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Gene Expression Profiles of Human Granulosa Cells Treated With Bioequivalent Doses of Corifollitrophin Alfa or Recombinant Human Follicle-Stimulating Hormone

ABSTRACT: Using recombinant DNA technologies, a new chimeric gene containing the coding sequences of the follicle stimulating hormone (FSH) β-subunit and the C-terminal peptide of the human chorionic gonadotrophin (hCG) β-subunit have been designed to generate a new gonadotrophin named corifollitrophin alfa. Corifollitrophin alfa has longer elimination half-life and a slower rate of absorption compared with FSH, which makes corifollitrophin alfa a long-acting hormone employed as a substitute of the recombinant FSH (recFSH) in the controlled ovarian stimulation (COS). The purpose of this study is to compare the gene expression profiles elicited by bioequivalent doses of corifollitrophin alfa or recFSH in primary cultures of human granulosa cells (hGCs). Both gonadotrophins exert their functions by binding FSH receptors (FSHRs), activating signaling pathways that increase the cyclic adenosine monophosphate (cAMP) intracellular content. Bioequivalence has been defined as the dose/duration of gonadotrophin treatment able to promote the same amount of intracellular cAMP. Primary cultured hGCs were treated with different doses of either gonadotrophin and the cAMP was measured after different incubation times to establish the bioequivalence. Results obtained by comparing the bioequivalent treatments, showed that corifollitrophin alfa is more effective than recFSH in inducing aromatase gene expression after 6 and 24 hours from the initial stimulation in agreement with its long-acting characteristic’s.

ORGANISM(S): Homo sapiens

PROVIDER: GSE115846 | GEO | 2020/06/01

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 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).

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Gene Expression Profiles of Human Granulosa Cells Treated With Bioequivalent Doses of Corifollitrophin Alfa or Recombinant Human Follicle-Stimulating Hormone

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