Journal: Environmental Health Perspectives

Article Title: Neonatal Genistein Exposure and Glucocorticoid Signaling in the Adult Mouse Uterus

doi: 10.1289/EHP1575

Figure Lengend Snippet: Effect of neonatal genistein exposure on immediate glucocorticoid target gene expression. ( A ) Schematic representation of dosing schedule and experimental end points. Body and uterine weights (g) were determined on PND5 for control and genistein-treated mice. Data represent mean of 6 – 7 mice ± SEM . ** p < 0.01 determined by Student’s t -test. ( B and C ) Relative mRNA expression of Ppib , Gilz , Fkbp5 , Sgk1 , Klf13 , Per1 , 11 β h s d I I , and Il13ra2 as measured by qRT-PCR 4 h following subcutaneous injection of vehicle or 1 mg / kg Dex on PND5. ( B ) Basal gene expression was determined by comparing the vehicle-treated genistein group to vehicle-treated controls. ( C ) Fold change was determined by reporting values relative to vehicle-treated controls. Values are normalized to the reference gene peptidylprolyl isomerase B ( Ppib ). Bar graphs show the mean ± SEM from 3 to 5 animals. ** p < 0.01 as determined by ANOVA with Tukey’s post hoc analysis. Dex, dexamethasone; PND, postnatal day; qRT-PCR, quantitative real-time polymerase chain reaction; Treat, treatment; Veh, vehicle.

Article Snippet: Dexamethasone (Dex; 1,4 -pregnadien- 9 α -fluoro- 16 α -methyl- 11 β , 17, 21-triol-3, 20-dione; ≥ 98 % ) by thin layer chromatography (TLC) and E 2 ( 17 - β -estradiol ; ≥ 98 % TLC ) were purchased from Steraloids.

Techniques: Targeted Gene Expression, Control, Expressing, Quantitative RT-PCR, Injection, Gene Expression, Real-time Polymerase Chain Reaction

Journal: Environmental Health Perspectives

Article Title: Neonatal Genistein Exposure and Glucocorticoid Signaling in the Adult Mouse Uterus

doi: 10.1289/EHP1575

Figure Lengend Snippet: Effect of neonatal genistein exposure on glucocorticoid target gene expression prior to puberty. ( A ) Schematic representation of dosing schedule and experimental end points. ( B and C ) Relative mRNA expression of Ppib , Gilz , Fkbp5 , Sgk1 , Klf13 , Per1 , 11 β h s d I I , and Il13ra2 as measured by qRT-PCR 4 h following intraperitoneal injection of vehicle or 1 mg / kg Dex on PND21. ( B ) Basal gene expression was determined by comparing vehicle-treated genistein group to vehicle-treated controls. ( C ) Fold change was determined by reporting values relative to vehicle-treated controls. Values are normalized to the reference gene peptidylprolyl isomerase B ( Ppib ). Bar graphs show the mean ± SEM from 4 to 5 animals. ** p < 0.01 as determined by ANOVA with Tukey’s post hoc analysis. Dex, dexamethasone; PND, postnatal day; qRT-PCR, quantitative real-time polymerase chain reaction; Treat, treatment; Veh, vehicle.

Article Snippet: Dexamethasone (Dex; 1,4 -pregnadien- 9 α -fluoro- 16 α -methyl- 11 β , 17, 21-triol-3, 20-dione; ≥ 98 % ) by thin layer chromatography (TLC) and E 2 ( 17 - β -estradiol ; ≥ 98 % TLC ) were purchased from Steraloids.

Techniques: Targeted Gene Expression, Expressing, Quantitative RT-PCR, Injection, Gene Expression, Real-time Polymerase Chain Reaction

Journal: Environmental Health Perspectives

Article Title: Neonatal Genistein Exposure and Glucocorticoid Signaling in the Adult Mouse Uterus

doi: 10.1289/EHP1575

Figure Lengend Snippet: Effect of neonatal genistein exposure on glucocorticoid target gene expression in the adult uterus. ( A ) Schematic representation of dosing schedule, surgical intervention, and experimental end points. Body and uterine weight (g) was determined on PND56 for control and genistein-treated mice. Data represent mean of 17 – 21 mice ± SEM . ( B and C ) Relative mRNA expression of Ppib , Gilz , Fkbp5 , Sgk1 , Klf13 , Per1 , 11 β h s d I I , and Il13ra2 as measured by qRT-PCR 4 h following intraperitoneal injection of vehicle or 1 mg / kg Dex in adult ADV/OVX mice on PND56. ( B ) Basal expression was determined by comparing the vehicle-treated genistein group to vehicle-treated control group. ( C ) Fold change was determined by reporting values relative to vehicle-treated controls. Values are normalized to the reference gene Ppib . ( D ) Per1 , 11 β h s d I I , and Il13ra2 mRNA expression was determined by reporting values relative to vehicle-treated controls. Bar graphs show the mean ± SEM from 3 to 5 animals. * p < 0.05 , ** p < 0.01 as determined by ANOVA with Tukey’s post hoc analysis. ADX/OVX, adrenalectomized/ovariectomized; Dex, dexamethasone; PND, postnatal day; qRT-PCR, quantitative real-time polymerase chain reaction; Treat, treatment; Veh, vehicle.

