Effect of the Methoxychlor Metabolite HPTE on the Rat Ovarian Granulosa Cell Transcriptome in Vitro
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ABSTRACT:
ORGANISM(S): Rattus norvegicus
PROVIDER: GSE13883 | GEO | 2009/06/10
SECONDARY ACCESSION(S): PRJNA110355
REPOSITORIES: GEO
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Project description:Ovarian granulosa cells play a central role in steroidogenesis, which is critical for female reproduction. Follicle-stimulating hormone (FSH) promotes cAMPâmediated signaling to regulate granulosa cell steroidogenesis. We have shown previously that 2, 2-bis-(p-hydroxyphenyl)-1, 1, 1-trichloroethane (HPTE) inhibits FSH- and dibutyryl cAMP-stimulated steroidogenesis, and affects the mRNA levels of steroidogenic pathway enzymes in rat granulosa cells. However, HPTE showed a differential effect in FSH- and cAMP-stimulated cells in that HPTE more completely blocked FSH- when compared to cAMP-driven steroidogenesis. The objective of this study was to analyze the effects of HPTE on global gene expression profiles in untreated granulosa cells and those challenged with FSH or cAMP. Granulosa cells from immature rats were cultured with 0, 1, 5, or 10 µM HPTE in the presence and absence of either 3 ng FSH/ml or 1 mM cAMP for 48 h. Total RNA was isolated for microarray analysis using the GeneChip Rat Genome 230 2.0 and ArrayAssist Microarray Suite. An investigation of changes in gene expression across all HPTE treatments showed that HPTE altered more genes in FSH- (~670 genes) than in cAMP-stimulated cells (~366 genes). Analysis confirmed that HPTE more effectively inhibited FSH- than cAMP-induced steroid pathway gene expression and steroidogenesis. Furthermore, expression patterns of novel genes regulating signal transduction, transport, cell cycle, adhesion, differentiation, motility and growth, apoptosis, development, and metabolism were all altered by HPTE. This study further established that HPTE exerts differential effects within the granulosa cell steroidogenic pathway, and revealed that these effects include broader changes in gene expression. Experiment Overall Design: Ovaries were dissected from immature (21-27 days old), Sprague-Dawley rats and were placed into ice cold Hamâs F12 medium. Granulosa cells were isolated from ovaries free of fat pads and surrounding connective tissue by performing non-enzymatic needle puncture method . Following two washes by centrifugation at 1400rpm/250xg for 5 min at 4 CË, the cells were plated and cultured at approximately 3-4x105 viable cells/mL/well in DMEM/F-12 medium containing 5% FCS at 37 CË in 5% CO2 in a 24-well culture plate for 24 hours. The cells were then cultured in serum-free DMEM/F12 containing 0.1 μM androstenedione as a substrate for aromatization into E2. Experiment Overall Design: Cells were treated in triplicate with increasing doses of HPTE (0, 1, 5, and 10 µM) 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). Similar doses of HPTE were previously used (Zachow and Uzumcu, 2006). Cultures were terminated at 48 h following the addition of treatments. Experiment Overall Design: Cell lysate was collected for total RNA isolation for DNA microarray analysis. Total RNA were extracted using the RNeasy Mini Kit (Qiagen, Cat # 74106) followed by DNase I treatment. RNA qualities were assessed by electrophoresis using the Agilent Bioanalyzer 2100 and spectrophotometric analysis prior to cDNA synthesis. Forty nanograms of total RNA from each sample was used to generate a high fidelity cDNA for array hybridization using NuGen Ovation Biotin RNA Amplification and Labeling system (NuGen, Cat#2300-60). After fragmentation and biotin labeling, the samples were hybridized to Affymetrix Rat Genome 230 2.0 arrays. Washing and staining of all arrays were carried out in the Affymetrix fluidics module as per the manufacturerâs protocol. The detection and quantization of target hybridization was performed with an Affymetrix GeneChip Scanner. Data were assessed for array performance prior to analysis and analyzed using ArrayAssist (Stratagene).
