Project description:Methoxyacetic acid (MAA) is the active metabolite of the widely used industrial chemical ethylene glycol monomethyl ether, an established testicular toxicant. MAA induces the degradation of testicular germ cells in association with changes in gene expression in both germ cells and Sertoli cells of the testis. This study investigates the impact of MAA on gene expression in testicular Leydig cells, which play a critical role in germ cell survival and male reproductive function. Cultured mouse TM3 Leydig cells were treated with MAA for 3, 8, and 24 h and global gene expression was monitored by microarray analysis. A total of 3,912 MAA-responsive genes were identified. Ingenuity Pathway analysis identified reproductive system disease, inflammatory disease and connective tissue disorder as the top biological functions affected by MAA. The MAA-responsive genes were classified into 1,366 early responders, 1,387 mid-responders, and 1,138 late responders, based on the time required for MAA to elicit a response. Analysis of enriched functional clusters for each subgroup identified 106 MAA early response genes involved in transcription regulation, including 32 genes associated with developmental processes and 60 DNA-binding proteins that responded to MAA rapidly but transiently, and which may contribute to the downstream effects of MAA seen for large numbers of mid and late response genes. Genes within the phosphatidylinositol/phospholipase C/calcium signaling pathway, whose activity is required for potentiation of nuclear receptor signaling by MAA, were also enriched in the set of early MAA response genes. These findings on the progressive changes in gene expression induced by MAA in Leydig cells may help elucidate the signaling pathways perturbed by this testicular toxicant and explain its mechanism of toxicity at the gene level.
Project description:To study the potential tumorigenic effect of two activating mutations of hLHR identified in patients, we generated in vitro cell model using mouse Leydig MA-10 cells. The two mutations, Asp578His and Asp578Gly, were stably transfected to MA-10 cell to create MA-10-Asp578His and MA-10-Asp578Gly line. The profile of expressed genes in cells expressing the mutated hLHR was compared with that of control cells using cDNA microarrays with NIA mouse clone sets. Keywords: Genetic modification analysis
Project description:The purpose of this study was to identify differentially-expressed genes between WT MA-10 mouse tumor Leydig cells and MA-10 cells in which the steroidogenic acute regulatory protein (STAR) is knocked out.
Project description:Methoxyacetic acid (MAA) is the active metabolite of the widely used industrial chemical ethylene glycol monomethyl ether, an established testicular toxicant. MAA induces the degradation of testicular germ cells in association with changes in gene expression in both germ cells and Sertoli cells of the testis. This study investigates the impact of MAA on gene expression in testicular Leydig cells, which play a critical role in germ cell survival and male reproductive function. Cultured mouse TM3 Leydig cells were treated with MAA for 3, 8, and 24 h and global gene expression was monitored by microarray analysis. A total of 3,912 MAA-responsive genes were identified. Ingenuity Pathway analysis identified reproductive system disease, inflammatory disease and connective tissue disorder as the top biological functions affected by MAA. The MAA-responsive genes were classified into 1,366 early responders, 1,387 mid-responders, and 1,138 late responders, based on the time required for MAA to elicit a response. Analysis of enriched functional clusters for each subgroup identified 106 MAA early response genes involved in transcription regulation, including 32 genes associated with developmental processes and 60 DNA-binding proteins that responded to MAA rapidly but transiently, and which may contribute to the downstream effects of MAA seen for large numbers of mid and late response genes. Genes within the phosphatidylinositol/phospholipase C/calcium signaling pathway, whose activity is required for potentiation of nuclear receptor signaling by MAA, were also enriched in the set of early MAA response genes. These findings on the progressive changes in gene expression induced by MAA in Leydig cells may help elucidate the signaling pathways perturbed by this testicular toxicant and explain its mechanism of toxicity at the gene level. Mouse TM3 Leydig cells (American Type Culture Collection, Manassas, VA) were grown in DMEM-F12 medium containing 5% horse serum and 2.5% FBS. Cells were grown to ~60% confluence and treated with culture medium alone, or with culture medium containing 1 mM or 5 mM MAA for either 3, 8 or 24 h. Total RNA was then isolated using TRIzol reagent, followed by incubation with RQ1 RNAse-free DNAse for 1 h at 37°C and then heating at 75°C for 5 min using the manufacturerâs protocol. A total of 6 cultures of TM3 cells were independently treated with MAA under each of the 6 treatment conditions specified above (i.e., 1 mM or 5 mM MAA for either 3, 8 or 24 h), and the corresponding 6 sets of RNA samples were validated by RNA integrity analysis (Agilent Bioanalyzer). Each RNA sample was also validated by qPCR analysis using SYBR Green I-based chemistry and primers specific for 3 genes known to respond to MAA (Cyp17a1, Shbg, and Igfbp3) to verify consistency of the MAA responses. The 6 RNA samples were then used to prepare two independent pools (n=3 RNA samples each) for microarray analysis with dye swaps. Sample labeling, hybridization to microarrays, scanning and calculation of normalized expression ratios were carried out at the Wayne State University Institute of Environmental Health Sciences microarray facility using Alexa 555 and Alexa 647 aminoallyl-aRNA samples
Project description:In males, Leydig cells are the main producers of testosterone and insulin-like 3, hormones which are both essential for sex differentiation and reproductive functions. Nuclear receptor chicken ovalbumin upstream promoter-transcription factors II (COUP-TFII) is expressed in the cells committed to give rise to the fully functional steroidogenic adult Leydig cells and has a major role in their function and differentiation. Up to date, only handful of COUP-TFII gene targets have been reported. A transcriptomic approach was used to identify additional genes affected by depletion of COUP-TFII in mouse MA-10 Leydig cell line.
Project description:Testosterone production by Leydig cells is a tightly regulated process requiring synchronized expression of several steroidogenic genes by numerous transcription factors. Myocyte enhancer factor 2 (MEF2) is a transcription factor recently identified in somatic cells of the male gonad. In other tissues, MEF2 is an essential regulator of organogenesis and cell differentiation. So far in the testis, MEF2 was found to regulate Leydig cell steroidogenesis by controlling Nr4a1 and Star gene expression. To expand our understanding of the role of MEF2 in Leydig cells, we performed microarray analyses of MA-10 Leydig cells depleted in MEF2 and results were analyzed using the Partek and IPA softwares. Several genes were differentially expressed in MEF2-depleted Leydig cells and 15 were validated by qPCR. A large number of these genes are known to be involved in fertility, gonad morphology and steroidogenesis and include Pde8a, Por, Ahr, Bmal1, Cyp1a1, Cyp1b1, Map2k1, Tsc22d3, Nr0b2, Smad4, and Star, which were all downregulated in the absence of MEF2. In silico analyses revealed the presence of MEF2 binding sites within the first 2 kb upstream the transcription start site of the Por, Bmal1, and Nr0b2 promoters, which suggests a direct regulation by MEF2. Using transient transfections in MA-10 Leydig cells, siRNA knockdown, and a MEF2-Engrailed dominant negative, we found that MEF2 activates the Por, Bmal1 and Nr0b2 promoters and that this requires an intact MEF2 element. Our results identify novel target genes for MEF2 and define MEF2 as an important regulator of Leydig cell function and male reproduction. MA-10 Leydig cells were treated with siRNA MEF2A/2D (siRNA MEF2) or scrambled siRNA as control (siRNA Ctrl) 48h before total RNA extraction.
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