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Search results for the GEO ID: GSE13883
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GSM349598
GPL1355
HPTE_0_Basal 1 Granulosa cells from post-natal days 21-27 female Sprague Dawley Rats Charles River outbred immature female rats Briefly, intact, non-primed, immature female rats (21-27 days old) were sacrificed by cervical dislocation. Ovaries were removed from the animals, cleaned free of associated fat, oviduct, and bursa ovary, and then placed in ice-cold DMEM/F-12. Granulosa cells were isolated using a non-enzymatic needle puncture method with a sterile bundle of beading needles to release the cells from follicles. Cell viability was determined by the trypan blue exclusion method. The cells were plated and cultured (24-well plates) at approximately 3-4 x 105 viable cells/ml/well and incubated at 37°C for 24 h in the DMEM/F-12 containing FBS (5%) in an atmosphere of 5% CO2 in air. Following the 24 h acclimation period, the medium was replaced with serum-free DMEM/F12 containing androstenedione (0.1 M) as a substrate for aromatization. Cells were treated with increasing doses of HPTE (0, 1, 5, and 10µM) in the absence (basal) or presence of 3 ng FSH/ml or 1 mM cAMP for 48 h. The doses of FSH and cAMP were selected based on a dose-response curve that provided a maximum level of stimulation for E2 production (data not shown). Cultures were terminated at 48 h following the addition of treatments. Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Cat # 74106) followed by DNase I treatment. RNA quality was assessed by electrophoresis using the Agilent Bioanalyzer 2100 and spectrophotometric analysis prior to cDNA synthesis (data not shown). Forty nanograms of total RNA from each sample were used to generate a high fidelity cDNA for array hybridization using the NuGen Ovation Biotin RNA Amplification and Labeling system (NuGen). After fragmentation and biotin labeling, the samples were hybridized to Affymetrix Rat Genome 230 2.0 arrays, which have eleven pairs of oligonucleotide probes per chip. Washing and staining of all arrays were carried out in the Affymetrix fluidics module as per the manufacturer’s protocol. The detection and quantification of target hybridization was performed with an Affymetrix GeneChip Scanner. Three separate samples were analyzed for each of the treatment groups.Initially, raw data were analyzed using Robust Multichip Analysis (RMA) with a logarithmic base 2 conversion utilizing the Affymetrix ArrayAssist Suite (Affymetrix, Inc., Santa Clara, CA). One-way ANOVA analysis of each group (basal, FSH, or cAMP) was used to determine genes that are significantly affected by HPTE. This was followed by an unpaired t-test to determine changes in global gene expression between HPTE-treated groups and the control groups to determine the levels of fold change. All statistical analyses were performed using P < 0.005 as the level of statistical significance.
GSM349599
GPL1355
HPTE_0_FSH 1 Granulosa cells from post-natal days 21-27 female Sprague Dawley Rats Charles River outbred immature female rats Briefly, intact, non-primed, immature female rats (21-27 days old) were sacrificed by cervical dislocation. Ovaries were removed from the animals, cleaned free of associated fat, oviduct, and bursa ovary, and then placed in ice-cold DMEM/F-12. Granulosa cells were isolated using a non-enzymatic needle puncture method with a sterile bundle of beading needles to release the cells from follicles. Cell viability was determined by the trypan blue exclusion method. The cells were plated and cultured (24-well plates) at approximately 3-4 x 105 viable cells/ml/well and incubated at 37°C for 24 h in the DMEM/F-12 containing FBS (5%) in an atmosphere of 5% CO2 in air. Following the 24 h acclimation period, the medium was replaced with serum-free DMEM/F12 containing androstenedione (0.1 M) as a substrate for aromatization. Cells were treated with increasing doses of HPTE (0, 1, 5, and 10µM) in the absence (basal) or presence of 3 ng FSH/ml or 1 mM cAMP for 48 h. The doses of FSH and cAMP were selected based on a dose-response curve that provided a maximum level of stimulation for E2 production (data not shown). Cultures were terminated at 48 h following the addition of treatments. Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Cat # 74106) followed by DNase I treatment. RNA quality was assessed by electrophoresis using the Agilent Bioanalyzer 2100 and spectrophotometric analysis prior to cDNA synthesis (data not shown). Forty nanograms of total RNA from each sample were used to generate a high fidelity cDNA for array hybridization using the NuGen Ovation Biotin RNA Amplification and Labeling system (NuGen). After fragmentation and biotin labeling, the samples were hybridized to Affymetrix Rat Genome 230 2.0 arrays, which have eleven pairs of oligonucleotide probes per chip. Washing and staining of all arrays were carried out in the Affymetrix fluidics module as per the manufacturer’s protocol. The detection and quantification of target hybridization was performed with an Affymetrix GeneChip Scanner. Three separate samples were analyzed for each of the treatment groups.Initially, raw data were analyzed using Robust Multichip Analysis (RMA) with a logarithmic base 2 conversion utilizing the Affymetrix ArrayAssist Suite (Affymetrix, Inc., Santa Clara, CA). One-way ANOVA analysis of each group (basal, FSH, or cAMP) was used to determine genes that are significantly affected by HPTE. This was followed by an unpaired t-test to determine changes in global gene expression between HPTE-treated groups and the control groups to determine the levels of fold change. All statistical analyses were performed using P < 0.005 as the level of statistical significance.
GSM349636
GPL1355
HPTE_0_cAMP 1 Granulosa cells from post-natal days 21-27 female Sprague Dawley Rats Charles River outbred immature female rats Briefly, intact, non-primed, immature female rats (21-27 days old) were sacrificed by cervical dislocation. Ovaries were removed from the animals, cleaned free of associated fat, oviduct, and bursa ovary, and then placed in ice-cold DMEM/F-12. Granulosa cells were isolated using a non-enzymatic needle puncture method with a sterile bundle of beading needles to release the cells from follicles. Cell viability was determined by the trypan blue exclusion method. The cells were plated and cultured (24-well plates) at approximately 3-4 x 105 viable cells/ml/well and incubated at 37°C for 24 h in the DMEM/F-12 containing FBS (5%) in an atmosphere of 5% CO2 in air. Following the 24 h acclimation period, the medium was replaced with serum-free DMEM/F12 containing androstenedione (0.1 M) as a substrate for aromatization. Cells were treated with increasing doses of HPTE (0, 1, 5, and 10µM) in the absence (basal) or presence of 3 ng FSH/ml or 1 mM cAMP for 48 h. The doses of FSH and cAMP were selected based on a dose-response curve that provided a maximum level of stimulation for E2 production (data not shown). Cultures were terminated at 48 h following the addition of treatments. Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Cat # 74106) followed by DNase I treatment. RNA quality was assessed by electrophoresis using the Agilent Bioanalyzer 2100 and spectrophotometric analysis prior to cDNA synthesis (data not shown). Forty nanograms of total RNA from each sample were used to generate a high fidelity cDNA for array hybridization using the NuGen Ovation Biotin RNA Amplification and Labeling system (NuGen). After fragmentation and biotin labeling, the samples were hybridized to Affymetrix Rat Genome 230 2.0 arrays, which have eleven pairs of oligonucleotide probes per chip. Washing and staining of all arrays were carried out in the Affymetrix fluidics module as per the manufacturer’s protocol. The detection and quantification of target hybridization was performed with an Affymetrix GeneChip Scanner. Three separate samples were analyzed for each of the treatment groups.Initially, raw data were analyzed using Robust Multichip Analysis (RMA) with a logarithmic base 2 conversion utilizing the Affymetrix ArrayAssist Suite (Affymetrix, Inc., Santa Clara, CA). One-way ANOVA analysis of each group (basal, FSH, or cAMP) was used to determine genes that are significantly affected by HPTE. This was followed by an unpaired t-test to determine changes in global gene expression between HPTE-treated groups and the control groups to determine the levels of fold change. All statistical analyses were performed using P < 0.005 as the level of statistical significance.
GSM349637
GPL1355
HPTE_1_Basal 1 Granulosa cells from post-natal days 21-27 female Sprague Dawley Rats Charles River outbred immature female rats Briefly, intact, non-primed, immature female rats (21-27 days old) were sacrificed by cervical dislocation. Ovaries were removed from the animals, cleaned free of associated fat, oviduct, and bursa ovary, and then placed in ice-cold DMEM/F-12. Granulosa cells were isolated using a non-enzymatic needle puncture method with a sterile bundle of beading needles to release the cells from follicles. Cell viability was determined by the trypan blue exclusion method. The cells were plated and cultured (24-well plates) at approximately 3-4 x 105 viable cells/ml/well and incubated at 37°C for 24 h in the DMEM/F-12 containing FBS (5%) in an atmosphere of 5% CO2 in air. Following the 24 h acclimation period, the medium was replaced with serum-free DMEM/F12 containing androstenedione (0.1 M) as a substrate for aromatization. Cells were treated with increasing doses of HPTE (0, 1, 5, and 10µM) in the absence (basal) or presence of 3 ng FSH/ml or 1 mM cAMP for 48 h. The doses of FSH and cAMP were selected based on a dose-response curve that provided a maximum level of stimulation for E2 production (data not shown). Cultures were terminated at 48 h following the addition of treatments. Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Cat # 74106) followed by DNase I treatment. RNA quality was assessed by electrophoresis using the Agilent Bioanalyzer 2100 and spectrophotometric analysis prior to cDNA synthesis (data not shown). Forty nanograms of total RNA from each sample were used to generate a high fidelity cDNA for array hybridization using the NuGen Ovation Biotin RNA Amplification and Labeling system (NuGen). After fragmentation and biotin labeling, the samples were hybridized to Affymetrix Rat Genome 230 2.0 arrays, which have eleven pairs of oligonucleotide probes per chip. Washing and staining of all arrays were carried out in the Affymetrix fluidics module as per the manufacturer’s protocol. The detection and quantification of target hybridization was performed with an Affymetrix GeneChip Scanner. Three separate samples were analyzed for each of the treatment groups.Initially, raw data were analyzed using Robust Multichip Analysis (RMA) with a logarithmic base 2 conversion utilizing the Affymetrix ArrayAssist Suite (Affymetrix, Inc., Santa Clara, CA). One-way ANOVA analysis of each group (basal, FSH, or cAMP) was used to determine genes that are significantly affected by HPTE. This was followed by an unpaired t-test to determine changes in global gene expression between HPTE-treated groups and the control groups to determine the levels of fold change. All statistical analyses were performed using P < 0.005 as the level of statistical significance.