2009-06-23 | E-GEOD-13883 | biostudies-arrayexpress
Project description:Steroid hormones regulate essential physiological processes and inadequate levels are associated with various pathological conditions. In testosterone-producing Leydig cells, steroidogenesis is strongly stimulated by LH via its receptor leading to increased cAMP production and expression of the steroidogenic acute regulatory (STAR) protein, which is essential for the initiation of steroidogenesis. Leydig cell steroidogenesis then passively decreases following the rapid degradation of cAMP into AMP by phosphodiesterases. In this study, we show that AMP-activated protein kinase (AMPK) is activated following cAMP breakdown in MA-10 and MLTC-1 Leydig cells. Activated AMPK then actively inhibits cAMP-induced steroidogenesis by repressing the expression of key regulators of steroidogenesis including Star and Nr4a1. Similar results were obtained in Y-1 adrenal cells and in the constitutive steroidogenic cell line R2C. Our data identify AMPK as an active repressor of steroid hormone biosynthesis in steroidogenic cells that is essential to preserve cellular energy and prevent excess steroid production. Steroid hormones regulate essential physiological processes and inadequate levels are associated with various pathological conditions. In testosterone-producing Leydig cells, steroidogenesis is strongly stimulated by LH via its receptor leading to increased cAMP production and expression of the steroidogenic acute regulatory (STAR) protein, which is essential for the initiation of steroidogenesis. Leydig cell steroidogenesis then passively decreases following the rapid degradation of cAMP into AMP by phosphodiesterases. In this study, we show that AMP-activated protein kinase (AMPK) is activated following cAMP breakdown in MA-10 and MLTC-1 Leydig cells. Activated AMPK then actively inhibits cAMP-induced steroidogenesis by repressing the expression of key regulators of steroidogenesis including Star and Nr4a1. Similar results were obtained in Y-1 adrenal cells and in the constitutive steroidogenic cell line R2C. Our data identify AMPK as an active repressor of steroid hormone biosynthesis in steroidogenic cells that is essential to preserve cellular energy and prevent excess steroid production. MA-10 Leydig cells were treated with either DMSO (control), 10 uM forskolin or forskolin+Aicar (1 mM) for 1.5 h before total RNA extraction
2014-08-19 | E-GEOD-50118 | biostudies-arrayexpress
Project description:Steroid hormones regulate essential physiological processes and inadequate levels are associated with various pathological conditions. In testosterone-producing Leydig cells, steroidogenesis is strongly stimulated by LH via its receptor leading to increased cAMP production and expression of the steroidogenic acute regulatory (STAR) protein, which is essential for the initiation of steroidogenesis. Leydig cell steroidogenesis then passively decreases following the rapid degradation of cAMP into AMP by phosphodiesterases. In this study, we show that AMP-activated protein kinase (AMPK) is activated following cAMP breakdown in MA-10 and MLTC-1 Leydig cells. Activated AMPK then actively inhibits cAMP-induced steroidogenesis by repressing the expression of key regulators of steroidogenesis including Star and Nr4a1. Similar results were obtained in Y-1 adrenal cells and in the constitutive steroidogenic cell line R2C. Our data identify AMPK as an active repressor of steroid hormone biosynthesis in steroidogenic cells that is essential to preserve cellular energy and prevent excess steroid production. Steroid hormones regulate essential physiological processes and inadequate levels are associated with various pathological conditions. In testosterone-producing Leydig cells, steroidogenesis is strongly stimulated by LH via its receptor leading to increased cAMP production and expression of the steroidogenic acute regulatory (STAR) protein, which is essential for the initiation of steroidogenesis. Leydig cell steroidogenesis then passively decreases following the rapid degradation of cAMP into AMP by phosphodiesterases. In this study, we show that AMP-activated protein kinase (AMPK) is activated following cAMP breakdown in MA-10 and MLTC-1 Leydig cells. Activated AMPK then actively inhibits cAMP-induced steroidogenesis by repressing the expression of key regulators of steroidogenesis including Star and Nr4a1. Similar results were obtained in Y-1 adrenal cells and in the constitutive steroidogenic cell line R2C. Our data identify AMPK as an active repressor of steroid hormone biosynthesis in steroidogenic cells that is essential to preserve cellular energy and prevent excess steroid production.
2014-08-19 | GSE50118 | GEO
Project description:PKA activation by FSH is essential to transduce FSH-mediated effects on granulosa cell proliferation, differentiation and steroidogenesis. However, It is unknown whether activation of PKA is sufficient to account for the entire program of granulosa cell responses to FSH. We addressed this question by conducting a comprehensive comparative analysis of signaling pathways and gene expression profiles of granulosa cells stimulated with FSH or expressing a constitutively active PKA mutant, PKA-CQR.
