The rat uterus responds to acute estrogen treatment with a series of well characterized physiological responses; however, the gene expression changes required to elicit these responses have not been fully characterized.  In order to understand early events induced by estrogen exposure in vivo, we evaluated the temporal gene expression in the uterus of the immature rat after a single dose of 17 Alpha-ethynyl estradiol (EE) by microarray analysis, evaluating the expression of 15,923 genes.  Immature 20 day old rats were exposed to a single dose of EE (10 ug/kg) and the effect on uterine histology, weight and gene expression were determined after 1, 2, 8, 24, 48, 72 and 96 h.  EE induced changes in the expression of 3,867 genes, at least at one time point (pÂ¡Ã0.0001), and at least 1.5 fold (up- or down-regulated).  Specifically, the expression of 8, 116, 3030, 2076, 381, 445, and 125 genes was modified at 1, 2, 8, 24, 48, 72 or 96 hours after exposure to EE respectively (pÂ¡Ã0.0001, t Test).  At the tissue and organ level, a clear uterotrophic response was elicited by EE after only 8 h, reaching a maximum after 24 h and remaining detectable even after 96 h of exposure.  The uterine phenotypic changes were induced by sequential changes in the transcriptional status of a large number of genes, in a program that involves multiple molecular pathways.  Using the gene ontology to better understand the temporal response to estrogen exposure, we determined that the earliest changes were in the expression of genes whose products are involved in transcriptional regulation and signal transduction, followed by genes implicated in protein synthesis, energy utilization, solute transport, cell proliferation and differentiation, tissue remodeling and immunological responses among other pathways.  The compendium of genes here presented represents a comprehensive compilation of estrogen-responsive genes involved in the uterotrophic response.

Transcription profiling of rat uterus after exposure to 17 alpha-ethynyl estradiol for different lengths of time

Uterine temporal response to acute exposure to 17alpha-ethinyl estradiol in the immature rat

Affymetrix GeneChip Rat Expression Array RAE230A [RAE230A]

rawData

scan

processedDataMatrix

The rat uterus responds to acute estrogen treatment with a series of well characterized physiological responses; however, the gene expression changes required to elicit these responses have not been fully characterized.  In order to understand early events induced by estrogen exposure in vivo, we evaluated the temporal gene expression in the uterus of the immature rat after a single dose of 17 Alpha-ethynyl estradiol (EE) by microarray analysis, evaluating the expression of 15,923 genes.  Immature 20 day old rats were exposed to a single dose of EE (10 ug/kg) and the effect on uterine histology, weight and gene expression were determined after 1, 2, 8, 24, 48, 72 and 96 h.  EE induced changes in the expression of 3,867 genes, at least at one time point (p¡Ü0.0001), and at least 1.5 fold (up- or down-regulated).  Specifically, the expression of 8, 116, 3030, 2076, 381, 445, and 125 genes was modified at 1, 2, 8, 24, 48, 72 or 96 hours after exposure to EE respectively (p¡Ü0.0001, t Test).  At the tissue and organ level, a clear uterotrophic response was elicited by EE after only 8 h, reaching a maximum after 24 h and remaining detectable even after 96 h of exposure.  The uterine phenotypic changes were induced by sequential changes in the transcriptional status of a large number of genes, in a program that involves multiple molecular pathways.  Using the gene ontology to better understand the temporal response to estrogen exposure, we determined that the earliest changes were in the expression of genes whose products are involved in transcriptional regulation and signal transduction, followed by genes implicated in protein synthesis, energy utilization, solute transport, cell proliferation and differentiation, tissue remodeling and immunological responses among other pathways.  The compendium of genes here presented represents a comprehensive compilation of estrogen-responsive genes involved in the uterotrophic response.

compound

dose

time

Transcription profiling by array of rat uterus after treatment with 17 alpha-ethynyl estradiol

The rat uterus responds to acute estrogen treatment with a series of well characterized physiological responses; however, the gene expression changes required to elicit these responses have not been fully characterized.  In order to understand early events induced by estrogen exposure in vivo, we evaluated the temporal gene expression in the uterus of the immature rat after a single dose of 17 Alpha-ethynyl estradiol (EE) by microarray analysis, evaluating the expression of 15,923 genes.  Immature 20 day old rats were exposed to a single dose of EE (10 ug/kg) and the effect on uterine histology, weight and gene expression were determined after 1, 2, 8, 24, 48, 72 and 96 h.  EE induced changes in the expression of 3,867 genes, at least at one time point (p¡Ü0.0001), and at least 1.5 fold (up- or down-regulated).  Specifically, the expression of 8, 116, 3030, 2076, 381, 445, and 125 genes was modified at 1, 2, 8, 24, 48, 72 or 96 hours after exposure to EE respectively (p¡Ü0.0001, t Test).  At the tissue and organ level, a clear uterotrophic response was elicited by EE after only 8 h, reaching a maximum after 24 h and remaining detectable even after 96 h of exposure.  The uterine phenotypic changes were induced by sequential changes in the transcriptional status of a large number of genes, in a program that involves multiple molecular pathways.  Using the gene ontology to better understand the temporal response to estrogen exposure, we determined that the earliest changes were in the expression of genes whose products are involved in transcriptional regulation and signal transduction, followed by genes implicated in protein synthesis, energy utilization, solute transport, cell proliferation and differentiation, tissue remodeling and immunological responses among other pathways.  The compendium of genes here presented represents a comprehensive compilation of estrogen-responsive genes involved in the uterotrophic response.


Affymetrix:Protocol:ExpressionStat

Title: Affymetrix CHP Analysis (ExpressionStat). Description:

Affymetrix:Protocol:Hybridization-EukGE-WS2v4_450

Title: Affymetrix EukGE-WS2v4_450 Hybridization. Description:

Affymetrix:Protocol:Hybridization-EukGE-WS2v5_450

(Parameters: Extracted product = total_RNA, Amplification = none)

P-AFFY-6

Title: Affymetrix CEL analysis. Description:

P-MEXP-10382

Total RNA was extracted from uterus and ovaries from individual animals (combining only the tissues from the same animal).

P-MEXP-44884

On PND 20, the animals were randomly assigned to 14 groups of 10 animals each (7 control and 7 EE-treated groups, respectively), and dosed once by subcutaneous injection, with 0 or 10.0 ug EE/kg in peanut oil.  Animals received 5 ml/kg body weight of dose solution.  The dose was administered to each animal in a staggered fashion, within the group and between groups, to ensure that the time of exposure was within 5% of the reported exposure length.  Body weights (nearest 1.0 g) were recorded daily and before sacrifice.  The animals were sacrificed by CO2 asphyxiation at 1, 2, 8, 24, 48, 72 or 96 hours after dosing.  The body of the uterus was cut just above its junction with the cervix and leaving the ovaries attached to it, carefully dissected free of adhering fat and mesentery and weighed as a whole.  Then the ovaries were dissected free, and the uterine and ovarian wet weight were recorded.  The individual uteri were then placed into RNAlater (50-100 mg/ml of solution; Ambion, Austin, TX), at room temperature.

P-MEXP-8351

P-MEXP-8354

P-MEXP-8356

Five month old male and female Sprague Dawley rats weighing approximately 300 g (females) or 350-400 g (males) were used (Charles River VAF/Plus, Raleigh, NC).  The rats were acclimated to the local vivarium conditions (24oC; 12-h light/12-h dark cycle) for two weeks.  All rats were singly housed in 20 x 32 x 20-cm cages (stainless steel) during the experimental phase of the protocol.  Animals were allowed free access to water and were fed a casein-based diet (soy and alfalfa-free diet, Purina 5K96; Purina Mills).  The experimental protocol was carried out according to Procter and Gamble's animal care approved protocols and animals were maintained in accordance with the NIH Guide for the Care and Use of Laboratory Animals. Breeding was carried out by co-housing one male and one female overnight.  Successful mating was confirmed the following morning by the presence of sperm in vaginal smears.  Sperm positive animals were considered to be at gestation day (GD) 0.  The dams were randomized into 6 groups per chemical, and housed in individual cages.  Each treatment group had a minimum of 8 pregnant females.

P-MEXP-8436

Standard Affymetrix Scan Protocol  Before you scan the probe array, follow the directions in this section on handling the probe array. If necessary, clean the glass surface of the probe array with a non-abrasive towel or tissue before scanning. Do not use alcohol to clean glass.  Before scanning the probe array cartridge, apply Tough-Spots™ to each of the two septa on the probe array cartridge to prevent the leaking of fluids from the cartridge during scanning.  1. On the back of the probe array cartridge, clean excess fluid from around septa. 2. Carefully apply one Tough-Spots to each of the two septa. Press to ensure that the spots remain flat. If the Tough-Spots do not apply smoothly, that is, if you observe bumps,bubbles, tears, or curled edges, do not attempt to smooth out the spot. Remove the spot and apply a new spot.   Scanning the Probe Array 1. Select Run start Scanner from the menu bar. Alternatively, click the Start Scan icon inthe tool bar.  AutoLoader with Barcoded GeneChips:  The Scanner dialog box and will ask you if the first 4 arrays are at room temperature. Click yes. Do not select the re-scan option. Before clicking OK make sure that all of the arrays are loaded into the autoloader in the proper position. The autoloader will review the chip inventory and count the number of  GeneChips loaded. (Max 48)  The Scanner begins scanning the probe array and acquiring data. When Scan inProgress is selected from the View menu, the probe array image appears on the screen as the scan progresses.   AutoLoader with Non Barcoded GeneChips 1. Select the experiment name that corresponds to the probe array to be scanned.A previously run experiment can also be selected by using the Include Scanned Experiments option box. After selecting this option, previously scanned experiments appear in the drop-down list. 2. By default, GeneChip® Scanner 3000, only one scan is required.  3. Once the experiment has been selected, click the Start button.   A dialog box prompts you to load an array into the scanner. 4. When you are using the GeneChip Scanner 3000, pixel resolution and wavelength are preset and cannot be changed. 5. Open the sample door on the scanner and insert the probe array into the holder. Do not force the probe array into the holder. Close the sample door of the scanner. 6. The scanner begins scanning the probe array and acquiring data. When Scan in Progress is selected from the View menu, the probe array image appears on the screen as the scan progresses. 

Transcription profiling by array of rat uterus after treatment with 17 alpha-ethynyl estradiol

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