GSM349638
GPL1355
HPTE_1_FSH 1 Granulosa cells from post-natal days 21-27 female Sprague Dawley Rats Charles River outbred immature female rats Briefly, intact, non-primed, immature female rats (21-27 days old) were sacrificed by cervical dislocation. Ovaries were removed from the animals, cleaned free of associated fat, oviduct, and bursa ovary, and then placed in ice-cold DMEM/F-12. Granulosa cells were isolated using a non-enzymatic needle puncture method with a sterile bundle of beading needles to release the cells from follicles. Cell viability was determined by the trypan blue exclusion method. The cells were plated and cultured (24-well plates) at approximately 3-4 x 105 viable cells/ml/well and incubated at 37°C for 24 h in the DMEM/F-12 containing FBS (5%) in an atmosphere of 5% CO2 in air. Following the 24 h acclimation period, the medium was replaced with serum-free DMEM/F12 containing androstenedione (0.1 M) as a substrate for aromatization. Cells were treated with increasing doses of HPTE (0, 1, 5, and 10µM) in the absence (basal) or presence of 3 ng FSH/ml or 1 mM cAMP for 48 h. The doses of FSH and cAMP were selected based on a dose-response curve that provided a maximum level of stimulation for E2 production (data not shown). Cultures were terminated at 48 h following the addition of treatments. Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Cat # 74106) followed by DNase I treatment. RNA quality was assessed by electrophoresis using the Agilent Bioanalyzer 2100 and spectrophotometric analysis prior to cDNA synthesis (data not shown). Forty nanograms of total RNA from each sample were used to generate a high fidelity cDNA for array hybridization using the NuGen Ovation Biotin RNA Amplification and Labeling system (NuGen). After fragmentation and biotin labeling, the samples were hybridized to Affymetrix Rat Genome 230 2.0 arrays, which have eleven pairs of oligonucleotide probes per chip. Washing and staining of all arrays were carried out in the Affymetrix fluidics module as per the manufacturer’s protocol. The detection and quantification of target hybridization was performed with an Affymetrix GeneChip Scanner. Three separate samples were analyzed for each of the treatment groups.Initially, raw data were analyzed using Robust Multichip Analysis (RMA) with a logarithmic base 2 conversion utilizing the Affymetrix ArrayAssist Suite (Affymetrix, Inc., Santa Clara, CA). One-way ANOVA analysis of each group (basal, FSH, or cAMP) was used to determine genes that are significantly affected by HPTE. This was followed by an unpaired t-test to determine changes in global gene expression between HPTE-treated groups and the control groups to determine the levels of fold change. All statistical analyses were performed using P < 0.005 as the level of statistical significance.
GSM349639
GPL1355
HPTE_1_cAMP 1 Granulosa cells from post-natal days 21-27 female Sprague Dawley Rats Charles River outbred immature female rats Briefly, intact, non-primed, immature female rats (21-27 days old) were sacrificed by cervical dislocation. Ovaries were removed from the animals, cleaned free of associated fat, oviduct, and bursa ovary, and then placed in ice-cold DMEM/F-12. Granulosa cells were isolated using a non-enzymatic needle puncture method with a sterile bundle of beading needles to release the cells from follicles. Cell viability was determined by the trypan blue exclusion method. The cells were plated and cultured (24-well plates) at approximately 3-4 x 105 viable cells/ml/well and incubated at 37°C for 24 h in the DMEM/F-12 containing FBS (5%) in an atmosphere of 5% CO2 in air. Following the 24 h acclimation period, the medium was replaced with serum-free DMEM/F12 containing androstenedione (0.1 M) as a substrate for aromatization. Cells were treated with increasing doses of HPTE (0, 1, 5, and 10µM) in the absence (basal) or presence of 3 ng FSH/ml or 1 mM cAMP for 48 h. The doses of FSH and cAMP were selected based on a dose-response curve that provided a maximum level of stimulation for E2 production (data not shown). Cultures were terminated at 48 h following the addition of treatments. Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Cat # 74106) followed by DNase I treatment. RNA quality was assessed by electrophoresis using the Agilent Bioanalyzer 2100 and spectrophotometric analysis prior to cDNA synthesis (data not shown). Forty nanograms of total RNA from each sample were used to generate a high fidelity cDNA for array hybridization using the NuGen Ovation Biotin RNA Amplification and Labeling system (NuGen). After fragmentation and biotin labeling, the samples were hybridized to Affymetrix Rat Genome 230 2.0 arrays, which have eleven pairs of oligonucleotide probes per chip. Washing and staining of all arrays were carried out in the Affymetrix fluidics module as per the manufacturer’s protocol. The detection and quantification of target hybridization was performed with an Affymetrix GeneChip Scanner. Three separate samples were analyzed for each of the treatment groups.Initially, raw data were analyzed using Robust Multichip Analysis (RMA) with a logarithmic base 2 conversion utilizing the Affymetrix ArrayAssist Suite (Affymetrix, Inc., Santa Clara, CA). One-way ANOVA analysis of each group (basal, FSH, or cAMP) was used to determine genes that are significantly affected by HPTE. This was followed by an unpaired t-test to determine changes in global gene expression between HPTE-treated groups and the control groups to determine the levels of fold change. All statistical analyses were performed using P < 0.005 as the level of statistical significance.
GSM349640
GPL1355
HPTE_5_Basal 1 Granulosa cells from post-natal days 21-27 female Sprague Dawley Rats Charles River outbred immature female rats Briefly, intact, non-primed, immature female rats (21-27 days old) were sacrificed by cervical dislocation. Ovaries were removed from the animals, cleaned free of associated fat, oviduct, and bursa ovary, and then placed in ice-cold DMEM/F-12. Granulosa cells were isolated using a non-enzymatic needle puncture method with a sterile bundle of beading needles to release the cells from follicles. Cell viability was determined by the trypan blue exclusion method. The cells were plated and cultured (24-well plates) at approximately 3-4 x 105 viable cells/ml/well and incubated at 37°C for 24 h in the DMEM/F-12 containing FBS (5%) in an atmosphere of 5% CO2 in air. Following the 24 h acclimation period, the medium was replaced with serum-free DMEM/F12 containing androstenedione (0.1 M) as a substrate for aromatization. Cells were treated with increasing doses of HPTE (0, 1, 5, and 10µM) in the absence (basal) or presence of 3 ng FSH/ml or 1 mM cAMP for 48 h. The doses of FSH and cAMP were selected based on a dose-response curve that provided a maximum level of stimulation for E2 production (data not shown). Cultures were terminated at 48 h following the addition of treatments. Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Cat # 74106) followed by DNase I treatment. RNA quality was assessed by electrophoresis using the Agilent Bioanalyzer 2100 and spectrophotometric analysis prior to cDNA synthesis (data not shown). Forty nanograms of total RNA from each sample were used to generate a high fidelity cDNA for array hybridization using the NuGen Ovation Biotin RNA Amplification and Labeling system (NuGen). After fragmentation and biotin labeling, the samples were hybridized to Affymetrix Rat Genome 230 2.0 arrays, which have eleven pairs of oligonucleotide probes per chip. Washing and staining of all arrays were carried out in the Affymetrix fluidics module as per the manufacturer’s protocol. The detection and quantification of target hybridization was performed with an Affymetrix GeneChip Scanner. Three separate samples were analyzed for each of the treatment groups.Initially, raw data were analyzed using Robust Multichip Analysis (RMA) with a logarithmic base 2 conversion utilizing the Affymetrix ArrayAssist Suite (Affymetrix, Inc., Santa Clara, CA). One-way ANOVA analysis of each group (basal, FSH, or cAMP) was used to determine genes that are significantly affected by HPTE. This was followed by an unpaired t-test to determine changes in global gene expression between HPTE-treated groups and the control groups to determine the levels of fold change. All statistical analyses were performed using P < 0.005 as the level of statistical significance.
GSM349641
GPL1355
HPTE_5_FSH 1 Granulosa cells from post-natal days 21-27 female Sprague Dawley Rats Charles River outbred immature female rats Briefly, intact, non-primed, immature female rats (21-27 days old) were sacrificed by cervical dislocation. Ovaries were removed from the animals, cleaned free of associated fat, oviduct, and bursa ovary, and then placed in ice-cold DMEM/F-12. Granulosa cells were isolated using a non-enzymatic needle puncture method with a sterile bundle of beading needles to release the cells from follicles. Cell viability was determined by the trypan blue exclusion method. The cells were plated and cultured (24-well plates) at approximately 3-4 x 105 viable cells/ml/well and incubated at 37°C for 24 h in the DMEM/F-12 containing FBS (5%) in an atmosphere of 5% CO2 in air. Following the 24 h acclimation period, the medium was replaced with serum-free DMEM/F12 containing androstenedione (0.1 M) as a substrate for aromatization. Cells were treated with increasing doses of HPTE (0, 1, 5, and 10µM) in the absence (basal) or presence of 3 ng FSH/ml or 1 mM cAMP for 48 h. The doses of FSH and cAMP were selected based on a dose-response curve that provided a maximum level of stimulation for E2 production (data not shown). Cultures were terminated at 48 h following the addition of treatments. Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Cat # 74106) followed by DNase I treatment. RNA quality was assessed by electrophoresis using the Agilent Bioanalyzer 2100 and spectrophotometric analysis prior to cDNA synthesis (data not shown). Forty nanograms of total RNA from each sample were used to generate a high fidelity cDNA for array hybridization using the NuGen Ovation Biotin RNA Amplification and Labeling system (NuGen). After fragmentation and biotin labeling, the samples were hybridized to Affymetrix Rat Genome 230 2.0 arrays, which have eleven pairs of oligonucleotide probes per chip. Washing and staining of all arrays were carried out in the Affymetrix fluidics module as per the manufacturer’s protocol. The detection and quantification of target hybridization was performed with an Affymetrix GeneChip Scanner. Three separate samples were analyzed for each of the treatment groups.Initially, raw data were analyzed using Robust Multichip Analysis (RMA) with a logarithmic base 2 conversion utilizing the Affymetrix ArrayAssist Suite (Affymetrix, Inc., Santa Clara, CA). One-way ANOVA analysis of each group (basal, FSH, or cAMP) was used to determine genes that are significantly affected by HPTE. This was followed by an unpaired t-test to determine changes in global gene expression between HPTE-treated groups and the control groups to determine the levels of fold change. All statistical analyses were performed using P < 0.005 as the level of statistical significance.
GSM349642
GPL1355
HPTE_5_cAMP 1 Granulosa cells from post-natal days 21-27 female Sprague Dawley Rats Charles River outbred immature female rats Briefly, intact, non-primed, immature female rats (21-27 days old) were sacrificed by cervical dislocation. Ovaries were removed from the animals, cleaned free of associated fat, oviduct, and bursa ovary, and then placed in ice-cold DMEM/F-12. Granulosa cells were isolated using a non-enzymatic needle puncture method with a sterile bundle of beading needles to release the cells from follicles. Cell viability was determined by the trypan blue exclusion method. The cells were plated and cultured (24-well plates) at approximately 3-4 x 105 viable cells/ml/well and incubated at 37°C for 24 h in the DMEM/F-12 containing FBS (5%) in an atmosphere of 5% CO2 in air. Following the 24 h acclimation period, the medium was replaced with serum-free DMEM/F12 containing androstenedione (0.1 M) as a substrate for aromatization. Cells were treated with increasing doses of HPTE (0, 1, 5, and 10µM) in the absence (basal) or presence of 3 ng FSH/ml or 1 mM cAMP for 48 h. The doses of FSH and cAMP were selected based on a dose-response curve that provided a maximum level of stimulation for E2 production (data not shown). Cultures were terminated at 48 h following the addition of treatments. Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Cat # 74106) followed by DNase I treatment. RNA quality was assessed by electrophoresis using the Agilent Bioanalyzer 2100 and spectrophotometric analysis prior to cDNA synthesis (data not shown). Forty nanograms of total RNA from each sample were used to generate a high fidelity cDNA for array hybridization using the NuGen Ovation Biotin RNA Amplification and Labeling system (NuGen). After fragmentation and biotin labeling, the samples were hybridized to Affymetrix Rat Genome 230 2.0 arrays, which have eleven pairs of oligonucleotide probes per chip. Washing and staining of all arrays were carried out in the Affymetrix fluidics module as per the manufacturer’s protocol. The detection and quantification of target hybridization was performed with an Affymetrix GeneChip Scanner. Three separate samples were analyzed for each of the treatment groups.Initially, raw data were analyzed using Robust Multichip Analysis (RMA) with a logarithmic base 2 conversion utilizing the Affymetrix ArrayAssist Suite (Affymetrix, Inc., Santa Clara, CA). One-way ANOVA analysis of each group (basal, FSH, or cAMP) was used to determine genes that are significantly affected by HPTE. This was followed by an unpaired t-test to determine changes in global gene expression between HPTE-treated groups and the control groups to determine the levels of fold change. All statistical analyses were performed using P < 0.005 as the level of statistical significance.
