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Series GSE4782

Query DataSets for GSE4782

Status

Public on Sep 30, 2007

Title

The effects of estrogen and testosterone on gene expression in the rat mesenteric arteries

Organism

Rattus norvegicus

Experiment type

Expression profiling by array

Summary

Goal of the experiment: To examine the effects of estrogen and testosterone on gene expression in the rat mesenteric arteries.

Brief description of the experiment: A dramatic difference exists in the timing of development of cardiovascular disease in men vs. women. The primary candidates underlying the cause of gender differences in cardiovascular disease are the sex steroids, estrogen and testosterone. The effect of estrogen on the cardiovascular system has been expected to be protective but this concept has become controversial. The effect of testosterone on the cardiovascular system is considered to be deleterious. In spite of these concepts there is little data on the direct effects of estrogen and testosterone on gene expression in the vasculature. Since estrogen and testosterone exert many of their effects through genomic mechanisms, the DNA microarray is an excellent tool for assessing their effects in the vasculature. In this study, ovariectomized Sprague Dawley rats were treated for 4 days with vehicle (sesame oil), estradiol benzoate (0.15 mg/kg/day), or testosterone (1 mg/kg/day). The mesenteric arteries were obtained, total RNA was extracted, and CodeLink Uniset Rat I DNA microarrays (GE) were used to identify differential gene expression. Seven genes were identified as differentially expressed from the DNA microarray data and confirmed by real time RT-PCR. The expression of D site albumin promoter binding protein and fatty acid synthase were increased in response to both estrogen and testosterone. 3alpha-hydroxysteroid dehydrogenase, interleukin 4 receptor, JunB and c-Fos expression were increased by estrogen but not by testosterone. The aryl hydrocarbon nuclear translocator-like was reduced by testosterone. These data identify genes not previously known to be responsive to estrogen and testosterone in the vasculature.

Keywords: hormone treatment, vasculature, gender differences

Experimental factors: hormone treatment
Keywords: hormone treatment

Overall design

Experimental design: 3 treatments were given (vehicle (control), estradiol benzoate, and testosterone) and 4 animals received each treatment. The mesenteric arteries were obtained from each of the 12 animals treated after euthanasia, total RNA was extracted from each mesenteric artery preparation, and cRNA was synthesized from each total RNA preparation (see below). Samples from vehicle-treated animals are designated C for control: C3, C4, C5, and C8. Samples from estradiol benzoate-treated animals are designated E: E2, E3, E5, and E8. Samples from testosterone-treated animals are designated T: T2, T4, T6, and T8. Samples labelled AA and AB (i.e., C3AA, C3AB) are technical replicates derived from the same cRNA.

Quality control steps: The cRNA that was synthesized from each mesenteric artery sample was used for hybridization to duplicate CodeLink (Amersham/GE) microarrays. 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: All samples were obtained from ovariectomized 5.5-week old female Sprague Dawley rats (Rattus norvegicus).

Manipulations of biological samples and protocols used: The rats were ovariectomized at 3 weeks of age. Hormone treatments were begun after a two week recovery period. The rats were injected intraperitoneally with vehicle (sesame oil), estradiol benzoate (0.15 mg/kg/day), or testosterone (1 mg/kg/day) for 4 days. The animals were euthanized after the treatment on the fourth day. The mesenteric vasculature was dissected free from the gastrointestinal tract and placed in RNAlater (Ambion). The mesenteric arteries were dissected free from the surrounding fat, mesenteric veins, and connective tissues while immersed in RNAlater (Rodrigo et al., J Pharm Tox Meth 47(2):87-92, 2002).

Experimental factor: hormone treatment.

Technical protocols: The mesenteric arteries were homogenized in TRI reagent (MRC) using a Polytron (Brinkmann). 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 was evaluated by spectrophotometry and agarose gel electrophoresis.
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 micrograms of total mesenteric artery 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.

Contributor(s)

Eyster KM, Mark CJ, Gayle R, Martin DS

Citation(s)

17652031

Submission date

May 05, 2006

Last update date

Apr 17, 2012

Contact name

Kathleen M Eyster

E-mail(s)

Kathleen.Eyster@usd.edu