Article Snippet: Dexamethasone (Dex; 1,4 -pregnadien- 9 α -fluoro- 16 α -methyl- 11 β , 17, 21-triol-3, 20-dione; ≥ 98 % ) by thin layer chromatography (TLC) and E 2 ( 17 - β -estradiol ; ≥ 98 % TLC ) were purchased from Steraloids.

Techniques: Targeted Gene Expression, Control, Expressing, Quantitative RT-PCR, Injection, Real-time Polymerase Chain Reaction

Journal: Environmental Health Perspectives

Article Title: Neonatal Genistein Exposure and Glucocorticoid Signaling in the Adult Mouse Uterus

doi: 10.1289/EHP1575

Figure Lengend Snippet: Effect of genistein exposure on genome-wide regulation by glucocorticoids. ( A ) Adult ADX/OVX (PND56) mice, exposed as neonates to control or genistein, were treated with vehicle (saline) or 1 mg / kg Dex for 4 h. Whole genome microarray analysis was performed on total RNA isolated from the uterus, with three biological replicates evaluated for each treatment group. Heat map and hierarchical clustering of significantly regulated genes ( p < 0.01 ), where red represents induced genes and green represents repressed genes. ( B ) The number of significantly regulated genes by treatment ( p < 0.01 ) were compared between the control and genistein groups by Venn diagram. ( C ) Significantly regulated genes were annotated with IPA software. The top five regulated canonical pathways are reported for the control and genistein groups. The total number of genes in the IPA-annotated pathway is listed on the right side of the graph. The total percentage of significantly regulated genes is depicted as the sum of induced and repressed genes. ( D ) Genes uniquely regulated by Dex in the control (2,799) and genistein (1,320) groups. The top five canonical pathways and diseases and biological functions were ranked and assigned an activation z -score (orange indicates induced; blue indicates repressed). ADX/OVX, adrenalectomized/ovariectomized; Con, control; Dex, dexamethasone; IPA, Ingenuity Pathway Analysis; Neo, neonatal; Veh, vehicle.

Article Snippet: Dexamethasone (Dex; 1,4 -pregnadien- 9 α -fluoro- 16 α -methyl- 11 β , 17, 21-triol-3, 20-dione; ≥ 98 % ) by thin layer chromatography (TLC) and E 2 ( 17 - β -estradiol ; ≥ 98 % TLC ) were purchased from Steraloids.

Techniques: Genome Wide, Control, Saline, Microarray, Isolation, Software, Activation Assay

Journal: Environmental Health Perspectives

Article Title: Neonatal Genistein Exposure and Glucocorticoid Signaling in the Adult Mouse Uterus

doi: 10.1289/EHP1575

Figure Lengend Snippet: Effect of neonatal genistein exposure on glucocorticoid-responsive genes differentially regulated in genistein-exposed mice. Adult ADX/OVX (PND56) mice, exposed as neonates to control, 50 mg / kg genistein or 10 μ g / kg E 2 on PND1–5, were treated with vehicle (saline) or 1 mg / kg Dex for 4 h. Relative mRNA expression in the uterus of Adamtsl2 , Ihh , Retn , and Ovgp1 were measured by qRT-PCR and values were normalized to the reference gene Ppib . Dex treatment for each neonatal exposure group (vehicle, genistein, or E 2 ) was set relative to vehicle treatment for that group (i.e., E 2 -exposed treated with Dex set relative to E 2 -exposed vehicle). The reported fold change from microarray analysis is listed below the qRT-PCR results. The results represent the mean of 4 – 5 animals ± SEM . ** p < 0.01 according to ANOVA with Turkey’s post hoc analysis. ADX/OVX, adrenalectomized/ovariectomized; Dex, dexamethasone; E 2 , estradiol; Gen, genistein; N/D, not determined to be significantly regulated by microarray analysis; PND, postnatal day; qRT-PCR, quantitative real-time polymerase chain reaction; Veh, vehicle.

Article Snippet: Dexamethasone (Dex; 1,4 -pregnadien- 9 α -fluoro- 16 α -methyl- 11 β , 17, 21-triol-3, 20-dione; ≥ 98 % ) by thin layer chromatography (TLC) and E 2 ( 17 - β -estradiol ; ≥ 98 % TLC ) were purchased from Steraloids.