GSM349643
GPL1355
HPTE_10_Basal 1 Granulosa cells from post-natal days 21-27 female Sprague Dawley Rats Charles River outbred immature female rats Briefly, intact, non-primed, immature female rats (21-27 days old) were sacrificed by cervical dislocation. Ovaries were removed from the animals, cleaned free of associated fat, oviduct, and bursa ovary, and then placed in ice-cold DMEM/F-12. Granulosa cells were isolated using a non-enzymatic needle puncture method with a sterile bundle of beading needles to release the cells from follicles. Cell viability was determined by the trypan blue exclusion method. The cells were plated and cultured (24-well plates) at approximately 3-4 x 105 viable cells/ml/well and incubated at 37°C for 24 h in the DMEM/F-12 containing FBS (5%) in an atmosphere of 5% CO2 in air. Following the 24 h acclimation period, the medium was replaced with serum-free DMEM/F12 containing androstenedione (0.1 M) as a substrate for aromatization. Cells were treated with increasing doses of HPTE (0, 1, 5, and 10µM) in the absence (basal) or presence of 3 ng FSH/ml or 1 mM cAMP for 48 h. The doses of FSH and cAMP were selected based on a dose-response curve that provided a maximum level of stimulation for E2 production (data not shown). Cultures were terminated at 48 h following the addition of treatments. Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Cat # 74106) followed by DNase I treatment. RNA quality was assessed by electrophoresis using the Agilent Bioanalyzer 2100 and spectrophotometric analysis prior to cDNA synthesis (data not shown). Forty nanograms of total RNA from each sample were used to generate a high fidelity cDNA for array hybridization using the NuGen Ovation Biotin RNA Amplification and Labeling system (NuGen). After fragmentation and biotin labeling, the samples were hybridized to Affymetrix Rat Genome 230 2.0 arrays, which have eleven pairs of oligonucleotide probes per chip. Washing and staining of all arrays were carried out in the Affymetrix fluidics module as per the manufacturer’s protocol. The detection and quantification of target hybridization was performed with an Affymetrix GeneChip Scanner. Three separate samples were analyzed for each of the treatment groups.Initially, raw data were analyzed using Robust Multichip Analysis (RMA) with a logarithmic base 2 conversion utilizing the Affymetrix ArrayAssist Suite (Affymetrix, Inc., Santa Clara, CA). One-way ANOVA analysis of each group (basal, FSH, or cAMP) was used to determine genes that are significantly affected by HPTE. This was followed by an unpaired t-test to determine changes in global gene expression between HPTE-treated groups and the control groups to determine the levels of fold change. All statistical analyses were performed using P < 0.005 as the level of statistical significance.
GSM349651
GPL1355
HPTE_10_FSH 1 Granulosa cells from post-natal days 21-27 female Sprague Dawley Rats Charles River outbred immature female rats Briefly, intact, non-primed, immature female rats (21-27 days old) were sacrificed by cervical dislocation. Ovaries were removed from the animals, cleaned free of associated fat, oviduct, and bursa ovary, and then placed in ice-cold DMEM/F-12. Granulosa cells were isolated using a non-enzymatic needle puncture method with a sterile bundle of beading needles to release the cells from follicles. Cell viability was determined by the trypan blue exclusion method. The cells were plated and cultured (24-well plates) at approximately 3-4 x 105 viable cells/ml/well and incubated at 37°C for 24 h in the DMEM/F-12 containing FBS (5%) in an atmosphere of 5% CO2 in air. Following the 24 h acclimation period, the medium was replaced with serum-free DMEM/F12 containing androstenedione (0.1 M) as a substrate for aromatization. Cells were treated with increasing doses of HPTE (0, 1, 5, and 10µM) in the absence (basal) or presence of 3 ng FSH/ml or 1 mM cAMP for 48 h. The doses of FSH and cAMP were selected based on a dose-response curve that provided a maximum level of stimulation for E2 production (data not shown). Cultures were terminated at 48 h following the addition of treatments. Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Cat # 74106) followed by DNase I treatment. RNA quality was assessed by electrophoresis using the Agilent Bioanalyzer 2100 and spectrophotometric analysis prior to cDNA synthesis (data not shown). Forty nanograms of total RNA from each sample were used to generate a high fidelity cDNA for array hybridization using the NuGen Ovation Biotin RNA Amplification and Labeling system (NuGen). After fragmentation and biotin labeling, the samples were hybridized to Affymetrix Rat Genome 230 2.0 arrays, which have eleven pairs of oligonucleotide probes per chip. Washing and staining of all arrays were carried out in the Affymetrix fluidics module as per the manufacturer’s protocol. The detection and quantification of target hybridization was performed with an Affymetrix GeneChip Scanner. Three separate samples were analyzed for each of the treatment groups.Initially, raw data were analyzed using Robust Multichip Analysis (RMA) with a logarithmic base 2 conversion utilizing the Affymetrix ArrayAssist Suite (Affymetrix, Inc., Santa Clara, CA). One-way ANOVA analysis of each group (basal, FSH, or cAMP) was used to determine genes that are significantly affected by HPTE. This was followed by an unpaired t-test to determine changes in global gene expression between HPTE-treated groups and the control groups to determine the levels of fold change. All statistical analyses were performed using P < 0.005 as the level of statistical significance.
GSM349652
GPL1355
HPTE_10_cAMP 1 Granulosa cells from post-natal days 21-27 female Sprague Dawley Rats Charles River outbred immature female rats Briefly, intact, non-primed, immature female rats (21-27 days old) were sacrificed by cervical dislocation. Ovaries were removed from the animals, cleaned free of associated fat, oviduct, and bursa ovary, and then placed in ice-cold DMEM/F-12. Granulosa cells were isolated using a non-enzymatic needle puncture method with a sterile bundle of beading needles to release the cells from follicles. Cell viability was determined by the trypan blue exclusion method. The cells were plated and cultured (24-well plates) at approximately 3-4 x 105 viable cells/ml/well and incubated at 37°C for 24 h in the DMEM/F-12 containing FBS (5%) in an atmosphere of 5% CO2 in air. Following the 24 h acclimation period, the medium was replaced with serum-free DMEM/F12 containing androstenedione (0.1 M) as a substrate for aromatization. Cells were treated with increasing doses of HPTE (0, 1, 5, and 10µM) in the absence (basal) or presence of 3 ng FSH/ml or 1 mM cAMP for 48 h. The doses of FSH and cAMP were selected based on a dose-response curve that provided a maximum level of stimulation for E2 production (data not shown). Cultures were terminated at 48 h following the addition of treatments. Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Cat # 74106) followed by DNase I treatment. RNA quality was assessed by electrophoresis using the Agilent Bioanalyzer 2100 and spectrophotometric analysis prior to cDNA synthesis (data not shown). Forty nanograms of total RNA from each sample were used to generate a high fidelity cDNA for array hybridization using the NuGen Ovation Biotin RNA Amplification and Labeling system (NuGen). After fragmentation and biotin labeling, the samples were hybridized to Affymetrix Rat Genome 230 2.0 arrays, which have eleven pairs of oligonucleotide probes per chip. Washing and staining of all arrays were carried out in the Affymetrix fluidics module as per the manufacturer’s protocol. The detection and quantification of target hybridization was performed with an Affymetrix GeneChip Scanner. Three separate samples were analyzed for each of the treatment groups.Initially, raw data were analyzed using Robust Multichip Analysis (RMA) with a logarithmic base 2 conversion utilizing the Affymetrix ArrayAssist Suite (Affymetrix, Inc., Santa Clara, CA). One-way ANOVA analysis of each group (basal, FSH, or cAMP) was used to determine genes that are significantly affected by HPTE. This was followed by an unpaired t-test to determine changes in global gene expression between HPTE-treated groups and the control groups to determine the levels of fold change. All statistical analyses were performed using P < 0.005 as the level of statistical significance.
GSM349691
GPL1355
HPTE_0_ Basal 2 Granulosa cells from post-natal days 21-27 female Sprague Dawley Rats Charles River outbred immature female rats Briefly, intact, non-primed, immature female rats (21-27 days old) were sacrificed by cervical dislocation. Ovaries were removed from the animals, cleaned free of associated fat, oviduct, and bursa ovary, and then placed in ice-cold DMEM/F-12. Granulosa cells were isolated using a non-enzymatic needle puncture method with a sterile bundle of beading needles to release the cells from follicles. Cell viability was determined by the trypan blue exclusion method. The cells were plated and cultured (24-well plates) at approximately 3-4 x 105 viable cells/ml/well and incubated at 37°C for 24 h in the DMEM/F-12 containing FBS (5%) in an atmosphere of 5% CO2 in air. Following the 24 h acclimation period, the medium was replaced with serum-free DMEM/F12 containing androstenedione (0.1 M) as a substrate for aromatization. Cells were treated with increasing doses of HPTE (0, 1, 5, and 10µM) in the absence (basal) or presence of 3 ng FSH/ml or 1 mM cAMP for 48 h. The doses of FSH and cAMP were selected based on a dose-response curve that provided a maximum level of stimulation for E2 production (data not shown). Cultures were terminated at 48 h following the addition of treatments. Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Cat # 74106) followed by DNase I treatment. RNA quality was assessed by electrophoresis using the Agilent Bioanalyzer 2100 and spectrophotometric analysis prior to cDNA synthesis (data not shown). Forty nanograms of total RNA from each sample were used to generate a high fidelity cDNA for array hybridization using the NuGen Ovation Biotin RNA Amplification and Labeling system (NuGen). After fragmentation and biotin labeling, the samples were hybridized to Affymetrix Rat Genome 230 2.0 arrays, which have eleven pairs of oligonucleotide probes per chip. Washing and staining of all arrays were carried out in the Affymetrix fluidics module as per the manufacturer’s protocol. The detection and quantification of target hybridization was performed with an Affymetrix GeneChip Scanner. Three separate samples were analyzed for each of the treatment groups.Initially, raw data were analyzed using Robust Multichip Analysis (RMA) with a logarithmic base 2 conversion utilizing the Affymetrix ArrayAssist Suite (Affymetrix, Inc., Santa Clara, CA). One-way ANOVA analysis of each group (basal, FSH, or cAMP) was used to determine genes that are significantly affected by HPTE. This was followed by an unpaired t-test to determine changes in global gene expression between HPTE-treated groups and the control groups to determine the levels of fold change. All statistical analyses were performed using P < 0.005 as the level of statistical significance.
