Unknown,Transcriptomics,Genomics,Proteomics

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Influence of lysophosphatidic acid (LPA) and LPA together with interferon tau (IFNt) on transcriptional profiling of bovine endometrial cells.

ABSTRACT: The endometrium plays a crucial role in the reproductive organs in the aspect of embryo-maternal communication and pregnancy. This study investigated transcriptome profiles of endometrial cells stimulated with PBS, LPA and LPA in combination with IFNt. LPA, one of the signaling molecule, is locally produced and released from the bovine endometrium during estrous cycle and early pregnancy. The highest concentration of LPA and expression of its active receptor (LPAR1) were detected in bovine endometrium at the time of maternal recognition of pregnancy, when the conceptus announces its presence by increased IFNt production. Using transcriptomic approach we compared the influence of LPA and LPA together with IFNt on the gene expression profiles in bovine endometrial cells. A total of nine normally cycling Holstein/Polish Black and White (75/25% respectively) cows were used in this study. Global transcriptional profiling was performed using co-cultured stromal and epithelial cells (ratio - 3:1) isolated from bovine endometrium. Three experimental conditions (control (PBS), LPA and LPA plus IFNt) with three replicates per condition were prepared. Total RNAs were extracted from 9 pooled samples (n=3 for each sample) amplified and hybridized onto Affymetrix microarrays.

ORGANISM(S): Bos taurus

SUBMITTER: Dorota Boruszewska

PROVIDER: E-GEOD-53457 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Brown adipose tissue (BAT) is a thermogenic organ that dissipates stored energy as heat to maintain body temperature in infants and small mammals. This process may also provide protection from development of diet-induced obesity. We found that the bioactive lipid mediator lysophosphatidic acid (LPA) markedly decreases differentiation of cultured primary brown adipocyte precursors, while potent selective inhibitors of the LPA-generating enzyme autotaxin (ATX) promote differentiation. Transgenic mice overexpressing ATX exhibited reduced expression of BAT-related genes in peripheral white adipose tissue and accumulated significantly more fat than wild-type controls when fed a high fat diet. Our results indicate that ATX and its product LPA are physiologically relevant negative regulators of brown fat adipogenesis and suggest that a decrease in peripheral brown adipose tissue results in increased susceptibility to diet-induced obesity in mice. Primary BAT cell cultures were established as previously reported with some minor modifications (Cannon B., 2001; NÃ©chad M., 1983). Four- to 6-week-old mice were euthanized under aseptic conditions and interscapular BAT (IBAT) dissected, finely minced in digestion buffer containing 123 mM NaCl, 5mM HCl, 1.3 mM CaCl2, 5mM glucose, 1.5% (w/v) crude bovine serum albumin fraction V, 100 mM HEPES, 0.2% collagenase type II (Sigma-Aldrich Corp., St. Louis, MO), pH 7.4, and digested in 10 ml of the same buffer for 30 min at 37Â°C. The supernatant obtained was then filtered using a 250 Âµm nylon mesh and kept in ice for 15 minutes to allow fat droplets and mature cells to float. The resulting infranatant was filtered through a 30 Âµm nylon mesh to enrich the fraction with precursor cells and centrifuged at 700g for 10 min. The pellet obtained was washed once in warm DMEM and resuspended in DMEM medium containing 10% newborn calf serum, 10 mM HEPES, 4mM glutamine, 25 Âµg/ml sodium ascorbate (Sigma-Aldrich Corp., St. Louis, MO)., 100U/ml penicillin and 100mg/ml streptomycin. Cells were seeded onto 35mm dish plates. Insulin treatment was started on day 1 at 50 nM and the concentration increased to 100 nM at day 5 and 200 nM at day 7. Differentiated/confluent cells were collected for biochemical analysis (~day 9). To identify genes regulated by ATX and LPA signaling, 9 DIV primary BAT cultures were treated with the vehicle (DMSO), specific ATX inhibitor HA155 (2.5ÂµM), or LPA (1-Oleoly-LPA, 5ÂµM) (Avanti Polar Lipids, AL, USA) during differentiation. Treatment of the BAT cultures did not affect confluence or markers of proliferation. RNA was extracted from three cultures per condition using TRIzol reagent (Invitrogen, Carlsbad, CA) as described previously (Blalock EM., 2003).

Project description:Interferon tau (IFNT), a Type I IFN similar to alpha IFNs (IFNA), is the pregnancy recognition signal, produced by the ruminant conceptus. To elucidate specific effects of bovine IFNT and of other conceptus-derived factors, endometrial gene expression changes during early pregnancy were compared to gene expression changes after intrauterine application of human IFNA2. In study one, endometrial tissue samples were obtained on days (D) 12, 15, and 18 post-mating from nonpregnant or pregnant heifers. In study two, heifers were treated from D14 to D16 of the estrous cycle with an intrauterine device releasing IFNA2 or placebo lipid extrudates or PBS only as controls. Endometrial biopsies were collected after flushing the uterus. All samples from both experiments were analyzed with an Affymetrix Bovine Genome Array. Study one revealed differential gene expression between pregnant and nonpregnant endometria on D15 and D18. In study two, IFNA2 treatment resulted in differential gene expression in the bovine endometrium. Comparison of the datasets from both studies identified genes that were differentially expressed in response to IFNA2 but not in response to pregnancy on D15 or D18. Vice versa, genes were found as differentially expressed during pregnancy but not after IFNA2 treatment. In study three, spatiotemporal alterations in expression of selected genes were determined in uteri from nonpregnant and early pregnant heifers using in situ hybridization. The findings of this study suggest differential effects of bovine IFNT compared to human IFNA2 and that some pregnancy-specific changes in the endometrium are elicited by conceptus-derived factors other than IFNT. Study I: Early pregnancy; day 12 of pregnancy (n=5 heifers), day 15 of pregnancy (n=3), day 18 of pregnancy (n=4), day 12 cyclic controls (n=5), day 15 cyclic controls (n=3), day 18 cyclic controls (n=4). Study II: Treatment with human interferon alpha (IFNA); IFNA treatment group (IFNA, n=3 heifers), placebo group (PLAC, n=3 heifers), control group (CONT, n=3 heifers).

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Influence of lysophosphatidic acid (LPA) and LPA together with interferon tau (IFNt) on transcriptional profiling of bovine endometrial cells.

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