Project description:We performed global scale microarray analysis to identify detailed mechanisms by which nonpermissive temperature induces cell differentiation in testicular Sertoli TTE3 cells harboring temperature-sensitive SV40 large T-antigen by using an Affymetrix GeneChip system. Testicular Sertoli TTE3 cells used in the present study were derived from transgenic mice harboring a temperature-sensitive simian virus 40 large T-antigen. In the TTE3 cells, inactivation of the T-antigen by a nonpermissive temperature at 39˚C led to cell differentiation accompanying elevation of transferrin and cyclin-dependent kinase inhibitor CDKN1A. Of the 22, 690 probe sets analyzed, nonpermissive temperature up-regulated 729 probe sets and down-regulated 471 probe sets by >2.0-fold. Keywords: Sertoli cell; differentiation; temperature-sensitive SV40 large T-antigen; nonpermissive temperature
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: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:We performed global scale microarray analysis to identify detailed mechanisms by which nonpermissive temperature induces cell differentiation in testicular Sertoli TTE3 cells harboring temperature-sensitive SV40 large T-antigen by using an Affymetrix GeneChip system. Testicular Sertoli TTE3 cells used in the present study were derived from transgenic mice harboring a temperature-sensitive simian virus 40 large T-antigen. In the TTE3 cells, inactivation of the T-antigen by a nonpermissive temperature at 39ËC led to cell differentiation accompanying elevation of transferrin and cyclin-dependent kinase inhibitor CDKN1A. Of the 22, 690 probe sets analyzed, nonpermissive temperature up-regulated 729 probe sets and down-regulated 471 probe sets by >2.0-fold. Experiment Overall Design: TTE3 cells were cultured at 39ËC for 0, 6, 12, and 24 h. Total RNA samples were prepared from the cells. Gene expression was analyzed by an Affymetrix GeneChip® system with the Mouse Expression Array 430A which was spotted with 22,690 probe sets. Sample preparation for array hybridization was carried out as described in the manufactureâs instructions.
Project description:To study effect of VRK1 deletion on spermatogenesis of the mouse, transciptomic analysis of genes in postnatal 8-day testicular cells of wild type and VRK1-deficient Mus musculus was performed.
Project description:We performed global scale microarray analysis to identify detailed mechanisms by which bisphenol A (BPA) induce cell death by using an Affymetrix GeneChip system. Testicular Sertoli TTE3 cells used in the present study were derived from transgenic mice harboring a temperature-sensitive simian virus 40 large T-antigen. Cell death accompanying endoplasmic reticulum stress was observed in the cells treated with 0.2 mM BPA. Of the 22,690 probe sets analyzed, approximately 1,300 genes were down- and up-regulated by a factor of 2.0 or greater in the cells treated with BPA. Keywords: bisphenol A, gene expression, testicular Sertoli cell
Project description:Sertoli cells, omnipresent, somatic cells within the seminiferous tubules of the mammalian testis are essential to male fertility. Sertoli cells maintain the integrity of the testicular microenvironment, regulate hormone synthesis, and of particular importance, synthesize the active derivative of vitamin A, all trans retinoic acid (atRA), which is required for germ cell differentiation and the commitment of male germ cells to meiosis. Stages VII-IX, when atRA synthesis occurs in the testis, coincides with multiple germ cell development and testicular restructuring events that rely on Sertoli cell gene products to proceed normally. In this study, we have synchronized and captured the mouse testis at four recurrent points of atRA synthesis to observe transcriptomic changes within Sertoli cells as mice age and the Sertoli cells are exposed to increasingly developed germ cell subtypes. This work provides comprehensive, high-resolution characterization of when known, functional Sertoli cell genes are induced across the first wave of spermatogenesis, and outlines in silico predictions of germ cell derived signaling mechanisms targeting Sertoli cells.