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

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.

Project description:Evidence that persistent environmental pollutants may target the male reproductive system is increasing. The male reproductive system is regulated by secretion of testosterone by testicular Leydig cells, and perturbation of Leydig cells function may have ultimate consequences. 3-methylsulfonyl-DDE (3-MeSO2-DDE) is a potent adrenal toxicants formed from the persistent insecticide DDT. Although studies have revealed endocrine disruptive effect of 3-MeSO2-DDE, the underlying mechanisms at cellular level in steroidogenic Leydig cells remains to be established. The current study addresses the effect of 3-MeSO2-DDE viability, hormone production and proteome response of primary neonatal porcine Leydig cells. The AlamarBlue™ assay was used to evaluate cell viability. Solid phase radioimmunoassay was used to measure concentration of hormones produced by both unstimulated and luteinizing hormone (LH)-stimulated Leydig cells following 48 h exposure. Protein samples from Leydig cells exposed to a non-cytotoxic concentration of 3-MeSO2-DDE (10µM) were subjected to nano-LC-MS/MS and analyzed on a Q Exactive mass spectrometer and quantified using label-free quantitative algorithm. Gene Ontology (GO) and Ingenuity Pathway Analysis (IPA) were carried out for functional annotation and identification of protein interaction networks. 3-MeSO2-DDE regulated Leydig cell steroidogenesis differentially depending on cell culture condition. Whereas its effect on testosterone secretion at basal condition was stimulatory, the effect on LH-stimulated cells was inhibitory. From triplicate experiments, a total of 7540 proteins were identified in which the abundance of 87 proteins in unstimulated Leydig cells and 146 proteins in LH-stimulated Leydig cells were found to be significantly regulated in response to 3-MeSO2-DDE exposure. These proteins not only are the first reported in relation to 3-MeSO2-DDE exposure, but also display small number of proteins shared between culture conditions, suggesting the action of 3-MeSO2-DDE on several targeted pathways, including mitochondrial dysfunction, oxidative phosphorylation, EIF2-signaling, and glutathion-mediated detoxification. Further identification and characterization of these proteins and pathways may build our understanding to the molecular basis of 3-MeSO2-DDE induced endocrine disruption in Leydig cells.

Project description:MAMLD1 (mastermind-like domain containing 1, alias CXorf6) on human chromosome Xq28 is a causative gene for hypospadias, a mild form of 46,XY disorders of sex development. To date, multiple mutations have been identified in patients with various types of hypospadias. In this regard, the mouse homologous gene Mamld1 is transiently expressed in fetal Sertoli and Leydig cells around the critical period for sex development, and transient Mamld1 knockdown using small interfering RNAs (siRNAs) reduces testosterone production in cultured mouse Leydig tumor cells (MLTCs). We performed detailed analyses in Mamld1 knockdown experiments using MLTCs. We used microarrays to display the global gene expression profile change, after knockdown of Mamld1 expression in MLTCs, to find the gene set regulated by Mamld1. To compare changes in gene expression profile in MLTCs, MLTCs were transfected with control siRNA or Mamld1 siRNA, 48h later, total RNA was subjected to Q-PCR to confirm a significantly decreased Mamld1 mRNA, and then these total RNA samples were for microarray analysis. The microarray data was analyzed to explore gene sets/pathways, which were significantly impacted by knockdown of Mamld1. Real-time RT-PCR and microarray analyses showed significantly decreased Cyp17a1 expression (~70%) in both siRNA1- and siRNA2-transfected MLTCs. The siRNAs knockdown did not affect the expressions of Nr5a1 (Sf1), Star, Por, and Insl3. 47 genes including a Notch-related gene Hey1 were significantly up-regulated (fold change >2.0) and 38 genes were significantly down-regulated (fold change <0.5) in both siRNA1- and siRNA2-transfected MLTCs (Table S1 and Table S2). However, Mamld1 knockdown had no discernible effect on the Hes3 expression level (siRNA1: fold change 0.92, P=0.80; siRNA2: fold change 1.43, P=0.35). MLTCs were maintained in RPMI 1640 supplemented with 10% fetal bovine serum, and were transiently transfected with two siRNAs against Mamld1 or with non-targeting control RNA. To compare changes in gene expression profile in MLTCs, 48h later, total RNA was subjected to Q-PCR to confirm a significantly decreased Mamld1 mRNA, and then these total RNA samples were for microarray analysis. Supplementary file: t-test unpaired [i1] Vs [control], [i2] Vs [control], P <= 0.05 FC >=2.0

Dataset Information

Novel targets for the transcription factors MEF2 in MA-10 Leydig cells

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets