generations. The current study was designed to determine the impact of an altered sperm epigenome on the subsequent development of an adult somatic cell (Sertoli cell) that influences the onset of a specific disease (male infertility). A gestating female rat (F0 generation) was exposed to the agriculture fungicide vinclozolin during gonadal sex determination and then the subsequent F3 generation progeny used for the isolation of Sertoli cells and assessment of testis disease. The Sertoli cells provide the physical and nutritional support for the spermatogenic cells in the testis. The F3 generation Sertoli cells have an altered transcriptome and epigenome associated with adult onset testis disease. The environmentally induced epigenetic transgenerational inheritance of Sertoli cell abnormalities appears to be a component of the molecular etiology of male infertility. RNA samples from Sertoli cell of 3 F3-control lineage groups are compared to Sertoli cell of 3 F3-vinclozolin lineage groups
Project description:Mammalian spermatogenesis is a complex biological process that occurs within a highly organized tissue, the seminiferous epithelium. The coordinated maturation of spermatogonia, spermatocytes and spermatids suggests the existence of precise programs of gene expression in these cells as well as in their neighboring somatic Sertoli cells. The objective of this study was to elucidate genes encoding the proteins that execute these programs. Rat seminiferous tubules at stages I, II-III, IV-V, VI, VIIa,b, VIIc,d, VIII, IX-XI, XII, XIII-XIV of the cycle were isolated by microdissection and Sertoli cells, spermatogonia plus early spermatocytes, pachytene spermatocytes and spermatids were purified from enzymatically-dispersed testes. Microarray analysis using Rat Genome 230 2.0 arrays identified a total of 16,971 probe sets that recognized transcripts. A comparison with the transcriptome of other tissues identified 398 testis-specific probe sets, which therefore are potential targets for the development of new contraceptives. Sequential waves of cell and stage-specific gene expression are associated with progression of germ cells through the stages of the cycle of the seminiferous epithelium and 1612 probe sets recognized transcripts whose expressions varied at least 4-fold across the stages of the cycle. Pathway analyses reveal that entire biological processes are regulated cyclically in testicular cells. Important among these are cell cycle and DNA repair. Thus, stage-specific gene expression is a widespread and fundamental characteristic of spermatogenic cells and Sertoli cells. Experiment Overall Design: Seminiferous tubules at the following stages or groups of stages were isolated by transillumination-assisted microdissection: I, II-III, IV-V, VI, VIIa,b, VIIc,d, VIII, IX-XI, XII, XIII-XIV. Twenty-five segments of tubules, 2 m in length, were collected at each stage or group of stages from each rat. Tubules from two separate animals were pooled to form one sample from each stage or groups of stages. A total of 5 independent samples per stage or groups of stages were analyzed. Mature Sertoli cells were isolated to about 85% purity (n=4). Three groups of germ cells were isolated by centrifugal elutriation from enzyme-dispersed testes: round spermatids (n=4), pachytene spermatocytes (n=4) and spermatogonia plus early spermatocytes (n=3) . Purity of these cells has been shown to vary between 85% to 95%
Project description:The aim of this experiment was to analyze the transcription profile in Sertoli cells from wild type and Fbxo38 knockout mice by RNA-seq methodology.
