Lacrimal Gland Gene expression in Aromatase Knockout Mice and Wild Type Controls
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
SUBMITTER: David Sullivan
PROVIDER: E-GEOD-5878 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:Prior to the onset of autoimmune destruction, type 1 diabetic patients and an animal model thereof, the nonobese diabetic (NOD) mouse, show morphological and functional abnormalities in target organs, which may act as inciting events for leukocyte infiltration. To better understand these abnormalities, but without the complications associated with inflammatory infiltrates, we examined genes expressed in autoimmune target tissues (pancreas, submandibular glands, and lacrimal glands) of NOD/scid mice and of autoimmune-resistant C57BL6/scid mice. Experiment Overall Design: Pancreata (6 weeks old mice), submandibular (9 and 15 weeks), and lacrimal glands (15 weeks) from individual NOD-scid and B6-scid mice were isolated for RNA extraction and hybridization on Affymetrix microarrays.
2007-11-29 | E-GEOD-4953 | biostudies-arrayexpress
Project description:Goal of the experiment: To examine differential gene expression in granulosa cells of women undergoing ovarian stimulation with either recombinant human follicle stimulating hormone or purified human menopausal gonadotropin for in vitro fertilization. Brief description of the experiment: The study was designed to test the hypothesis that human granulosa cell (GC) gene expression response differs between pure recombinant FSH and human menopausal gonadotropin (hMG) stimulation regimens. Women < 35 years-old undergoing in vitro fertilization for tubal or male factor infertility were prospectively randomized to one of two stimulation protocols, Gonadotropin releasing hormone (GnRH) agonist long protocol plus individualized dosages of (1) recombinant follicle stimulating hormone (rFSH) (Gonal-F®) (n=4) or (2) purified human menopausal gonadotropin (hMG; Menopur®; 75 IU FSH/75 IU LH per vial) (n=3). Follicle development was monitored by ultrasound and serum estradiol levels. When two follicles were ≥ 17mm, human chorionic gonadotropin (hCG) (10,000 IU; Novarel®) was administered. Oocytes were retrieved 35 h post-hCG. Following oocyte recovery, GC were immediately collected in RNALater®. Pooled GC from individual patients were washed, centrifuged over 40% Percoll®, resuspended in RNALater, and snap-frozen in LN. Total RNA was extracted and stored at -70C. Biotinylated cRNA was synthesized from total RNA and each sample was run individually on CodeLink Whole Human Genome Bioarrays (Applied Microarrays). Unnamed genes and genes with <2-fold difference in expression were excluded from further analysis. After exclusions, 1736 genes exhibited differential expression between groups. Over 400 were categorized as signal transduction genes, ~180 as transcriptional regulators, and ~175 as enzymes/metabolic genes. Expression of selected genes was confirmed by RT-PCR. Differentially expressed genes included A kinase anchor protein 11 (AKAP11), bone morphogenic protein receptor II (BMPR2), epidermal growth factor (EGF), insulin-like growth factor binding protein (IGFBP)-4, IGFBP-5, basigin, and hypoxia-inducible factor (HIF)-1 alpha. The results suggest that major differences exist in the mechanism by which pure FSH alone versus FSH/LH regulate gene expression in preovulatory GC that could impact oocyte maturity and developmental competence. Experimental design: Women < 35 years-old undergoing in vitro fertilization for tubal or male factor infertility were prospectively randomized to one of two stimulation protocols, Gonadotropin releasing hormone (GnRH) agonist long protocol plus individualized dosages of (1) recombinant follicle stimulating hormone (rFSH) (Gonal-F®) (n=4) or (2) purified human menopausal gonadotropin (hMG; Menopur®; 75 IU FSH/75 IU LH per vial) (n=3). Follicle development was monitored by ultrasound and serum estradiol levels. When two follicles were ≥ 17mm, human chorionic gonadotropin (hCG) (10,000 IU; Novarel®) was administered. Oocytes were retrieved 35 h post-hCG. Following oocyte recovery, GC were immediately collected in RNALater®. Pooled GC from individual patients were washed, centrifuged over 40% Percoll®, resuspended in RNALater, and snap-frozen in LN. Total RNA was extracted and stored at -70C. Biotinylated cRNA was synthesized from total RNA and each sample was run individually on CodeLink Whole Human Genome Bioarrays (Applied Microarrays Quality control steps: The cRNA that was synthesized from the granulosa cells from individual patients was used for hybridization to a single CodeLink (Applied Microarrays) Whole Human Genome microarray. Only one sample was hybridized with each slide and only one dye (Alexa 647) was used so no dye swaps were necessary. Bacterial control spikes were used as per manufacturer's instructions. Samples used, extract preparation and labelling: The origin of each biological sample: Granulosa cells from women < 35 years-old undergoing in vitro fertilization. Manipulations of biological samples and protocols used: Female patients were treated with gonadotropin releasing hormone (GnRH) agonist long protocol plus individualized dosages of (1) recombinant follicle stimulating hormone (rFSH) (Gonal-F®) (n=4) or (2) purified human menopausal gonadotropin (hMG; Menopur®; 75 IU FSH/75 IU LH per vial) (n=3). Follicle development was monitored by ultrasound and serum estradiol levels. When two follicles were ≥ 17mm, human chorionic gonadotropin (hCG) (10,000 IU; Novarel®) was administered. Oocytes were retrieved 35 h post-hCG. Following oocyte recovery, granulosa cells were immediately collected in RNALater®. Pooled GC from individual patients were washed, centrifuged over 40% Percoll®, resuspended in RNALater, and snap-frozen in LN. Experimental factor: treatment with recombinant FSH versus purified human menopausal gonadotropin Technical protocols: Granulosa cells were collected into RNAlater (Ambion) and stored at -80C until processed. The granulosa cells were pelleted by centrifugation to remove the RNAlater. Each cell pellet was homogenized in TRI reagent (Molecular Research Centers). Bromochloropropane and sodium acetate were added, and the samples were centrifuged to separate the phases. The RNA-containing layer was removed and the RNA purified on an RNeasy extraction column (Qiagen). The sample was treated with an on-column DNase treatment (RNase-free DNase, Qiagen). The purity and quantity were evaluated by an Agilent Bioanalyzer using the RNA 6000 Nanoassay LabChip. Labelled cRNA was prepared using the manufacturer's Instruction Protocol (www1.amershambiosciences.com, CodeLink Gene Expression System: Manual Labelled cRNA Target Preparation). The source of reagents was the CodeLink Expression Assay Reagent Kit, Manual Prep, except where specified otherwise. 0.285 micrograms of total granulosa cell RNA was mixed with bacterial control RNA spikes and primed with T7 oligo(dT) primer for 10 min at 70C. (The bacterial control spikes included araB, entF, fixB, gnd, hisB, and leuB.) The first strand of cDNA was synthesized with first strand buffer, dNTP mix, RNase inhibitor, and reverse transcriptase for 2 h at 42C. The second strand cDNA synthesis reaction was prepared using second strand buffer, dNTP mix, DNA polymerase mix, and RNase H; the reaction was carried out for 2h at 16C. The double-stranded cDNA was purified on QIAquick columns (Qiagen) and the eluent was dried down in a SpeedVac concentrator. The double-stranded cDNA was resuspended in a mixture containing T7 reaction buffer, T7 ATP, T7 GTP, T7 UTP, T7 CTP, biotin-11-UTP, and T7 enzyme mix for the synthesis of cRNA. 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2008-04-14 | E-GEOD-10815 | biostudies-arrayexpress
Project description:The present experiments were performed to determine the roles of estrogen receptors M-NM-1 and M-NM-2 (ERM-NM-1 and ERM-NM-2) in normal and neoplastic development in the mouse mammary gland. In wild-type mice, in vivo administration of estradiol (E) + progesterone (P) stimulated mammary ductal growth and alveolar differentiation. Mammary glands from mice in which the ERM-NM-2 gene has been deleted (M-NM-2ERKO mice) demonstrated normal ductal growth and differentiation in response to E + P. By contrast, mammary glands from mice in which the ERM-NM-1 gene has been deleted (M-NM-1ERKO mice) demonstrated only rudimentary ductal structures that did not differentiate in response to E + P. EGF demonstrates estrogen-like activity in the mammary glands of M-NM-1ERKO mice: treatment of M-NM-1ERKO mice with EGF + P (without E) supported normal mammary gland development, induced expression of progesterone receptor (PR), and increased levels of G- protein-coupled receptor (GPR30) protein. Mammary gland development in M-NM-2ERKO mice treated with EGF + P was comparable to that of wild-type mice receiving EGF + P; EGF had no statistically significant effects on the induction of PR or expression of GPR30 in mammary glands harvested from either wild-type mice or M-NM-2ERKO mice. In vitro exposure of mammary glands to 7,12-dimethylbenz[a]anthracene (DMBA) induced preneoplastic mammary alveolar lesions (MAL) in glands from wild-type mice and M-NM-2ERKO mice, but failed to induce MAL in mammary glands from M-NM-1ERKO mice. Microarray analysis of DMBA-treated mammary glands identified 28 functional pathways whose expression was significantly different in M-NM-1ERKO mice versus both M-NM-2ERKO and wild-type mice; key functions that were differentially expressed in M-NM-1ERKO mice included cell division, cell proliferation, and apoptosis. The data demonstrate distinct roles for ERM-NM-1 and ERM-NM-2 in normal and neoplastic development in the mouse mammary gland, and suggest that EGF can mimic the ERM-NM-1-mediated effects of E in this organ. Gene expression of mammary gland organ culture and DMBA-induced lesions from 4 mouse strains.
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