Temporal Profiling of Rat Transcriptomes in Retinol-Replenished Vitamin A-Deficient Testis
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ABSTRACT:
ORGANISM(S): Rattus norvegicus
SUBMITTER: Timothy Doyle
PROVIDER: E-GEOD-14962 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:The objective of this study was to understand the genetic mechanisms of Vitamin-A-Deficiency (VAD)-induced arrest of spermatogonial stem-cell differentiation. Vitamin A and its derivatives (the retinoids) participate in many physiological processes including vision, cellular differentiation and reproduction. VAD affects spermatogenesis, the subject of our present study. Spermatogenesis is a highly regulated process of differentiation and complex morphologic alterations that, in the postnatal testis, leads to the formation of sperm in the seminiferous epithelium. VAD causes early cessation of spermatogenesis, characterized by degeneration of meiotic germ cells, leading to seminiferous tubules containing mostly type A spermatogonia and Sertoli cells. In this study, we investigated the molecular basis of VAD on spermatogenesis in mice. We used adult Balb/C mice fed with a Control or VAD diet for an extended period of time (8-28 weeks) and selected two time points (18 and 25 weeks) for microarray analysis. To understand the effect of VAD on the spermatogonial stem cell transcriptome, we studied isolated pure populations of spermatogonia from control and vitamin-A-deficient mice from two representative time points (18 and 25 weeks) using Affymetrix GeneChip microarrays. We identified target genes involved in the arrest of spermatogonial differentiation and spermatogenesis. Our results establish a better understanding of the chronology and magnitude of the consequences of VAD on mouse testes and add to the current knowledge of the molecular regulatory mechanisms of germ cell development. Spermatogonia were isolated by the STATPUT procedure with minor modifications. VAD mice were sacrificed at 18 and 25 weeks of VAD treatment for spermatogonia isolation. Decapsulated testes from 2 mice were suspended in RPMI medium containing collagenase (0.6 mg/ml), Hyaluronidase (0.12 mg/ml) and DNAse I (1.25 mg/ml) and incubated at 37ºC for 30 minutes in a shaking water bath. The tissues were then allowed to come down the tube and the supernatant (containing interstitial cells) was removed. The pellet was incubated with 3 ml of 0.25% Trypsin /EDTA (GIBCO, Invitrogen, USA), in the presence of DNAse I (0.2 mg/ml) at 37ºC for 15 minutes in a shaking water bath. The dispersed cells were washed twice with RPMI medium containing 10% heat inactivated FBS to neutralize the protease activity, and filtered through a sterile 0.22 nylon to remove any undigested fragments. Cells of the dissociated seminiferous epithelium were then plated overnight in a 34ºC 3% CO2 incubator. The following day, germ cells, in suspension, were separated by sedimentation with use of a 2-4% BSA gradient. The cells were allowed to sediment for a standard period of 2.5 h, and fractions of 2-ml volume were collected. The cells of each fraction were examined under a phase contrast microscope, and fractions containing cells of similar size and morphology were pooled and spun down by low-speed centrifugation. Purity of spermatogonia was estimated and was routinely higher than 90%. Total RNA was extracted from the isolated germ cells using TRIzol ® Reagent, and cleaned with RNeasy minicolumns. RNA content was determined by measurement of optical density at 260 nm. Only the RNA samples showing an OD 260/280 ratio higher than 1.8 were used for microarray hybridization. Raw expression values in Affymetrix CEL file format were generated by GeneChip Operating Software.
2010-08-03 | E-GEOD-23119 | biostudies-arrayexpress
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.
2011-06-23 | E-GEOD-23200 | biostudies-arrayexpress
Project description:Vitamin A deficiencies and insufficiencies affect hundreds of millions of people in both developing and developed countries. These individuals experience higher rates of mortality and increased morbidity during enteric and respiratory infections compared to those who are vitamin A sufficient. Previously, our laboratory has demonstrated that vitamin A-deficient (VAD) mice have significantly impaired virus-specific IgA and CD8+ T cell responses in the airways. Here we demonstrate that VAD mice experience enhanced cytokine/chemokine gene expression and release in the respiratory tract 10 days following virus infection compared to control vitamin A-sufficient animals. Cytokines/chemokines that are reproducibly upregulated at the gene expression and protein levels include IFNγ and IL-6. Despite previous indications that cytokine dysregulation in VAD animals might reflect low forkhead box (Fox)P3+ T cell frequencies, we found no reduction in FoxP3+ T cells in VAD respiratory tissues. As an alternative explanation for the high cytokine levels, we found that virus infection and viral antigen persistence were enhanced on day 10 post-infection in VAD animals compared to controls, and that respiratory tract tissues had an increased potential to activate virus-specific T cells. Results encourage cautious management of virus infections in patients with VAD; efforts to enhance FoxP3+ T cell frequencies and quell immune effectors might in some cases exacerbate disease if virus has not been cleared. We used microarrays to explore the gene expression profiles differentially expressed in nasal-associated lymph tissue (NALT) lymphocytes from vitamin A-sufficient and vitamin A-deficient mice following infection with Sendai virus.