GSM349692
GPL1355
HPTE_0_FSH 2 Granulosa cells from post-natal days 21-27 female Sprague Dawley Rats Charles River outbred immature female rats Briefly, intact, non-primed, immature female rats (21-27 days old) were sacrificed by cervical dislocation. Ovaries were removed from the animals, cleaned free of associated fat, oviduct, and bursa ovary, and then placed in ice-cold DMEM/F-12. Granulosa cells were isolated using a non-enzymatic needle puncture method with a sterile bundle of beading needles to release the cells from follicles. Cell viability was determined by the trypan blue exclusion method. The cells were plated and cultured (24-well plates) at approximately 3-4 x 105 viable cells/ml/well and incubated at 37°C for 24 h in the DMEM/F-12 containing FBS (5%) in an atmosphere of 5% CO2 in air. Following the 24 h acclimation period, the medium was replaced with serum-free DMEM/F12 containing androstenedione (0.1 M) as a substrate for aromatization. Cells were treated with increasing doses of HPTE (0, 1, 5, and 10µM) in the absence (basal) or presence of 3 ng FSH/ml or 1 mM cAMP for 48 h. The doses of FSH and cAMP were selected based on a dose-response curve that provided a maximum level of stimulation for E2 production (data not shown). Cultures were terminated at 48 h following the addition of treatments. Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Cat # 74106) followed by DNase I treatment. RNA quality was assessed by electrophoresis using the Agilent Bioanalyzer 2100 and spectrophotometric analysis prior to cDNA synthesis (data not shown). Forty nanograms of total RNA from each sample were used to generate a high fidelity cDNA for array hybridization using the NuGen Ovation Biotin RNA Amplification and Labeling system (NuGen). After fragmentation and biotin labeling, the samples were hybridized to Affymetrix Rat Genome 230 2.0 arrays, which have eleven pairs of oligonucleotide probes per chip. Washing and staining of all arrays were carried out in the Affymetrix fluidics module as per the manufacturer’s protocol. The detection and quantification of target hybridization was performed with an Affymetrix GeneChip Scanner. Three separate samples were analyzed for each of the treatment groups.Initially, raw data were analyzed using Robust Multichip Analysis (RMA) with a logarithmic base 2 conversion utilizing the Affymetrix ArrayAssist Suite (Affymetrix, Inc., Santa Clara, CA). One-way ANOVA analysis of each group (basal, FSH, or cAMP) was used to determine genes that are significantly affected by HPTE. This was followed by an unpaired t-test to determine changes in global gene expression between HPTE-treated groups and the control groups to determine the levels of fold change. All statistical analyses were performed using P < 0.005 as the level of statistical significance.
GSM349693
GPL1355
HPTE_0_cAMP 2 Granulosa cells from post-natal days 21-27 female Sprague Dawley Rats Charles River outbred immature female rats Briefly, intact, non-primed, immature female rats (21-27 days old) were sacrificed by cervical dislocation. Ovaries were removed from the animals, cleaned free of associated fat, oviduct, and bursa ovary, and then placed in ice-cold DMEM/F-12. Granulosa cells were isolated using a non-enzymatic needle puncture method with a sterile bundle of beading needles to release the cells from follicles. Cell viability was determined by the trypan blue exclusion method. The cells were plated and cultured (24-well plates) at approximately 3-4 x 105 viable cells/ml/well and incubated at 37°C for 24 h in the DMEM/F-12 containing FBS (5%) in an atmosphere of 5% CO2 in air. Following the 24 h acclimation period, the medium was replaced with serum-free DMEM/F12 containing androstenedione (0.1 M) as a substrate for aromatization. Cells were treated with increasing doses of HPTE (0, 1, 5, and 10µM) in the absence (basal) or presence of 3 ng FSH/ml or 1 mM cAMP for 48 h. The doses of FSH and cAMP were selected based on a dose-response curve that provided a maximum level of stimulation for E2 production (data not shown). Cultures were terminated at 48 h following the addition of treatments. Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Cat # 74106) followed by DNase I treatment. RNA quality was assessed by electrophoresis using the Agilent Bioanalyzer 2100 and spectrophotometric analysis prior to cDNA synthesis (data not shown). Forty nanograms of total RNA from each sample were used to generate a high fidelity cDNA for array hybridization using the NuGen Ovation Biotin RNA Amplification and Labeling system (NuGen). After fragmentation and biotin labeling, the samples were hybridized to Affymetrix Rat Genome 230 2.0 arrays, which have eleven pairs of oligonucleotide probes per chip. Washing and staining of all arrays were carried out in the Affymetrix fluidics module as per the manufacturer’s protocol. The detection and quantification of target hybridization was performed with an Affymetrix GeneChip Scanner. Three separate samples were analyzed for each of the treatment groups.Initially, raw data were analyzed using Robust Multichip Analysis (RMA) with a logarithmic base 2 conversion utilizing the Affymetrix ArrayAssist Suite (Affymetrix, Inc., Santa Clara, CA). One-way ANOVA analysis of each group (basal, FSH, or cAMP) was used to determine genes that are significantly affected by HPTE. This was followed by an unpaired t-test to determine changes in global gene expression between HPTE-treated groups and the control groups to determine the levels of fold change. All statistical analyses were performed using P < 0.005 as the level of statistical significance.
GSM349694
GPL1355
HPTE_1_Basal 2 Granulosa cells from post-natal days 21-27 female Sprague Dawley Rats Charles River outbred immature female rats Briefly, intact, non-primed, immature female rats (21-27 days old) were sacrificed by cervical dislocation. Ovaries were removed from the animals, cleaned free of associated fat, oviduct, and bursa ovary, and then placed in ice-cold DMEM/F-12. Granulosa cells were isolated using a non-enzymatic needle puncture method with a sterile bundle of beading needles to release the cells from follicles. Cell viability was determined by the trypan blue exclusion method. The cells were plated and cultured (24-well plates) at approximately 3-4 x 105 viable cells/ml/well and incubated at 37°C for 24 h in the DMEM/F-12 containing FBS (5%) in an atmosphere of 5% CO2 in air. Following the 24 h acclimation period, the medium was replaced with serum-free DMEM/F12 containing androstenedione (0.1 M) as a substrate for aromatization. Cells were treated with increasing doses of HPTE (0, 1, 5, and 10µM) in the absence (basal) or presence of 3 ng FSH/ml or 1 mM cAMP for 48 h. The doses of FSH and cAMP were selected based on a dose-response curve that provided a maximum level of stimulation for E2 production (data not shown). Cultures were terminated at 48 h following the addition of treatments. Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Cat # 74106) followed by DNase I treatment. RNA quality was assessed by electrophoresis using the Agilent Bioanalyzer 2100 and spectrophotometric analysis prior to cDNA synthesis (data not shown). Forty nanograms of total RNA from each sample were used to generate a high fidelity cDNA for array hybridization using the NuGen Ovation Biotin RNA Amplification and Labeling system (NuGen). After fragmentation and biotin labeling, the samples were hybridized to Affymetrix Rat Genome 230 2.0 arrays, which have eleven pairs of oligonucleotide probes per chip. Washing and staining of all arrays were carried out in the Affymetrix fluidics module as per the manufacturer’s protocol. The detection and quantification of target hybridization was performed with an Affymetrix GeneChip Scanner. Three separate samples were analyzed for each of the treatment groups.Initially, raw data were analyzed using Robust Multichip Analysis (RMA) with a logarithmic base 2 conversion utilizing the Affymetrix ArrayAssist Suite (Affymetrix, Inc., Santa Clara, CA). One-way ANOVA analysis of each group (basal, FSH, or cAMP) was used to determine genes that are significantly affected by HPTE. This was followed by an unpaired t-test to determine changes in global gene expression between HPTE-treated groups and the control groups to determine the levels of fold change. All statistical analyses were performed using P < 0.005 as the level of statistical significance.
GSM349695
GPL1355
HPTE_1_FSH 2 Granulosa cells from post-natal days 21-27 female Sprague Dawley Rats Charles River outbred immature female rats Briefly, intact, non-primed, immature female rats (21-27 days old) were sacrificed by cervical dislocation. Ovaries were removed from the animals, cleaned free of associated fat, oviduct, and bursa ovary, and then placed in ice-cold DMEM/F-12. Granulosa cells were isolated using a non-enzymatic needle puncture method with a sterile bundle of beading needles to release the cells from follicles. Cell viability was determined by the trypan blue exclusion method. The cells were plated and cultured (24-well plates) at approximately 3-4 x 105 viable cells/ml/well and incubated at 37°C for 24 h in the DMEM/F-12 containing FBS (5%) in an atmosphere of 5% CO2 in air. Following the 24 h acclimation period, the medium was replaced with serum-free DMEM/F12 containing androstenedione (0.1 M) as a substrate for aromatization. Cells were treated with increasing doses of HPTE (0, 1, 5, and 10µM) in the absence (basal) or presence of 3 ng FSH/ml or 1 mM cAMP for 48 h. The doses of FSH and cAMP were selected based on a dose-response curve that provided a maximum level of stimulation for E2 production (data not shown). Cultures were terminated at 48 h following the addition of treatments. Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Cat # 74106) followed by DNase I treatment. RNA quality was assessed by electrophoresis using the Agilent Bioanalyzer 2100 and spectrophotometric analysis prior to cDNA synthesis (data not shown). Forty nanograms of total RNA from each sample were used to generate a high fidelity cDNA for array hybridization using the NuGen Ovation Biotin RNA Amplification and Labeling system (NuGen). After fragmentation and biotin labeling, the samples were hybridized to Affymetrix Rat Genome 230 2.0 arrays, which have eleven pairs of oligonucleotide probes per chip. Washing and staining of all arrays were carried out in the Affymetrix fluidics module as per the manufacturer’s protocol. The detection and quantification of target hybridization was performed with an Affymetrix GeneChip Scanner. Three separate samples were analyzed for each of the treatment groups.Initially, raw data were analyzed using Robust Multichip Analysis (RMA) with a logarithmic base 2 conversion utilizing the Affymetrix ArrayAssist Suite (Affymetrix, Inc., Santa Clara, CA). One-way ANOVA analysis of each group (basal, FSH, or cAMP) was used to determine genes that are significantly affected by HPTE. This was followed by an unpaired t-test to determine changes in global gene expression between HPTE-treated groups and the control groups to determine the levels of fold change. All statistical analyses were performed using P < 0.005 as the level of statistical significance.
GSM349696
GPL1355
HPTE_1_cAMP 2 Granulosa cells from post-natal days 21-27 female Sprague Dawley Rats Charles River outbred immature female rats Briefly, intact, non-primed, immature female rats (21-27 days old) were sacrificed by cervical dislocation. Ovaries were removed from the animals, cleaned free of associated fat, oviduct, and bursa ovary, and then placed in ice-cold DMEM/F-12. Granulosa cells were isolated using a non-enzymatic needle puncture method with a sterile bundle of beading needles to release the cells from follicles. Cell viability was determined by the trypan blue exclusion method. The cells were plated and cultured (24-well plates) at approximately 3-4 x 105 viable cells/ml/well and incubated at 37°C for 24 h in the DMEM/F-12 containing FBS (5%) in an atmosphere of 5% CO2 in air. Following the 24 h acclimation period, the medium was replaced with serum-free DMEM/F12 containing androstenedione (0.1 M) as a substrate for aromatization. Cells were treated with increasing doses of HPTE (0, 1, 5, and 10µM) in the absence (basal) or presence of 3 ng FSH/ml or 1 mM cAMP for 48 h. The doses of FSH and cAMP were selected based on a dose-response curve that provided a maximum level of stimulation for E2 production (data not shown). Cultures were terminated at 48 h following the addition of treatments. Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Cat # 74106) followed by DNase I treatment. RNA quality was assessed by electrophoresis using the Agilent Bioanalyzer 2100 and spectrophotometric analysis prior to cDNA synthesis (data not shown). Forty nanograms of total RNA from each sample were used to generate a high fidelity cDNA for array hybridization using the NuGen Ovation Biotin RNA Amplification and Labeling system (NuGen). After fragmentation and biotin labeling, the samples were hybridized to Affymetrix Rat Genome 230 2.0 arrays, which have eleven pairs of oligonucleotide probes per chip. Washing and staining of all arrays were carried out in the Affymetrix fluidics module as per the manufacturer’s protocol. The detection and quantification of target hybridization was performed with an Affymetrix GeneChip Scanner. Three separate samples were analyzed for each of the treatment groups.Initially, raw data were analyzed using Robust Multichip Analysis (RMA) with a logarithmic base 2 conversion utilizing the Affymetrix ArrayAssist Suite (Affymetrix, Inc., Santa Clara, CA). One-way ANOVA analysis of each group (basal, FSH, or cAMP) was used to determine genes that are significantly affected by HPTE. This was followed by an unpaired t-test to determine changes in global gene expression between HPTE-treated groups and the control groups to determine the levels of fold change. All statistical analyses were performed using P < 0.005 as the level of statistical significance.
