Project name: Mus musculus

Description: Mice possessing the lethal yellow (LY) agouti mutation (C57BL/6J Ay/a) exhibit adult-onset obesity, altered metabolic regulation, and early reproductive senescence. The present study was designed to test the hypothesis that aging obese mice possess differences in expression of ovarian genes involved in reproductive function relative to age-matched lean mice. To synchronize ovarian state and exclude gonadotropin-mediated effects, 90- and 180-day old diestrous LY and lean black (C57BL/6J a/a) mice were suppressed with GnRH antagonist (Antide®) for five days, then injected with 5 IU equine chorionic gonadotropin (eCG). Complementary RNA derived from RNA extracts of whole ovarian homogenates collected 36h post eCG were run individually on CodeLink Mouse Whole Genome Bioarrays (Applied Microarrays). Unidentified genes, EST, genes whose expression was below sensitivity limits, and those with less than a 2-fold difference in expression between groups were excluded from analysis. After exclusions, 52 genes had a significant (p<0.05) difference in expression between 180-day-old obese LY and lean black mice. LY exhibited elevated ovarian expression of agouti (350x), leptin (6.5x), and numerous genes involved in cholesterol/lipid transport and metabolism, e.g., lanosterol synthase, CYP51, and steroidogenic acute regulatory protein (StAR). Fewer genes showed lower expression in LY mice, e.g. angiotensinogen. In contrast, none of these genes showed differential expression in 90-day-old LY and black mice, which are of similar body weight. Interestingly, 180-day-old LY mice had a 2-fold greater expression of 11β-hydroxysteroid dehydrogenase (HSD) type 1 and a 2-fold lesser expression of 11β-HSD type 2, differences not seen in 90-day-old mice. Consistent with altered 11β-HSD expression, ovarian concentrations of corticosterone (C) were elevated in aging (180-day) LY mice relative to black mice, but C levels were similar in young (90-day) LY and black mice. The data suggest that reproductive dysfunction in aging obese mice is related to modified intraovarian gene expression that is directly related to acquired obesity, but independent of gonadotropic state.Overall design: Experiment Design:Goal of the experiment: To examine differential gene expression the ovaries of obese Lethal Yellow mice (C57BL/6 Ay/a) mice at 90 and 180 days of age compared to their lean black littermates (C57BL6 a/a).Brief description of the experiment: Mice possessing the lethal yellow (LY) agouti mutation (C57BL/6J Ay/a) exhibit adult-onset obesity, altered metabolic regulation, and early reproductive senescence. The present study was designed to test the hypothesis that aging obese mice possess differences in expression of ovarian genes involved in reproductive function relative to age-matched lean mice. To synchronize ovarian state and exclude gonadotropin-mediated effects, 90- and 180-day old diestrous LY and lean black (C57BL/6J a/a) mice were suppressed with GnRH antagonist (Antide®) for five days, then injected with 5 IU equine chorionic gonadotropin (eCG). Complementary RNA derived from RNA extracts of whole ovarian homogenates collected 36h post eCG were run individually on CodeLink Mouse Whole Genome Bioarrays (Applied Microarrays). Unidentified genes, EST, genes whose expression was below sensitivity limits, and those with less than a 2-fold difference in expression between groups were excluded from analysis. After exclusions, 52 genes had a significant (p<0.05) difference in expression between 180-day-old obese LY and lean black mice. LY exhibited elevated ovarian expression of agouti (350x), leptin (6.5x), and numerous genes involved in cholesterol/lipid transport and metabolism, e.g., lanosterol synthase, CYP51, and steroidogenic acute regulatory protein (StAR). Fewer genes showed lower expression in LY mice, e.g. angiotensinogen. In contrast, none of these genes showed differential expression in 90-day-old LY and black mice, which are of similar body weight. Interestingly, 180-day-old LY mice had a 2-fold greater expression of 11β-hydroxysteroid dehydrogenase (HSD) type 1 and a 2-fold lesser expression of 11β-HSD type 2, differences not seen in 90-day-old mice. Consistent with altered 11β-HSD expression, ovarian concentrations of corticosterone (C) were elevated in aging (180-day) LY mice relative to black mice, but C levels were similar in young (90-day) LY and black mice. The data suggest that reproductive dysfunction in aging obese mice is related to modified intraovarian gene expression that is directly related to acquired obesity, but independent of gonadotropic state.Keywords: obesity, ovary, agingExperimental factors: age, presence vs absence of LY mutationExperimental design: The ovarian cycles of 90- and 180-day old mice with the lethal yellow mutation in the agouti gene (C57BL/6J Ay/a) and their non-mutated black littermates (C57BL6 a/a) (n=3 per group) were synchronized with the GnRH antagonist, Antide. At 36 hours after the Antide injections, the mice were stimulated to ovulate with an injection of eCG. At 36 hours after eCG injection the mice were euthanized and the ovaries collected. One ovary from each animal was preserved in RNAlater for DNA microarray analysis.Quality control steps: The cRNA that was synthesized from each ovary was used for hybridization to a single CodeLink (Applied Microarrays) Whole Mouse 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: Ovaries of mice with the lethal yellow mutation in the agouti gene (C57BL/6J Ay/a) and their non-mutated black littermates (C57BL6 a/a) (n=3 per group).Manipulations of biological samples and protocols used: The ovarian cycles of 90- and 180-day old mice with the lethal yellow mutation in the agouti gene (C57BL/6J Ay/a) and their non-mutated black littermates (C57BL6 a/a) (n=3 per group) were synchronized with the GnRH antagonist, Antide. At 36 hours after the Antide injections, the mice were stimulated to ovulate with an injection of eCG. At 36 hours after eCG injection the mice were euthanized and the ovaries collected. One ovary from each animal was preserved in RNAlater for DNA microarray analysis.Experimental factor: age, presence vs absence of LY mutationTechnical protocols: The ovaries were collected into RNAlater (Ambion) and stored at -80C until processed. The RNAlater was blotted from the ovaries and each was individually homogenized in TRI reagent (MRC). 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. 2.0 micrograms of total endometrial 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. The cRNA synthesis reaction was terminated after 14h at 37C by purifying the cRNA on RNeasy columns (Qiagen). The concentration of cRNA was determined by spectrophotometry.Hybridization procedures and parameters: 10 micrograms of cRNA was mixed with fragmentation buffer and heated to 94C for 20 min. The fragmented cRNA was mixed with CodeLink hybridization buffer, loaded on the microarray slides, and hybridized for 18 hours at 37C.The slides were washed in 0.75x TNT (Tris-HCl, NaCl, Tween-20) at 46C for 1h then incubated with streptavidin-Alexa 647 fluorescent dye for 30 min at room temperature. The Alexa fluor was prepared in TNB blocking buffer (0.1M Tris-HCl, 0.15M NaCL, 0.5% NEN Blocking Reagent-PerkinElmer) The slides were then washed 4 times for 5 min each in 1x TNT and twice in 0.05% Tween 20 for 5 sec each. The slides were dried by centrifugation and scanned in an Axon GenePix 4000B scanner.

Data type: Transcriptome or Gene expression

Sample scope: Multiisolate

Relevance: ModelOrganism

Release date: 2010-02-18

Last updated: 2009-02-20