2016-02-11 | E-GEOD-65805 | biostudies-arrayexpress
Project description:Mechanical unloading by ventricular assist devices (VAD) leads to significant gene-expression changes often summarized as reverse remodeling. However, little is known on individual transcriptome changes during VAD-support and its relationship to non-failing hearts (NF). In addition no data are available for the transcriptome regulation during non-pulsatile VAD-support. Therefore we analysed the gene-expression patterns of 30 paired samples from VAD-supported (including 8 non-pulsatile VADs) and 8 non-failing control hearts (NF) using the first total human genome-array available. Transmural myocardial samples were collected for RNA-isolation. RNA was isolated by commercial methods and processed according to chip-manufacturer recommendations. cRNA were hybridized on Affymetrix HG-U133 Plus 2.0 arrays, providing coverage of the whole human genome Array. Data was analyzed using Microarray Analysis Suite 5.0 (Affymetrix) and clustered by Expressionist software (Genedata). 352 transcripts were differentially regulated between samples from VAD-implantation and NF, whereas 510 were significantly regulated between VAD-transplantation and NF (paired t-test p<0.001, fold change >=1.6). Remarkably, only a minor fraction of 111 transcripts was regulated in heart failure (HF) and during VAD-support. Unsupervised hierarchical clustering of paired VAD- and NF-samples revealed separation of HF- and NF- samples, however individual differentiation of VAD-implantation and VAD-transplantation was not accomplished. Clustering of pulsatile and non-pulsatile VAD did not lead to robust separation of gene expression patterns. During VAD-support myocardial gene expression changes do not indicate reversal of the HF-phenotype, but reveal a distinct HF-related pattern. Transcriptome analysis of pulsatile and non-pulsatile VAD-supported hearts did not provide evidence for a pump-mode specific transcriptome pattern. Microarrays were used to elucidate the differences between non-failing control hearts and those, suffering from end-stage heart failure pre and post mechanical unloading.
2010-07-29 | E-GEOD-21610 | biostudies-arrayexpress
Project description:We used microarrays to examine the temporal transcriptome changes of vitamin a deficient rats testes replenished with retinol
2009-02-27 | GSE14962 | GEO
Project description:Vitamin A deficiencies and insufficiencies affect hundreds of millions of people in both developing and developed countries. These individuals experience higher rates of mortality and increased morbidity during enteric and respiratory infections compared to those who are vitamin A sufficient. Previously, our laboratory has demonstrated that vitamin A-deficient (VAD) mice have significantly impaired virus-specific IgA and CD8+ T cell responses in the airways. Here we demonstrate that VAD mice experience enhanced cytokine/chemokine gene expression and release in the respiratory tract 10 days following virus infection compared to control vitamin A-sufficient animals. Cytokines/chemokines that are reproducibly upregulated at the gene expression and protein levels include IFNγ and IL-6. Despite previous indications that cytokine dysregulation in VAD animals might reflect low forkhead box (Fox)P3+ T cell frequencies, we found no reduction in FoxP3+ T cells in VAD respiratory tissues. As an alternative explanation for the high cytokine levels, we found that virus infection and viral antigen persistence were enhanced on day 10 post-infection in VAD animals compared to controls, and that respiratory tract tissues had an increased potential to activate virus-specific T cells. Results encourage cautious management of virus infections in patients with VAD; efforts to enhance FoxP3+ T cell frequencies and quell immune effectors might in some cases exacerbate disease if virus has not been cleared.