GSM349697
GPL1355
HPTE_5_Basal 2 Granulosa cells from post-natal days 21-27 female Sprague Dawley Rats Charles River outbred immature female rats Briefly, intact, non-primed, immature female rats (21-27 days old) were sacrificed by cervical dislocation. Ovaries were removed from the animals, cleaned free of associated fat, oviduct, and bursa ovary, and then placed in ice-cold DMEM/F-12. Granulosa cells were isolated using a non-enzymatic needle puncture method with a sterile bundle of beading needles to release the cells from follicles. Cell viability was determined by the trypan blue exclusion method. The cells were plated and cultured (24-well plates) at approximately 3-4 x 105 viable cells/ml/well and incubated at 37°C for 24 h in the DMEM/F-12 containing FBS (5%) in an atmosphere of 5% CO2 in air. Following the 24 h acclimation period, the medium was replaced with serum-free DMEM/F12 containing androstenedione (0.1 M) as a substrate for aromatization. Cells were treated with increasing doses of HPTE (0, 1, 5, and 10µM) in the absence (basal) or presence of 3 ng FSH/ml or 1 mM cAMP for 48 h. The doses of FSH and cAMP were selected based on a dose-response curve that provided a maximum level of stimulation for E2 production (data not shown). Cultures were terminated at 48 h following the addition of treatments. Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Cat # 74106) followed by DNase I treatment. RNA quality was assessed by electrophoresis using the Agilent Bioanalyzer 2100 and spectrophotometric analysis prior to cDNA synthesis (data not shown). Forty nanograms of total RNA from each sample were used to generate a high fidelity cDNA for array hybridization using the NuGen Ovation Biotin RNA Amplification and Labeling system (NuGen). After fragmentation and biotin labeling, the samples were hybridized to Affymetrix Rat Genome 230 2.0 arrays, which have eleven pairs of oligonucleotide probes per chip. Washing and staining of all arrays were carried out in the Affymetrix fluidics module as per the manufacturer’s protocol. The detection and quantification of target hybridization was performed with an Affymetrix GeneChip Scanner. Three separate samples were analyzed for each of the treatment groups.Initially, raw data were analyzed using Robust Multichip Analysis (RMA) with a logarithmic base 2 conversion utilizing the Affymetrix ArrayAssist Suite (Affymetrix, Inc., Santa Clara, CA). One-way ANOVA analysis of each group (basal, FSH, or cAMP) was used to determine genes that are significantly affected by HPTE. This was followed by an unpaired t-test to determine changes in global gene expression between HPTE-treated groups and the control groups to determine the levels of fold change. All statistical analyses were performed using P < 0.005 as the level of statistical significance.
GSM349699
GPL1355
HPTE_5_FSH 2 Granulosa cells from post-natal days 21-27 female Sprague Dawley Rats Charles River outbred immature female rats Briefly, intact, non-primed, immature female rats (21-27 days old) were sacrificed by cervical dislocation. Ovaries were removed from the animals, cleaned free of associated fat, oviduct, and bursa ovary, and then placed in ice-cold DMEM/F-12. Granulosa cells were isolated using a non-enzymatic needle puncture method with a sterile bundle of beading needles to release the cells from follicles. Cell viability was determined by the trypan blue exclusion method. The cells were plated and cultured (24-well plates) at approximately 3-4 x 105 viable cells/ml/well and incubated at 37°C for 24 h in the DMEM/F-12 containing FBS (5%) in an atmosphere of 5% CO2 in air. Following the 24 h acclimation period, the medium was replaced with serum-free DMEM/F12 containing androstenedione (0.1 M) as a substrate for aromatization. Cells were treated with increasing doses of HPTE (0, 1, 5, and 10µM) in the absence (basal) or presence of 3 ng FSH/ml or 1 mM cAMP for 48 h. The doses of FSH and cAMP were selected based on a dose-response curve that provided a maximum level of stimulation for E2 production (data not shown). Cultures were terminated at 48 h following the addition of treatments. Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Cat # 74106) followed by DNase I treatment. RNA quality was assessed by electrophoresis using the Agilent Bioanalyzer 2100 and spectrophotometric analysis prior to cDNA synthesis (data not shown). Forty nanograms of total RNA from each sample were used to generate a high fidelity cDNA for array hybridization using the NuGen Ovation Biotin RNA Amplification and Labeling system (NuGen). After fragmentation and biotin labeling, the samples were hybridized to Affymetrix Rat Genome 230 2.0 arrays, which have eleven pairs of oligonucleotide probes per chip. Washing and staining of all arrays were carried out in the Affymetrix fluidics module as per the manufacturer’s protocol. The detection and quantification of target hybridization was performed with an Affymetrix GeneChip Scanner. Three separate samples were analyzed for each of the treatment groups.Initially, raw data were analyzed using Robust Multichip Analysis (RMA) with a logarithmic base 2 conversion utilizing the Affymetrix ArrayAssist Suite (Affymetrix, Inc., Santa Clara, CA). One-way ANOVA analysis of each group (basal, FSH, or cAMP) was used to determine genes that are significantly affected by HPTE. This was followed by an unpaired t-test to determine changes in global gene expression between HPTE-treated groups and the control groups to determine the levels of fold change. All statistical analyses were performed using P < 0.005 as the level of statistical significance.
GSM349700
GPL1355
HPTE_5_cAMP 2 Granulosa cells from post-natal days 21-27 female Sprague Dawley Rats Charles River outbred immature female rats Briefly, intact, non-primed, immature female rats (21-27 days old) were sacrificed by cervical dislocation. Ovaries were removed from the animals, cleaned free of associated fat, oviduct, and bursa ovary, and then placed in ice-cold DMEM/F-12. Granulosa cells were isolated using a non-enzymatic needle puncture method with a sterile bundle of beading needles to release the cells from follicles. Cell viability was determined by the trypan blue exclusion method. The cells were plated and cultured (24-well plates) at approximately 3-4 x 105 viable cells/ml/well and incubated at 37°C for 24 h in the DMEM/F-12 containing FBS (5%) in an atmosphere of 5% CO2 in air. Following the 24 h acclimation period, the medium was replaced with serum-free DMEM/F12 containing androstenedione (0.1 M) as a substrate for aromatization. Cells were treated with increasing doses of HPTE (0, 1, 5, and 10µM) in the absence (basal) or presence of 3 ng FSH/ml or 1 mM cAMP for 48 h. The doses of FSH and cAMP were selected based on a dose-response curve that provided a maximum level of stimulation for E2 production (data not shown). Cultures were terminated at 48 h following the addition of treatments. Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Cat # 74106) followed by DNase I treatment. RNA quality was assessed by electrophoresis using the Agilent Bioanalyzer 2100 and spectrophotometric analysis prior to cDNA synthesis (data not shown). Forty nanograms of total RNA from each sample were used to generate a high fidelity cDNA for array hybridization using the NuGen Ovation Biotin RNA Amplification and Labeling system (NuGen). After fragmentation and biotin labeling, the samples were hybridized to Affymetrix Rat Genome 230 2.0 arrays, which have eleven pairs of oligonucleotide probes per chip. Washing and staining of all arrays were carried out in the Affymetrix fluidics module as per the manufacturer’s protocol. The detection and quantification of target hybridization was performed with an Affymetrix GeneChip Scanner. Three separate samples were analyzed for each of the treatment groups.Initially, raw data were analyzed using Robust Multichip Analysis (RMA) with a logarithmic base 2 conversion utilizing the Affymetrix ArrayAssist Suite (Affymetrix, Inc., Santa Clara, CA). One-way ANOVA analysis of each group (basal, FSH, or cAMP) was used to determine genes that are significantly affected by HPTE. This was followed by an unpaired t-test to determine changes in global gene expression between HPTE-treated groups and the control groups to determine the levels of fold change. All statistical analyses were performed using P < 0.005 as the level of statistical significance.
GSM349701
GPL1355
HPTE_10_Basal 2 Granulosa cells from post-natal days 21-27 female Sprague Dawley Rats Charles River outbred immature female rats Briefly, intact, non-primed, immature female rats (21-27 days old) were sacrificed by cervical dislocation. Ovaries were removed from the animals, cleaned free of associated fat, oviduct, and bursa ovary, and then placed in ice-cold DMEM/F-12. Granulosa cells were isolated using a non-enzymatic needle puncture method with a sterile bundle of beading needles to release the cells from follicles. Cell viability was determined by the trypan blue exclusion method. The cells were plated and cultured (24-well plates) at approximately 3-4 x 105 viable cells/ml/well and incubated at 37°C for 24 h in the DMEM/F-12 containing FBS (5%) in an atmosphere of 5% CO2 in air. Following the 24 h acclimation period, the medium was replaced with serum-free DMEM/F12 containing androstenedione (0.1 M) as a substrate for aromatization. Cells were treated with increasing doses of HPTE (0, 1, 5, and 10µM) in the absence (basal) or presence of 3 ng FSH/ml or 1 mM cAMP for 48 h. The doses of FSH and cAMP were selected based on a dose-response curve that provided a maximum level of stimulation for E2 production (data not shown). Cultures were terminated at 48 h following the addition of treatments. Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Cat # 74106) followed by DNase I treatment. RNA quality was assessed by electrophoresis using the Agilent Bioanalyzer 2100 and spectrophotometric analysis prior to cDNA synthesis (data not shown). Forty nanograms of total RNA from each sample were used to generate a high fidelity cDNA for array hybridization using the NuGen Ovation Biotin RNA Amplification and Labeling system (NuGen). After fragmentation and biotin labeling, the samples were hybridized to Affymetrix Rat Genome 230 2.0 arrays, which have eleven pairs of oligonucleotide probes per chip. Washing and staining of all arrays were carried out in the Affymetrix fluidics module as per the manufacturer’s protocol. The detection and quantification of target hybridization was performed with an Affymetrix GeneChip Scanner. Three separate samples were analyzed for each of the treatment groups.Initially, raw data were analyzed using Robust Multichip Analysis (RMA) with a logarithmic base 2 conversion utilizing the Affymetrix ArrayAssist Suite (Affymetrix, Inc., Santa Clara, CA). One-way ANOVA analysis of each group (basal, FSH, or cAMP) was used to determine genes that are significantly affected by HPTE. This was followed by an unpaired t-test to determine changes in global gene expression between HPTE-treated groups and the control groups to determine the levels of fold change. All statistical analyses were performed using P < 0.005 as the level of statistical significance.
