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.

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 ÂµM 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 Î¼M 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 ÂµM) 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).

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.

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Comparison of Gene expression profiling of granulosa cells treated with follicle stimulating hormone or constitutively active protein kinase A

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