2016-02-11 | GSE65805 | GEO
Project description:Observational studies in human suggest involvement of vitamin A/retinoic acid (RA) signaling in the regulation of airway smooth muscle (ASM) function, but the precise mechanisms by which RA impacts ASM phenotype is not clear. Here, we generated trascriptional profiles from two different models of RA-sufficient and RA-deficient mouse ASM in order to determine the molecular targets of RA in ASM (VAS/VAD, CTR/BMS) Mouse ASMs were isolated from Acta2-hrGFP;Cspg4-DsRED adult mice using flow cytometry. VAS mice were given normal, vitamin A-sufficient diet (15 IU vitamin/g) while the VAD mice were given 3 days of vitamin A-deficient diet (< 0.1 IU vitamin/g). CTR/BMS mouse ASMs were also isolated from Acta2-hrGFP;Cspg4-DsRED adult mice using flow cytometry. CTR mice were given 3 days of powdered, vitamin A sufficient diet mixed with vehicles (corn oil) while the BMS diet were given 3 days of powdered, vitamin A sufficient diet mixed with a potent pan-RA-receptor antagonist (BMS, 5 mcg/g).
2018-12-25 | GSE116846 | GEO
Project description:The objective of this study was to understand the genetic mechanisms of Vitamin-A-Deficiency (VAD)-induced arrest of spermatogonial stem-cell differentiation. Vitamin A and its derivatives (the retinoids) participate in many physiological processes including vision, cellular differentiation and reproduction. VAD affects spermatogenesis, the subject of our present study. Spermatogenesis is a highly regulated process of differentiation and complex morphologic alterations that, in the postnatal testis, leads to the formation of sperm in the seminiferous epithelium. VAD causes early cessation of spermatogenesis, characterized by degeneration of meiotic germ cells, leading to seminiferous tubules containing mostly type A spermatogonia and Sertoli cells. In this study, we investigated the molecular basis of VAD on spermatogenesis in mice. We used adult Balb/C mice fed with a Control or VAD diet for an extended period of time (8-28 weeks) and selected two time points (18 and 25 weeks) for microarray analysis. To understand the effect of VAD on the spermatogonial stem cell transcriptome, we studied isolated pure populations of spermatogonia from control and vitamin-A-deficient mice from two representative time points (18 and 25 weeks) using Affymetrix GeneChip microarrays. We identified target genes involved in the arrest of spermatogonial differentiation and spermatogenesis. Our results establish a better understanding of the chronology and magnitude of the consequences of VAD on mouse testes and add to the current knowledge of the molecular regulatory mechanisms of germ cell development.
2010-08-03 | GSE23119 | GEO
Project description:Vitamin A (retinol) is an essential precursor for the production of retinoic acid (RA), which in turn is a major regulator of gene expression, affecting cell differentiation throughout the body. Understanding how vitamin A nutritional status, as well as therapeutic retinoid treatment, regulates the expression of retinoid homeostatic genes is important for improving dietary recommendations and therapeutic strategies using retinoids. This study investigated genes central to processes of retinoid uptake and storage, release to plasma, and oxidation in the liver of rats under steady-state conditions after different exposures to dietary vitamin A (deficient, marginal, adequate and supplemented), and acutely after administration of a therapeutic dose of all-trans-RA. Over a very wide range of dietary vitamin A, lecithin:retinol acyltransferase (LRAT) as well as multiple cytochrome P450s (CYP26A1, CYP26B1, and CYP2C22) differed by diet and were highly correlated with one another and with vitamin A status assessed by liver retinol concentration (all correlations, P<0.05). After acute treatment with RA, the same genes were rapidly and concomitantly induced, preceding RARß, a classical direct target of RA. CYP26A1 mRNA exhibited the greatest dynamic range (change of log26 in 3 h). Moreover, CYP26A1 increased more rapidly in the liver of RA-primed rats than naïve rats. By in situ hybridization, CYP26A1 mRNA was strongly regulated within hepatocytes, closely resembling RBP4 in location. Overall, whether RA is produced endogenously from retinol or administered exogenously, changes in retinoid homeostatic gene expression simultaneously favor both retinol esterification and RA oxidation, with CYP26A1 exhibiting the greatest dynamic change. All rats were housed in a room maintained at 22°C with a 12-h dark:light cycle, and food and water were freely available. For the Steady-State