GSM349703
GPL1355
HPTE_0_ Basal 3 Granulosa cells from post-natal days 21-27 female Sprague Dawley Rats Charles River outbred immature female rats Briefly, intact, non-primed, immature female rats (21-27 days old) were sacrificed by cervical dislocation. Ovaries were removed from the animals, cleaned free of associated fat, oviduct, and bursa ovary, and then placed in ice-cold DMEM/F-12. Granulosa cells were isolated using a non-enzymatic needle puncture method with a sterile bundle of beading needles to release the cells from follicles. Cell viability was determined by the trypan blue exclusion method. The cells were plated and cultured (24-well plates) at approximately 3-4 x 105 viable cells/ml/well and incubated at 37°C for 24 h in the DMEM/F-12 containing FBS (5%) in an atmosphere of 5% CO2 in air. Following the 24 h acclimation period, the medium was replaced with serum-free DMEM/F12 containing androstenedione (0.1 M) as a substrate for aromatization. Cells were treated with increasing doses of HPTE (0, 1, 5, and 10µM) in the absence (basal) or presence of 3 ng FSH/ml or 1 mM cAMP for 48 h. The doses of FSH and cAMP were selected based on a dose-response curve that provided a maximum level of stimulation for E2 production (data not shown). Cultures were terminated at 48 h following the addition of treatments. Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Cat # 74106) followed by DNase I treatment. RNA quality was assessed by electrophoresis using the Agilent Bioanalyzer 2100 and spectrophotometric analysis prior to cDNA synthesis (data not shown). Forty nanograms of total RNA from each sample were used to generate a high fidelity cDNA for array hybridization using the NuGen Ovation Biotin RNA Amplification and Labeling system (NuGen). After fragmentation and biotin labeling, the samples were hybridized to Affymetrix Rat Genome 230 2.0 arrays, which have eleven pairs of oligonucleotide probes per chip. Washing and staining of all arrays were carried out in the Affymetrix fluidics module as per the manufacturer’s protocol. The detection and quantification of target hybridization was performed with an Affymetrix GeneChip Scanner. Three separate samples were analyzed for each of the treatment groups.Initially, raw data were analyzed using Robust Multichip Analysis (RMA) with a logarithmic base 2 conversion utilizing the Affymetrix ArrayAssist Suite (Affymetrix, Inc., Santa Clara, CA). One-way ANOVA analysis of each group (basal, FSH, or cAMP) was used to determine genes that are significantly affected by HPTE. This was followed by an unpaired t-test to determine changes in global gene expression between HPTE-treated groups and the control groups to determine the levels of fold change. All statistical analyses were performed using P < 0.005 as the level of statistical significance.
GSM349704
GPL1355
HPTE_10_FSH 2 Granulosa cells from post-natal days 21-27 female Sprague Dawley Rats Charles River outbred immature female rats Briefly, intact, non-primed, immature female rats (21-27 days old) were sacrificed by cervical dislocation. Ovaries were removed from the animals, cleaned free of associated fat, oviduct, and bursa ovary, and then placed in ice-cold DMEM/F-12. Granulosa cells were isolated using a non-enzymatic needle puncture method with a sterile bundle of beading needles to release the cells from follicles. Cell viability was determined by the trypan blue exclusion method. The cells were plated and cultured (24-well plates) at approximately 3-4 x 105 viable cells/ml/well and incubated at 37°C for 24 h in the DMEM/F-12 containing FBS (5%) in an atmosphere of 5% CO2 in air. Following the 24 h acclimation period, the medium was replaced with serum-free DMEM/F12 containing androstenedione (0.1 M) as a substrate for aromatization. Cells were treated with increasing doses of HPTE (0, 1, 5, and 10µM) in the absence (basal) or presence of 3 ng FSH/ml or 1 mM cAMP for 48 h. The doses of FSH and cAMP were selected based on a dose-response curve that provided a maximum level of stimulation for E2 production (data not shown). Cultures were terminated at 48 h following the addition of treatments. Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Cat # 74106) followed by DNase I treatment. RNA quality was assessed by electrophoresis using the Agilent Bioanalyzer 2100 and spectrophotometric analysis prior to cDNA synthesis (data not shown). Forty nanograms of total RNA from each sample were used to generate a high fidelity cDNA for array hybridization using the NuGen Ovation Biotin RNA Amplification and Labeling system (NuGen). After fragmentation and biotin labeling, the samples were hybridized to Affymetrix Rat Genome 230 2.0 arrays, which have eleven pairs of oligonucleotide probes per chip. Washing and staining of all arrays were carried out in the Affymetrix fluidics module as per the manufacturer’s protocol. The detection and quantification of target hybridization was performed with an Affymetrix GeneChip Scanner. Three separate samples were analyzed for each of the treatment groups.Initially, raw data were analyzed using Robust Multichip Analysis (RMA) with a logarithmic base 2 conversion utilizing the Affymetrix ArrayAssist Suite (Affymetrix, Inc., Santa Clara, CA). One-way ANOVA analysis of each group (basal, FSH, or cAMP) was used to determine genes that are significantly affected by HPTE. This was followed by an unpaired t-test to determine changes in global gene expression between HPTE-treated groups and the control groups to determine the levels of fold change. All statistical analyses were performed using P < 0.005 as the level of statistical significance.
GSM349705
GPL1355
HPTE_10_cAMP 2 Granulosa cells from post-natal days 21-27 female Sprague Dawley Rats Charles River outbred immature female rats Briefly, intact, non-primed, immature female rats (21-27 days old) were sacrificed by cervical dislocation. Ovaries were removed from the animals, cleaned free of associated fat, oviduct, and bursa ovary, and then placed in ice-cold DMEM/F-12. Granulosa cells were isolated using a non-enzymatic needle puncture method with a sterile bundle of beading needles to release the cells from follicles. Cell viability was determined by the trypan blue exclusion method. The cells were plated and cultured (24-well plates) at approximately 3-4 x 105 viable cells/ml/well and incubated at 37°C for 24 h in the DMEM/F-12 containing FBS (5%) in an atmosphere of 5% CO2 in air. Following the 24 h acclimation period, the medium was replaced with serum-free DMEM/F12 containing androstenedione (0.1 M) as a substrate for aromatization. Cells were treated with increasing doses of HPTE (0, 1, 5, and 10µM) in the absence (basal) or presence of 3 ng FSH/ml or 1 mM cAMP for 48 h. The doses of FSH and cAMP were selected based on a dose-response curve that provided a maximum level of stimulation for E2 production (data not shown). Cultures were terminated at 48 h following the addition of treatments. Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Cat # 74106) followed by DNase I treatment. RNA quality was assessed by electrophoresis using the Agilent Bioanalyzer 2100 and spectrophotometric analysis prior to cDNA synthesis (data not shown). Forty nanograms of total RNA from each sample were used to generate a high fidelity cDNA for array hybridization using the NuGen Ovation Biotin RNA Amplification and Labeling system (NuGen). After fragmentation and biotin labeling, the samples were hybridized to Affymetrix Rat Genome 230 2.0 arrays, which have eleven pairs of oligonucleotide probes per chip. Washing and staining of all arrays were carried out in the Affymetrix fluidics module as per the manufacturer’s protocol. The detection and quantification of target hybridization was performed with an Affymetrix GeneChip Scanner. Three separate samples were analyzed for each of the treatment groups.Initially, raw data were analyzed using Robust Multichip Analysis (RMA) with a logarithmic base 2 conversion utilizing the Affymetrix ArrayAssist Suite (Affymetrix, Inc., Santa Clara, CA). One-way ANOVA analysis of each group (basal, FSH, or cAMP) was used to determine genes that are significantly affected by HPTE. This was followed by an unpaired t-test to determine changes in global gene expression between HPTE-treated groups and the control groups to determine the levels of fold change. All statistical analyses were performed using P < 0.005 as the level of statistical significance.
GSM349706
GPL1355
HPTE_0_FSH 3 Granulosa cells from post-natal days 21-27 female Sprague Dawley Rats Charles River outbred immature female rats Briefly, intact, non-primed, immature female rats (21-27 days old) were sacrificed by cervical dislocation. Ovaries were removed from the animals, cleaned free of associated fat, oviduct, and bursa ovary, and then placed in ice-cold DMEM/F-12. Granulosa cells were isolated using a non-enzymatic needle puncture method with a sterile bundle of beading needles to release the cells from follicles. Cell viability was determined by the trypan blue exclusion method. The cells were plated and cultured (24-well plates) at approximately 3-4 x 105 viable cells/ml/well and incubated at 37°C for 24 h in the DMEM/F-12 containing FBS (5%) in an atmosphere of 5% CO2 in air. Following the 24 h acclimation period, the medium was replaced with serum-free DMEM/F12 containing androstenedione (0.1 M) as a substrate for aromatization. Cells were treated with increasing doses of HPTE (0, 1, 5, and 10µM) in the absence (basal) or presence of 3 ng FSH/ml or 1 mM cAMP for 48 h. The doses of FSH and cAMP were selected based on a dose-response curve that provided a maximum level of stimulation for E2 production (data not shown). Cultures were terminated at 48 h following the addition of treatments. Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Cat # 74106) followed by DNase I treatment. RNA quality was assessed by electrophoresis using the Agilent Bioanalyzer 2100 and spectrophotometric analysis prior to cDNA synthesis (data not shown). Forty nanograms of total RNA from each sample were used to generate a high fidelity cDNA for array hybridization using the NuGen Ovation Biotin RNA Amplification and Labeling system (NuGen). After fragmentation and biotin labeling, the samples were hybridized to Affymetrix Rat Genome 230 2.0 arrays, which have eleven pairs of oligonucleotide probes per chip. Washing and staining of all arrays were carried out in the Affymetrix fluidics module as per the manufacturer’s protocol. The detection and quantification of target hybridization was performed with an Affymetrix GeneChip Scanner. Three separate samples were analyzed for each of the treatment groups.Initially, raw data were analyzed using Robust Multichip Analysis (RMA) with a logarithmic base 2 conversion utilizing the Affymetrix ArrayAssist Suite (Affymetrix, Inc., Santa Clara, CA). One-way ANOVA analysis of each group (basal, FSH, or cAMP) was used to determine genes that are significantly affected by HPTE. This was followed by an unpaired t-test to determine changes in global gene expression between HPTE-treated groups and the control groups to determine the levels of fold change. All statistical analyses were performed using P < 0.005 as the level of statistical significance.
GSM349707
GPL1355
HPTE_0_cAMP 3 Granulosa cells from post-natal days 21-27 female Sprague Dawley Rats Charles River outbred immature female rats Briefly, intact, non-primed, immature female rats (21-27 days old) were sacrificed by cervical dislocation. Ovaries were removed from the animals, cleaned free of associated fat, oviduct, and bursa ovary, and then placed in ice-cold DMEM/F-12. Granulosa cells were isolated using a non-enzymatic needle puncture method with a sterile bundle of beading needles to release the cells from follicles. Cell viability was determined by the trypan blue exclusion method. The cells were plated and cultured (24-well plates) at approximately 3-4 x 105 viable cells/ml/well and incubated at 37°C for 24 h in the DMEM/F-12 containing FBS (5%) in an atmosphere of 5% CO2 in air. Following the 24 h acclimation period, the medium was replaced with serum-free DMEM/F12 containing androstenedione (0.1 M) as a substrate for aromatization. Cells were treated with increasing doses of HPTE (0, 1, 5, and 10µM) in the absence (basal) or presence of 3 ng FSH/ml or 1 mM cAMP for 48 h. The doses of FSH and cAMP were selected based on a dose-response curve that provided a maximum level of stimulation for E2 production (data not shown). Cultures were terminated at 48 h following the addition of treatments. Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Cat # 74106) followed by DNase I treatment. RNA quality was assessed by electrophoresis using the Agilent Bioanalyzer 2100 and spectrophotometric analysis prior to cDNA synthesis (data not shown). Forty nanograms of total RNA from each sample were used to generate a high fidelity cDNA for array hybridization using the NuGen Ovation Biotin RNA Amplification and Labeling system (NuGen). After fragmentation and biotin labeling, the samples were hybridized to Affymetrix Rat Genome 230 2.0 arrays, which have eleven pairs of oligonucleotide probes per chip. Washing and staining of all arrays were carried out in the Affymetrix fluidics module as per the manufacturer’s protocol. The detection and quantification of target hybridization was performed with an Affymetrix GeneChip Scanner. Three separate samples were analyzed for each of the treatment groups.Initially, raw data were analyzed using Robust Multichip Analysis (RMA) with a logarithmic base 2 conversion utilizing the Affymetrix ArrayAssist Suite (Affymetrix, Inc., Santa Clara, CA). One-way ANOVA analysis of each group (basal, FSH, or cAMP) was used to determine genes that are significantly affected by HPTE. This was followed by an unpaired t-test to determine changes in global gene expression between HPTE-treated groups and the control groups to determine the levels of fold change. All statistical analyses were performed using P < 0.005 as the level of statistical significance.