Techniques: Control, Saline, Expressing, Quantitative RT-PCR, Microarray, Real-time Polymerase Chain Reaction

Journal: Environmental Health Perspectives

Article Title: Neonatal Genistein Exposure and Glucocorticoid Signaling in the Adult Mouse Uterus

doi: 10.1289/EHP1575

Figure Lengend Snippet: Effect of neonatal genistein exposure on ligand and GR expression. ( A ) Serum levels of corticosterone were measured in adult mice with intact ovaries and adrenal glands from serum collected between 0930 and 1130 hours in the morning. Data represent the mean of 11 – 12 animals ± SEM . ( B ) Relative mRNA expression of GR ( Nr3c1 ) was measured by qRT-PCR in adult ADX/OVX control and genistein-exposed mice. Expression was normalized to the reference gene Ppib and set relative to expression in control mice. GR protein levels, quantified by western blot analysis, were normalized to levels of the reference protein β -actin , which was not altered by treatment group (see Figure S5). GR protein levels were set relative to control mice. The results and image represent the mean of 4 – 5 animals ± SEM . ( C ) Representative images of GR expression and localization (red) in luminal epithelial and endometrial stromal cells. Hoescht 33342 was used to visualize nuclei (blue). Images were taken at 630 × . ADX/OVX, adrenalectomized/ovariectomized; Dex, dexamethasone; GR, glucocorticoid receptor; qRT-PCR, quantitative real-time polymerase chain reaction; Veh, vehicle.

Article Snippet: Dexamethasone (Dex; 1,4 -pregnadien- 9 α -fluoro- 16 α -methyl- 11 β , 17, 21-triol-3, 20-dione; ≥ 98 % ) by thin layer chromatography (TLC) and E 2 ( 17 - β -estradiol ; ≥ 98 % TLC ) were purchased from Steraloids.

Techniques: Expressing, Quantitative RT-PCR, Control, Western Blot, Real-time Polymerase Chain Reaction

Journal: Environmental Health Perspectives

Article Title: Neonatal Genistein Exposure and Glucocorticoid Signaling in the Adult Mouse Uterus

doi: 10.1289/EHP1575

Figure Lengend Snippet: Effect of neonatal genistein exposure on GR recruitment to specific GREs. Adult ADX/OVX PND56 mice, exposed neonatally to control or genistein, were treated with vehicle (saline) or 1 mg / kg Dex for 1.5 h. ChIP assays were performed with rabbit IgG or rabbit anti-GR antibody. Coimmunoprecipitated DNA was analyzed by qRT-PCR using primers to GREs and plotted relative to input DNA. ( A ) Relative fold enrichment of GR recruitment to Gilz , Sgk1 , and Klf13 as determined by ChIP assays. ( B ) Relative fold enrichment of GR recruitment to Per1 . The results represent the mean of 4 – 5 animals ± SEM . * p < 0.05 , ** p < 0.01 as determined by Student’s t -test. ADX/OVX, adrenalectomized/ovariectomized; ChIP, chromatin immunoprecipitation; Dex, dexamethasone; GR, glucocorticoid receptor; GRE, glucocorticoid response element; IgG, immunoglobulin G; PND, postnatal day; Veh, vehicle.

Article Snippet: Dexamethasone (Dex; 1,4 -pregnadien- 9 α -fluoro- 16 α -methyl- 11 β , 17, 21-triol-3, 20-dione; ≥ 98 % ) by thin layer chromatography (TLC) and E 2 ( 17 - β -estradiol ; ≥ 98 % TLC ) were purchased from Steraloids.

Techniques: Control, Saline, Quantitative RT-PCR, Chromatin Immunoprecipitation

Journal: Environmental Health Perspectives

Article Title: Neonatal Genistein Exposure and Glucocorticoid Signaling in the Adult Mouse Uterus

doi: 10.1289/EHP1575

Figure Lengend Snippet: Effect of disruption of glucocorticoid signaling on glucocorticoid antagonism of uterine estrogen signaling. ( A ) Representative images of adult uteri following 24-h treatment with vehicle (saline), 10 μ g / kg E 2 , or 10 μ g / kg E 2 and 1 mg / kg Dex. ( B ) Proliferative marker MKi67 (red) by immunofluorescence in cross-sections from the control and genistein groups following 24-h treatment with vehicle, E 2 , or E 2 and Dex. Hoescht 33342 staining of the nuclei is shown in blue. Images taken at 630 × . The percentage of Ki67 positive cells was determined by the ratio of Ki67 positive epithelial cells/total number of epithelial cells per image. ( C ) IPA identified genes involved in cellular growth and proliferation as significantly regulated by Dex. Glucocorticoid treatment regulated 175 genes associated with proliferation of connective tissue cells in the uterus of control mice. Green indicates repression and red indicates up-regulation of gene expression. Dex, dexamethasone; E 2 , estradiol; IPA, Ingenuity Pathway Analysis.

Article Snippet: Dexamethasone (Dex; 1,4 -pregnadien- 9 α -fluoro- 16 α -methyl- 11 β , 17, 21-triol-3, 20-dione; ≥ 98 % ) by thin layer chromatography (TLC) and E 2 ( 17 - β -estradiol ; ≥ 98 % TLC ) were purchased from Steraloids.

Techniques: Disruption, Saline, Marker, Immunofluorescence, Control, Staining, Gene Expression