2016-10-01 | GSE73955 | GEO
Project description:The forkhead box transcription factor FOXO1 is highly expressed in granulosa cells of growing follicles but is down-regulated by FSH in culture or by LH-induced luteinization in vivo. To analyze the function of FOXO1, we infected rat and mouse granulosa cells with adenoviral vectors expressing two FOXO1 mutants: a gain-of-function mutant FOXOA3 that has two serine residues and one threonine residue mutated to alanines rendering this protein constitutively active and nuclear, and a FOXOA3-mutant DNA-binding domain (mDBD) in which the DBD is mutated. The infected cells were then treated with vehicle or FSH for specific time intervals. Infection of the granulosa cells was highly efficient, caused only minimal apoptosis, and maintained FOXO1 protein at levels of the endogenous protein observed in cells before exposure to FSH. RNA was prepared from control and adenoviral infected cells exposed to vehicle or FSH for 12 and 24 h. Affymetrix microarray and database analyses identified, and real time RT-PCR verified, that genes within the lipid, sterol, and steroidogenic biosynthetic pathways (Hmgcs1, Hmgcr, Mvk, Sqle, Lss, Cyp51, Tm7sf2, Dhcr24 and Star, Cyp11a1, and Cyp19), including two key transcriptional regulators Srebf1 and Srebf2 of cholesterol biosynthesis and steroidogenesis (Nr5a1, Nr5a2), were major targets induced by FSH and suppressed by FOXOA3 and FOXOA3-mDBD in the cultured granulosa cells. By contrast, FOXOA3 and FOXOA3- mDBD induced expression of Cyp27a1 mRNA that encodes an enzyme involved in cholesterol catabolism to oxysterols. The genes up-regulated by FSH in cultured granulosa cells were also induced in granulosa cells of preovulatory follicles and corpora lutea collected from immature mice primed with FSH (equine choriogonadotropin) and LH (human choriogonadotropin), respectively. Conversely, Foxo1 and Cyp27a1 mRNAs were reduced by these same treatments. Collectively, these data provide novel evidence that FOXO1 may play a key role in granulosa cells to modulate lipid and sterol biosynthesis, thereby preventing elevated steroidogenesis during early stages of follicle development. Experiment Overall Design: Immature rat granulosa cells were affected with either control (GFP) or FOXO1 mutant adenovirus (FOXOA3 and FOXOA3-mDBD) for 4 hours. Then the cells were treated with either FSH or vehicle for 24 hours. The gene expression profiles for the four treatment samples (control, control +FSH, FOXOA3 + FSH, FOXOA3-mDBD + FSH) were compared using Rat Genome 230.2 Arrays with one array per sample.
2009-04-25 | E-GEOD-15727 | biostudies-arrayexpress
Project description:The forkhead box transcription factor FOXO1 is highly expressed in granulosa cells of growing follicles but is down-regulated by FSH in culture or by LH-induced luteinization in vivo. To analyze the function of FOXO1, we infected rat and mouse granulosa cells with adenoviral vectors expressing two FOXO1 mutants: a gain-of-function mutant FOXOA3 that has two serine residues and one threonine residue mutated to alanines rendering this protein constitutively active and nuclear, and a FOXOA3-mutant DNA-binding domain (mDBD) in which the DBD is mutated. The infected cells were then treated with vehicle or FSH for specific time intervals. Infection of the granulosa cells was highly efficient, caused only minimal apoptosis, and maintained FOXO1 protein at levels of the endogenous protein observed in cells before exposure to FSH. RNA was prepared from control and adenoviral infected cells exposed to vehicle or FSH for 12 and 24 h. Affymetrix microarray and database analyses identified, and real time RT-PCR verified, that genes within the lipid, sterol, and steroidogenic biosynthetic pathways (Hmgcs1, Hmgcr, Mvk, Sqle, Lss, Cyp51, Tm7sf2, Dhcr24 and Star, Cyp11a1, and Cyp19), including two key transcriptional regulators Srebf1 and Srebf2 of cholesterol biosynthesis and steroidogenesis (Nr5a1, Nr5a2), were major targets induced by FSH and suppressed by FOXOA3 and FOXOA3-mDBD in the cultured granulosa cells. By contrast, FOXOA3 and FOXOA3- mDBD induced expression of Cyp27a1 mRNA that encodes an enzyme involved in cholesterol catabolism to oxysterols. The genes up-regulated by FSH in cultured granulosa cells were also induced in granulosa cells of preovulatory follicles and corpora lutea collected from immature mice primed with FSH (equine choriogonadotropin) and LH (human choriogonadotropin), respectively. Conversely, Foxo1 and Cyp27a1 mRNAs were reduced by these same treatments. Collectively, these data provide novel evidence that FOXO1 may play a key role in granulosa cells to modulate lipid and sterol biosynthesis, thereby preventing elevated steroidogenesis during early stages of follicle development.
2009-04-17 | GSE15727 | GEO
Project description:FSH signaling is well known to lead to steroidogenesis, cellular proliferation, and follicular growth. The development of global transcriptomic allows to expand our understanding of the FSH effect on several additional functions of granulosa cells and to better understand how it controls or prevents several differentiation events in granulosa cells from medium size follicles
2016-05-01 | GSE74834 | GEO
Project description:In the ovary, proliferation and differentiation of granulosa cells (GCs) drive the growth of follicles. This is, in part, dependent on gonadotropins. Our immunohistochemical studies provided hints of mitochondrial biogenesis and intracellular redistribution in GCs of growing follicles. A cellular model, human KGN cells (granulosa cell tumor cells, derived from growing follicles) was used to study aspects of mitochondrial dynamics. To elevate cAMP and thereby mimic gonadotropin actions, forskolin (FSK) was used, which increased KGN cell size and mitochondrial DNA within 24 h. MitoTracker experiments and ultrastructural 3D reconstruction revealed that FSK treatment induced the formation of elaborate mitochondrial networks. H89, a protein kinase A (PKA) inhibitor, reduced network formation. A proteomic analysis indicated that FSK among others elevated levels of regulators of the cytoskeleton and the steroidogenic enzyme CYP11A1, located in mitochondria, was more than 3-fold increased, implying that cAMP/PKA-associated structural changes go in parallel with the acquisition of steroidogenic competence of mitochondria in KGN cells. In summary, in situ observations showed increases in mitochondria and suggested intracellular trafficking in GCs during follicular growth and indicated that they may partially be under the control of gonadotropins/cAMP. In line with this, elevation of cAMP in KGN profoundly affected mitochondria dynamics in a PKA-dependent manner and implicated cytoskeletal changes.
2022-10-14 | PXD032160 | Pride
Project description:CCR4-NOT deadenylase is a major regulator of mRNA turnover in eukaryotes. It contains 2 heterogeneous catalytic subunits encoded by Cnot7/8 and Cnot6/6l genes in vertebrates, respectively. The physiological function of each catalytic subunit was unclear due to potential gene redundancy in vivo. In this study Cnot6/6l double knockout mice were generated. These mice were viable and generally healthy, but females were infertile with irregular estrus cycle and poor ovarian responses to gonadotropins. Follicle-stimulating hormone (FSH) stimulated transcription and translation of mRNAs encoding CNOT6 and CNOT6L in ovarian granulosa cells through the activity of phosphoinositide 3-kinase signaling pathway. Results of transcriptome analyses indicated that FSH downregulated a large number of transcripts in granulosa cells during the transition from pre-antral to antral follicles in a CNOT6/6L-dependent manner. These two deadenylases functioned as key effectors of FSH in altering the developmental programs in granulosa cells and triggered clearance of specific transcripts in growing follicles, particularly those encoding repressing factors of granulosa cell proliferation and steroidogenesis. These results demonstrated that FSH modulates granulosa cells function by stimulating selective translational activation and degradation of existed mRNAs, in additional to inducing de novo gene transcription. Meanwhile, this study provides in vivo evidence that CCR4-NOTCNOT6/6L-mediated mRNA deadenylation is dispensable in most somatic cell types, but is essential for the female reproductive endocrine regulation.
2021-11-04 | GSE158773 | GEO
Project description:Spermatogenesis requires the presence of functional somatic Sertoli cells in the seminiferous tubules of the testis. Sertoli cells provide support and factors necessary for the successful progression of germ cells into spermatozoa. Sertoli cells are regulated to a large degree by the glycoprotein hormone FSH, which is required for the testis to acquire full size and spermatogenic capacity. Signaling events initiated by the binding of FSH to its receptor lead to an alteration of Sertoli cell gene expression. To characterize the changes in gene expression in FSH-treated Sertoli cells, we used the mRNA from these cells to screen Affymetrix U34A rat GeneChip oligonucleotide microarrays. Sertoli cells from 20-d-old rats were cultured in the presence of 25 ng/ml ovine FSH. At 0, 2, 4, 8, and 24 h after the addition of FSH, total RNA was purified and used to prepare biotinylated target, which was hybridized to the U34A rat microarray containing approximately 9000 rat genes. Analysis identified 100-300 transcripts at each time point that were up-regulated or down-regulated by 2-fold or greater. Genes previously reported to be FSH or cAMP regulated in rat Sertoli cells were identified, in addition to numerous genes not reported to be expressed or FSH regulated in Sertoli cells. The expression patterns of five of these genes, encoding nerve growth factor inducible gene B, PRL-1, PC3 nerve growth factor-inducible antiproliferative putative secreted protein, diacylglycerol acyltransferase, and an expressed sequence tag, in FSH- and N,O'-dibutyryl cAMP-treated rat Sertoli cells were confirmed and characterized by Northern blot analysis. Thus, we have begun to define the transcriptome induced and repressed by FSH in rat Sertoli cells, and we have generated datasets of genes available for further analysis in regard to spermatogenesis and Sertoli cell signaling.
2007-12-12 | E-GEOD-509 | biostudies-arrayexpress