GSM349708
GPL1355
HPTE_1_Basal 3 Granulosa cells from post-natal days 21-27 female Sprague Dawley Rats Charles River outbred immature female rats Briefly, intact, non-primed, immature female rats (21-27 days old) were sacrificed by cervical dislocation. Ovaries were removed from the animals, cleaned free of associated fat, oviduct, and bursa ovary, and then placed in ice-cold DMEM/F-12. Granulosa cells were isolated using a non-enzymatic needle puncture method with a sterile bundle of beading needles to release the cells from follicles. Cell viability was determined by the trypan blue exclusion method. The cells were plated and cultured (24-well plates) at approximately 3-4 x 105 viable cells/ml/well and incubated at 37°C for 24 h in the DMEM/F-12 containing FBS (5%) in an atmosphere of 5% CO2 in air. Following the 24 h acclimation period, the medium was replaced with serum-free DMEM/F12 containing androstenedione (0.1 M) as a substrate for aromatization. Cells were treated with increasing doses of HPTE (0, 1, 5, and 10µM) in the absence (basal) or presence of 3 ng FSH/ml or 1 mM cAMP for 48 h. The doses of FSH and cAMP were selected based on a dose-response curve that provided a maximum level of stimulation for E2 production (data not shown). Cultures were terminated at 48 h following the addition of treatments. Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Cat # 74106) followed by DNase I treatment. RNA quality was assessed by electrophoresis using the Agilent Bioanalyzer 2100 and spectrophotometric analysis prior to cDNA synthesis (data not shown). Forty nanograms of total RNA from each sample were used to generate a high fidelity cDNA for array hybridization using the NuGen Ovation Biotin RNA Amplification and Labeling system (NuGen). After fragmentation and biotin labeling, the samples were hybridized to Affymetrix Rat Genome 230 2.0 arrays, which have eleven pairs of oligonucleotide probes per chip. Washing and staining of all arrays were carried out in the Affymetrix fluidics module as per the manufacturer’s protocol. The detection and quantification of target hybridization was performed with an Affymetrix GeneChip Scanner. Three separate samples were analyzed for each of the treatment groups.Initially, raw data were analyzed using Robust Multichip Analysis (RMA) with a logarithmic base 2 conversion utilizing the Affymetrix ArrayAssist Suite (Affymetrix, Inc., Santa Clara, CA). One-way ANOVA analysis of each group (basal, FSH, or cAMP) was used to determine genes that are significantly affected by HPTE. This was followed by an unpaired t-test to determine changes in global gene expression between HPTE-treated groups and the control groups to determine the levels of fold change. All statistical analyses were performed using P < 0.005 as the level of statistical significance.
GSM349709
GPL1355
HPTE_1_FSH 3 Granulosa cells from post-natal days 21-27 female Sprague Dawley Rats Charles River outbred immature female rats Briefly, intact, non-primed, immature female rats (21-27 days old) were sacrificed by cervical dislocation. Ovaries were removed from the animals, cleaned free of associated fat, oviduct, and bursa ovary, and then placed in ice-cold DMEM/F-12. Granulosa cells were isolated using a non-enzymatic needle puncture method with a sterile bundle of beading needles to release the cells from follicles. Cell viability was determined by the trypan blue exclusion method. The cells were plated and cultured (24-well plates) at approximately 3-4 x 105 viable cells/ml/well and incubated at 37°C for 24 h in the DMEM/F-12 containing FBS (5%) in an atmosphere of 5% CO2 in air. Following the 24 h acclimation period, the medium was replaced with serum-free DMEM/F12 containing androstenedione (0.1 M) as a substrate for aromatization. Cells were treated with increasing doses of HPTE (0, 1, 5, and 10µM) in the absence (basal) or presence of 3 ng FSH/ml or 1 mM cAMP for 48 h. The doses of FSH and cAMP were selected based on a dose-response curve that provided a maximum level of stimulation for E2 production (data not shown). Cultures were terminated at 48 h following the addition of treatments. Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Cat # 74106) followed by DNase I treatment. RNA quality was assessed by electrophoresis using the Agilent Bioanalyzer 2100 and spectrophotometric analysis prior to cDNA synthesis (data not shown). Forty nanograms of total RNA from each sample were used to generate a high fidelity cDNA for array hybridization using the NuGen Ovation Biotin RNA Amplification and Labeling system (NuGen). After fragmentation and biotin labeling, the samples were hybridized to Affymetrix Rat Genome 230 2.0 arrays, which have eleven pairs of oligonucleotide probes per chip. Washing and staining of all arrays were carried out in the Affymetrix fluidics module as per the manufacturer’s protocol. The detection and quantification of target hybridization was performed with an Affymetrix GeneChip Scanner. Three separate samples were analyzed for each of the treatment groups.Initially, raw data were analyzed using Robust Multichip Analysis (RMA) with a logarithmic base 2 conversion utilizing the Affymetrix ArrayAssist Suite (Affymetrix, Inc., Santa Clara, CA). One-way ANOVA analysis of each group (basal, FSH, or cAMP) was used to determine genes that are significantly affected by HPTE. This was followed by an unpaired t-test to determine changes in global gene expression between HPTE-treated groups and the control groups to determine the levels of fold change. All statistical analyses were performed using P < 0.005 as the level of statistical significance.
GSM349710
GPL1355
HPTE_1_cAMP 3 Granulosa cells from post-natal days 21-27 female Sprague Dawley Rats Charles River outbred immature female rats Briefly, intact, non-primed, immature female rats (21-27 days old) were sacrificed by cervical dislocation. Ovaries were removed from the animals, cleaned free of associated fat, oviduct, and bursa ovary, and then placed in ice-cold DMEM/F-12. Granulosa cells were isolated using a non-enzymatic needle puncture method with a sterile bundle of beading needles to release the cells from follicles. Cell viability was determined by the trypan blue exclusion method. The cells were plated and cultured (24-well plates) at approximately 3-4 x 105 viable cells/ml/well and incubated at 37°C for 24 h in the DMEM/F-12 containing FBS (5%) in an atmosphere of 5% CO2 in air. Following the 24 h acclimation period, the medium was replaced with serum-free DMEM/F12 containing androstenedione (0.1 M) as a substrate for aromatization. Cells were treated with increasing doses of HPTE (0, 1, 5, and 10µM) in the absence (basal) or presence of 3 ng FSH/ml or 1 mM cAMP for 48 h. The doses of FSH and cAMP were selected based on a dose-response curve that provided a maximum level of stimulation for E2 production (data not shown). Cultures were terminated at 48 h following the addition of treatments. Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Cat # 74106) followed by DNase I treatment. RNA quality was assessed by electrophoresis using the Agilent Bioanalyzer 2100 and spectrophotometric analysis prior to cDNA synthesis (data not shown). Forty nanograms of total RNA from each sample were used to generate a high fidelity cDNA for array hybridization using the NuGen Ovation Biotin RNA Amplification and Labeling system (NuGen). After fragmentation and biotin labeling, the samples were hybridized to Affymetrix Rat Genome 230 2.0 arrays, which have eleven pairs of oligonucleotide probes per chip. Washing and staining of all arrays were carried out in the Affymetrix fluidics module as per the manufacturer’s protocol. The detection and quantification of target hybridization was performed with an Affymetrix GeneChip Scanner. Three separate samples were analyzed for each of the treatment groups.Initially, raw data were analyzed using Robust Multichip Analysis (RMA) with a logarithmic base 2 conversion utilizing the Affymetrix ArrayAssist Suite (Affymetrix, Inc., Santa Clara, CA). One-way ANOVA analysis of each group (basal, FSH, or cAMP) was used to determine genes that are significantly affected by HPTE. This was followed by an unpaired t-test to determine changes in global gene expression between HPTE-treated groups and the control groups to determine the levels of fold change. All statistical analyses were performed using P < 0.005 as the level of statistical significance.
GSM349711
GPL1355
HPTE_5_Basal 3 Granulosa cells from post-natal days 21-27 female Sprague Dawley Rats Charles River outbred immature female rats Briefly, intact, non-primed, immature female rats (21-27 days old) were sacrificed by cervical dislocation. Ovaries were removed from the animals, cleaned free of associated fat, oviduct, and bursa ovary, and then placed in ice-cold DMEM/F-12. Granulosa cells were isolated using a non-enzymatic needle puncture method with a sterile bundle of beading needles to release the cells from follicles. Cell viability was determined by the trypan blue exclusion method. The cells were plated and cultured (24-well plates) at approximately 3-4 x 105 viable cells/ml/well and incubated at 37°C for 24 h in the DMEM/F-12 containing FBS (5%) in an atmosphere of 5% CO2 in air. Following the 24 h acclimation period, the medium was replaced with serum-free DMEM/F12 containing androstenedione (0.1 M) as a substrate for aromatization. Cells were treated with increasing doses of HPTE (0, 1, 5, and 10µM) in the absence (basal) or presence of 3 ng FSH/ml or 1 mM cAMP for 48 h. The doses of FSH and cAMP were selected based on a dose-response curve that provided a maximum level of stimulation for E2 production (data not shown). Cultures were terminated at 48 h following the addition of treatments. Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Cat # 74106) followed by DNase I treatment. RNA quality was assessed by electrophoresis using the Agilent Bioanalyzer 2100 and spectrophotometric analysis prior to cDNA synthesis (data not shown). Forty nanograms of total RNA from each sample were used to generate a high fidelity cDNA for array hybridization using the NuGen Ovation Biotin RNA Amplification and Labeling system (NuGen). After fragmentation and biotin labeling, the samples were hybridized to Affymetrix Rat Genome 230 2.0 arrays, which have eleven pairs of oligonucleotide probes per chip. Washing and staining of all arrays were carried out in the Affymetrix fluidics module as per the manufacturer’s protocol. The detection and quantification of target hybridization was performed with an Affymetrix GeneChip Scanner. Three separate samples were analyzed for each of the treatment groups.Initially, raw data were analyzed using Robust Multichip Analysis (RMA) with a logarithmic base 2 conversion utilizing the Affymetrix ArrayAssist Suite (Affymetrix, Inc., Santa Clara, CA). One-way ANOVA analysis of each group (basal, FSH, or cAMP) was used to determine genes that are significantly affected by HPTE. This was followed by an unpaired t-test to determine changes in global gene expression between HPTE-treated groups and the control groups to determine the levels of fold change. All statistical analyses were performed using P < 0.005 as the level of statistical significance.
GSM349712
GPL1355
HPTE_5_FSH 3 Granulosa cells from post-natal days 21-27 female Sprague Dawley Rats Charles River outbred immature female rats Briefly, intact, non-primed, immature female rats (21-27 days old) were sacrificed by cervical dislocation. Ovaries were removed from the animals, cleaned free of associated fat, oviduct, and bursa ovary, and then placed in ice-cold DMEM/F-12. Granulosa cells were isolated using a non-enzymatic needle puncture method with a sterile bundle of beading needles to release the cells from follicles. Cell viability was determined by the trypan blue exclusion method. The cells were plated and cultured (24-well plates) at approximately 3-4 x 105 viable cells/ml/well and incubated at 37°C for 24 h in the DMEM/F-12 containing FBS (5%) in an atmosphere of 5% CO2 in air. Following the 24 h acclimation period, the medium was replaced with serum-free DMEM/F12 containing androstenedione (0.1 M) as a substrate for aromatization. Cells were treated with increasing doses of HPTE (0, 1, 5, and 10µM) in the absence (basal) or presence of 3 ng FSH/ml or 1 mM cAMP for 48 h. The doses of FSH and cAMP were selected based on a dose-response curve that provided a maximum level of stimulation for E2 production (data not shown). Cultures were terminated at 48 h following the addition of treatments. Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Cat # 74106) followed by DNase I treatment. RNA quality was assessed by electrophoresis using the Agilent Bioanalyzer 2100 and spectrophotometric analysis prior to cDNA synthesis (data not shown). Forty nanograms of total RNA from each sample were used to generate a high fidelity cDNA for array hybridization using the NuGen Ovation Biotin RNA Amplification and Labeling system (NuGen). After fragmentation and biotin labeling, the samples were hybridized to Affymetrix Rat Genome 230 2.0 arrays, which have eleven pairs of oligonucleotide probes per chip. Washing and staining of all arrays were carried out in the Affymetrix fluidics module as per the manufacturer’s protocol. The detection and quantification of target hybridization was performed with an Affymetrix GeneChip Scanner. Three separate samples were analyzed for each of the treatment groups.Initially, raw data were analyzed using Robust Multichip Analysis (RMA) with a logarithmic base 2 conversion utilizing the Affymetrix ArrayAssist Suite (Affymetrix, Inc., Santa Clara, CA). One-way ANOVA analysis of each group (basal, FSH, or cAMP) was used to determine genes that are significantly affected by HPTE. This was followed by an unpaired t-test to determine changes in global gene expression between HPTE-treated groups and the control groups to determine the levels of fold change. All statistical analyses were performed using P < 0.005 as the level of statistical significance.