Vitamin A Study (experiment 1) and the Retinoic Acid 16-hour Kinetic Study (experiment 2), lactating female Sprague-Dawley rats with 12 female pups (purchased from Charles River Laboratories, Willmington, MA) were fed a vitamin A-deficient purified diet [AIN-93G diet, prepared by Research Diets, New Brunswick, NJ] to reduce the transfer of vitamin A in milk from mother to pups prior to the start of the study. For experiment 1, from weaning, the offspring were fed the same diet modified to contain vitamin A at one of four levels: 0 (vitamin A deficient), 0.4 mg retinol/kg diet (vitamin A marginal), 4 mg retinol/kg diet (vitamin A adequate control), or 100 mg retinol/kg diet (vitamin A supplemented). All rats were studied at 8 weeks of age. Rats were euthanized by carbon dioxide asphyxiation and blood and liver were collected rapidly and frozen in liquid nitrogen for storage at -80°C before analysis. In experiment 2, at 8 weeks of age female vitamin A-deficient rats were treated with ~100 ug of All-trans-Retinoic acid (at-RA) for 0 (vehicle only), 3, 6, 10 or 16 h (n=3-4/group). Tissues were collected and RNA was prepared in the same manner as in experiment 1. In the 90-minute "first pass" kinetic study (experiment 3), female rats were purchased at 6 weeks of age and fed a stock rodent diet. When the rats were 8 weeks old they were assigned to a control (naïve) group. Rats in the naive group received an equal amount of vehicle only (vegetable oil/5% ethanol). Food was removed immediately. Sixteen hours after priming, each rat was lightly anesthetized by isoflurane-oxygen inhalation and treated with ~25 ug all-trans-RA bound to albumin [10 ug RA per 100 g bo dy weight], injected into the exposed left common iliac vein. The incision was closed with a surgical staple. The rats were allowed to recover from the anesthesia. Rats were killed at 0 minute (vehicle injection), and 30, 60, and 90 min (n = 3/group) after injection of the RA test dose. Rats were euthanized by carbon dioxide asphyxiation and blood and liver were collected rapidly and frozen in liquid nitrogen for storage at -80°C before analysis. For experiment 1, five biological repeats were Vitamin A deficient, seven biological repeats were Vitamin A marginal, six biological repeats were Vitamin A adequate, and two biological repeats were Vitamin A supplemented. In experiment 2, three biological repeats were performed for each of the following treatments: untreated, at-RA for 3 hours, at-RA for 6 hours and at-RA for 16 hours. Four biological repeats were treated with at-RA for 10 hours. For experiment 3, two biological repeats were untreated and three biological repeats were performed for each of the following at-RA treatment times: 30, 60 and 90 minutes. A total of 47 samples were analyzed.
2011-08-31 | E-GEOD-24104 | biostudies-arrayexpress
Project description:Vitamin A (retinol) is an essential precursor for the production of retinoic acid (RA), which in turn is a major regulator of gene expression, affecting cell differentiation throughout the body. Understanding how vitamin A nutritional status, as well as therapeutic retinoid treatment, regulates the expression of retinoid homeostatic genes is important for improving dietary recommendations and therapeutic strategies using retinoids. This study investigated genes central to processes of retinoid uptake and storage, release to plasma, and oxidation in the liver of rats under steady-state conditions after different exposures to dietary vitamin A (deficient, marginal, adequate and supplemented), and acutely after administration of a therapeutic dose of all-trans-RA. Over a very wide range of dietary vitamin A, lecithin:retinol acyltransferase (LRAT) as well as multiple cytochrome P450s (CYP26A1, CYP26B1, and CYP2C22) differed by diet and were highly correlated with one another and with vitamin A status assessed by liver retinol concentration (all correlations, P<0.05). After acute treatment with RA, the same genes were rapidly and concomitantly induced, preceding RARß, a classical direct target of RA. CYP26A1 mRNA exhibited the greatest dynamic range (change of log26 in 3 h). Moreover, CYP26A1 increased more rapidly in the liver of RA-primed rats than naïve rats. By in situ hybridization, CYP26A1 mRNA was strongly regulated within hepatocytes, closely resembling RBP4 in location. Overall, whether RA is produced endogenously from retinol or administered exogenously, changes in retinoid homeostatic gene expression simultaneously favor both retinol esterification and RA oxidation, with CYP26A1 exhibiting the greatest dynamic change.
2011-09-01 | GSE24104 | GEO