Project description:Environmental toxicants have been shown to induce the epigenetic transgenerational inheritance of adult onset disease, including testis disease and male infertility. The current study was designed to determine the impact of an altered sperm epigenome on the subsequent development of an adult somatic cell (Sertoli cell) that influences the onset of a specific disease (male infertility). A gestating female rat (F0 generation) was exposed to the agriculture fungicide vinclozolin during gonadal sex determination and then the subsequent F3 generation progeny used for the isolation of Sertoli cells and assessment of testis disease. As previously observed, enhanced spermatogenic cell apoptosis was observed. The Sertoli cells provide the physical and nutritional support for the spermatogenic cells. Over 400 genes were differentially expressed in the F3 generation control versus vinclozolin lineage Sertoli cells. A number of specific cellular pathways were identified to be transgenerationally altered. One of the key metabolic processes affected was pyruvate/lactate production that is directly linked to spermatogenic cell viability. The Sertoli cell epigenome was also altered with over 100 promoter differential DNA methylation regions (DMR) modified. The genomic features and overlap with the sperm DMR were investigated. Observations demonstrate that the transgenerational sperm epigenetic alterations subsequently alters the development of a specific somatic cell (Sertoli cell) epigenome and transcriptome that correlates with adult onset disease (male infertility). The environmentally induced epigenetic transgenerational inheritance of testis disease appears to be a component of the molecular etiology of male infertility. The isolated Sertoli cell populations from three different groups of animals that were ancestrally exposed to vinclozolin during days 8-14 (E8-14) of fetal development and cell isolates were used to obtain Sertoli cell RNA and DNA from F3 generation control and vinclozolin lineages. The control and vinclozolin lineage Sertoli cell DNA was used in a methylated DNA immunoprecipitation (MeDIP) and genome wide promoter tiling array (Chip) analysis (MeDIP-Chip). Three different comparative (amplified MeDIP vs. amplified MeDIP) hybridizations experiments included in three sub-arrays were performed by Nimblegen. Each comparative hybridization experiment contained one biological replicate of Sertoli cell Whole Genome Amplified-MeDIP-DNA sample from each lineage-treatment.
Project description:In order to understand whether sole Cre recombinase expression has an effect on stress signalling pathways and the peroxisomal or other subcellular compartments, we have analyzed total RNA isolated from Sertoli cell cultures of anti-Müllerian-hormone (AMH)-Cre/wild-type and wild-type/wild-type C57BL/6J mouse testes using microarrays. The Sertoli cells were isolated from individual 14 day-old mice and were cultivated for 7 days. Total RNA was isolated from the Sertoli cell culture (for AMH-Cre/WT 5 mice and WT/WT 3 mice) and the same genotypes were pooled together. For the microarray analysis two technical replicates for each genotype were generated.
Project description:We performed a proteomic analysis of EVs derived from human Sertoli cells, aiming to obtain the profiling of the proteins in these EVs to explore their possible roles in the development of testis.
Project description:Protein phosphatase 6 (PP6) is a member of the PP2A-like subfamily, which plays significant roles in numerous fundamental biological activities. We found that PPP6C plays important roles in male germ cells recently. Spermatogenesis is supported by the Sertoli cells in seminiferous epithelium. In this study, we crossed Ppp6cF/F mice with AMH-Cre mice to gain mutant mice with specific depletion of the Ppp6c gene in the Sertoli cells. We discovered that the PPP6C cKO male mice were absolutely infertile and germ cells were largely lost during spermatogenesis. By combing phosphoproteome with bioinformatics analysis, we showed that the phosphorylation status of β-catenin at S552 (a marker of adherens junctions) was significantly upregulated in mutant mice. Abnormal β-catenin accumulation resulted in impaired testicular junction integrity, thus led to abnormal structure and functions of BTB. Taken together, our study reveals a novel function for PPP6C in male germ cell survival and differentiation by regulating the cell-cell communication through dephosphorylating β-catenin at S552.
Project description:To compare the transcriptional profile of endogenous Sertoli cells from different Stage of Sertoli cell development (embryonic, immature, mature) to the transcriptionla profile of induced embryonic Sertoli cells derived from MEFs or TTFs we employed the agilent whole genome microarray Keywords: Expression profiling by array The following samples were analyzed in duplicates (MEFs, TTFs, ieSCs (derived from MEFs), ieSCs (derived from TTFs), 14.5 dpc male gonad, immature Sertoli (19 dpc embryo testis) and mature (8 week-old mouse testis))
Project description:The Wilms tumor gene, Wt1, is specifically expressed in Sertoli cells (SCs) which support spermatogenesis. Germ cell loss was found in Wt1 knockout mice. In vitro studies demonstrated that Wt1 was essential for cell polarity maintenance in SCs. We used Digital Gene Expression Tag Profiling to detail the global programme of gene expression after Wt1 deletion in sertoli cell and identified 710 gene expression level change more than 2 times. Digital Gene Expression Tag Profiling analysis were performed using mRNA from control and Wt1-deficient Primary Sertoli cells, every group primare sertoli cell were from more than 30 mice.