GSM349713
GPL1355
HPTE_5_cAMP 3 Granulosa cells from post-natal days 21-27 female Sprague Dawley Rats Charles River outbred immature female rats Briefly, intact, non-primed, immature female rats (21-27 days old) were sacrificed by cervical dislocation. Ovaries were removed from the animals, cleaned free of associated fat, oviduct, and bursa ovary, and then placed in ice-cold DMEM/F-12. Granulosa cells were isolated using a non-enzymatic needle puncture method with a sterile bundle of beading needles to release the cells from follicles. Cell viability was determined by the trypan blue exclusion method. The cells were plated and cultured (24-well plates) at approximately 3-4 x 105 viable cells/ml/well and incubated at 37°C for 24 h in the DMEM/F-12 containing FBS (5%) in an atmosphere of 5% CO2 in air. Following the 24 h acclimation period, the medium was replaced with serum-free DMEM/F12 containing androstenedione (0.1 M) as a substrate for aromatization. Cells were treated with increasing doses of HPTE (0, 1, 5, and 10µM) in the absence (basal) or presence of 3 ng FSH/ml or 1 mM cAMP for 48 h. The doses of FSH and cAMP were selected based on a dose-response curve that provided a maximum level of stimulation for E2 production (data not shown). Cultures were terminated at 48 h following the addition of treatments. Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Cat # 74106) followed by DNase I treatment. RNA quality was assessed by electrophoresis using the Agilent Bioanalyzer 2100 and spectrophotometric analysis prior to cDNA synthesis (data not shown). Forty nanograms of total RNA from each sample were used to generate a high fidelity cDNA for array hybridization using the NuGen Ovation Biotin RNA Amplification and Labeling system (NuGen). After fragmentation and biotin labeling, the samples were hybridized to Affymetrix Rat Genome 230 2.0 arrays, which have eleven pairs of oligonucleotide probes per chip. Washing and staining of all arrays were carried out in the Affymetrix fluidics module as per the manufacturer’s protocol. The detection and quantification of target hybridization was performed with an Affymetrix GeneChip Scanner. Three separate samples were analyzed for each of the treatment groups.Initially, raw data were analyzed using Robust Multichip Analysis (RMA) with a logarithmic base 2 conversion utilizing the Affymetrix ArrayAssist Suite (Affymetrix, Inc., Santa Clara, CA). One-way ANOVA analysis of each group (basal, FSH, or cAMP) was used to determine genes that are significantly affected by HPTE. This was followed by an unpaired t-test to determine changes in global gene expression between HPTE-treated groups and the control groups to determine the levels of fold change. All statistical analyses were performed using P < 0.005 as the level of statistical significance.
GSM349714
GPL1355
HPTE_10_Basal 3 Granulosa cells from post-natal days 21-27 female Sprague Dawley Rats Charles River outbred immature female rats Briefly, intact, non-primed, immature female rats (21-27 days old) were sacrificed by cervical dislocation. Ovaries were removed from the animals, cleaned free of associated fat, oviduct, and bursa ovary, and then placed in ice-cold DMEM/F-12. Granulosa cells were isolated using a non-enzymatic needle puncture method with a sterile bundle of beading needles to release the cells from follicles. Cell viability was determined by the trypan blue exclusion method. The cells were plated and cultured (24-well plates) at approximately 3-4 x 105 viable cells/ml/well and incubated at 37°C for 24 h in the DMEM/F-12 containing FBS (5%) in an atmosphere of 5% CO2 in air. Following the 24 h acclimation period, the medium was replaced with serum-free DMEM/F12 containing androstenedione (0.1 M) as a substrate for aromatization. Cells were treated with increasing doses of HPTE (0, 1, 5, and 10µM) in the absence (basal) or presence of 3 ng FSH/ml or 1 mM cAMP for 48 h. The doses of FSH and cAMP were selected based on a dose-response curve that provided a maximum level of stimulation for E2 production (data not shown). Cultures were terminated at 48 h following the addition of treatments. Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Cat # 74106) followed by DNase I treatment. RNA quality was assessed by electrophoresis using the Agilent Bioanalyzer 2100 and spectrophotometric analysis prior to cDNA synthesis (data not shown). Forty nanograms of total RNA from each sample were used to generate a high fidelity cDNA for array hybridization using the NuGen Ovation Biotin RNA Amplification and Labeling system (NuGen). After fragmentation and biotin labeling, the samples were hybridized to Affymetrix Rat Genome 230 2.0 arrays, which have eleven pairs of oligonucleotide probes per chip. Washing and staining of all arrays were carried out in the Affymetrix fluidics module as per the manufacturer’s protocol. The detection and quantification of target hybridization was performed with an Affymetrix GeneChip Scanner. Three separate samples were analyzed for each of the treatment groups.Initially, raw data were analyzed using Robust Multichip Analysis (RMA) with a logarithmic base 2 conversion utilizing the Affymetrix ArrayAssist Suite (Affymetrix, Inc., Santa Clara, CA). One-way ANOVA analysis of each group (basal, FSH, or cAMP) was used to determine genes that are significantly affected by HPTE. This was followed by an unpaired t-test to determine changes in global gene expression between HPTE-treated groups and the control groups to determine the levels of fold change. All statistical analyses were performed using P < 0.005 as the level of statistical significance.
GSM349715
GPL1355
HPTE_10_FSH 3 Granulosa cells from post-natal days 21-27 female Sprague Dawley Rats Charles River outbred immature female rats Briefly, intact, non-primed, immature female rats (21-27 days old) were sacrificed by cervical dislocation. Ovaries were removed from the animals, cleaned free of associated fat, oviduct, and bursa ovary, and then placed in ice-cold DMEM/F-12. Granulosa cells were isolated using a non-enzymatic needle puncture method with a sterile bundle of beading needles to release the cells from follicles. Cell viability was determined by the trypan blue exclusion method. The cells were plated and cultured (24-well plates) at approximately 3-4 x 105 viable cells/ml/well and incubated at 37°C for 24 h in the DMEM/F-12 containing FBS (5%) in an atmosphere of 5% CO2 in air. Following the 24 h acclimation period, the medium was replaced with serum-free DMEM/F12 containing androstenedione (0.1 M) as a substrate for aromatization. Cells were treated with increasing doses of HPTE (0, 1, 5, and 10µM) in the absence (basal) or presence of 3 ng FSH/ml or 1 mM cAMP for 48 h. The doses of FSH and cAMP were selected based on a dose-response curve that provided a maximum level of stimulation for E2 production (data not shown). Cultures were terminated at 48 h following the addition of treatments. Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Cat # 74106) followed by DNase I treatment. RNA quality was assessed by electrophoresis using the Agilent Bioanalyzer 2100 and spectrophotometric analysis prior to cDNA synthesis (data not shown). Forty nanograms of total RNA from each sample were used to generate a high fidelity cDNA for array hybridization using the NuGen Ovation Biotin RNA Amplification and Labeling system (NuGen). After fragmentation and biotin labeling, the samples were hybridized to Affymetrix Rat Genome 230 2.0 arrays, which have eleven pairs of oligonucleotide probes per chip. Washing and staining of all arrays were carried out in the Affymetrix fluidics module as per the manufacturer’s protocol. The detection and quantification of target hybridization was performed with an Affymetrix GeneChip Scanner. Three separate samples were analyzed for each of the treatment groups.Initially, raw data were analyzed using Robust Multichip Analysis (RMA) with a logarithmic base 2 conversion utilizing the Affymetrix ArrayAssist Suite (Affymetrix, Inc., Santa Clara, CA). One-way ANOVA analysis of each group (basal, FSH, or cAMP) was used to determine genes that are significantly affected by HPTE. This was followed by an unpaired t-test to determine changes in global gene expression between HPTE-treated groups and the control groups to determine the levels of fold change. All statistical analyses were performed using P < 0.005 as the level of statistical significance.
GSM349716
GPL1355
HPTE_10_cAMP 3 Granulosa cells from post-natal days 21-27 female Sprague Dawley Rats Charles River outbred immature female rats Briefly, intact, non-primed, immature female rats (21-27 days old) were sacrificed by cervical dislocation. Ovaries were removed from the animals, cleaned free of associated fat, oviduct, and bursa ovary, and then placed in ice-cold DMEM/F-12. Granulosa cells were isolated using a non-enzymatic needle puncture method with a sterile bundle of beading needles to release the cells from follicles. Cell viability was determined by the trypan blue exclusion method. The cells were plated and cultured (24-well plates) at approximately 3-4 x 105 viable cells/ml/well and incubated at 37°C for 24 h in the DMEM/F-12 containing FBS (5%) in an atmosphere of 5% CO2 in air. Following the 24 h acclimation period, the medium was replaced with serum-free DMEM/F12 containing androstenedione (0.1 M) as a substrate for aromatization. Cells were treated with increasing doses of HPTE (0, 1, 5, and 10µM) in the absence (basal) or presence of 3 ng FSH/ml or 1 mM cAMP for 48 h. The doses of FSH and cAMP were selected based on a dose-response curve that provided a maximum level of stimulation for E2 production (data not shown). Cultures were terminated at 48 h following the addition of treatments. Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Cat # 74106) followed by DNase I treatment. RNA quality was assessed by electrophoresis using the Agilent Bioanalyzer 2100 and spectrophotometric analysis prior to cDNA synthesis (data not shown). Forty nanograms of total RNA from each sample were used to generate a high fidelity cDNA for array hybridization using the NuGen Ovation Biotin RNA Amplification and Labeling system (NuGen). After fragmentation and biotin labeling, the samples were hybridized to Affymetrix Rat Genome 230 2.0 arrays, which have eleven pairs of oligonucleotide probes per chip. Washing and staining of all arrays were carried out in the Affymetrix fluidics module as per the manufacturer’s protocol. The detection and quantification of target hybridization was performed with an Affymetrix GeneChip Scanner. Three separate samples were analyzed for each of the treatment groups.Initially, raw data were analyzed using Robust Multichip Analysis (RMA) with a logarithmic base 2 conversion utilizing the Affymetrix ArrayAssist Suite (Affymetrix, Inc., Santa Clara, CA). One-way ANOVA analysis of each group (basal, FSH, or cAMP) was used to determine genes that are significantly affected by HPTE. This was followed by an unpaired t-test to determine changes in global gene expression between HPTE-treated groups and the control groups to determine the levels of fold change. All statistical analyses were performed using P < 0.005 as the level of statistical significance.
 
 
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