Project description:To better assess the impact of contaminants on fish reproduction, it is increasingly important to collect data at multiple levels of biological organization. However, a challenge in ecotoxicology is that three of the most commonly measured reproductive endpoints; gonad histology, ex vivo steroid production, and gene expression, can be difficult to obtain in small-bodied fish (e.g. zebrafish, Japanese medaka, fathead minnow) due to limited tissue availability. Using 5M-NM-1-dihydrotestosterone (DHT) as a test compound, the objectives of the current study were to assess the feasibility of measuring gene expression in tissues also used for ex vivo steroid production studies. A portion of the testes was also used for qualitative histological examination. Male mummichog were exposed to 0, 5, and 50 M-BM-5g/L DHT for 21 days. Following the exposure, testes were collected and separated into three pieces for histology, ex vivo incubation to measure steroidogenic capacity [Testosterone(T), 17M-NM-2-estradiol (E2), 11-ketotestosterone (11KT)] and gene expression analyses. Testis RNA at time of dissection and testis RNA at end of ex vivo incubation (18 h) was collected and assessed for RNA integrity. The expression of genes coding for estrogen receptor M-NM-2a (esrba) and estrogen receptor M-NM-2b (esrbb) as well as the key steroidogenic genes, steroidogenic acute regulatory protein (star), P450 side chain cleavage (p450scc) and 11M-NM-2-hydroxysteroid dehydrogenase (hsd11b3) were assessed in testis before and after ex vivo incubation. There were no significant differences in RNA quality between the two time points, however the expression of esrba, esrbb and p450scc was significantly lower post-incubation, although star, hsd11b3, and all four reference gene expression levels remained unchanged over time. DHT did not affect the expression of any of the measured genes at either concentration. Histological examination showed that all fish contained mature testes dominated by spermatozoa and spermatids. DHT did not affect T production but significantly decreased 11KT production in the high treatment group. This suggests a role for DHT in androgen regulation, and has Microarray analysis was performed with four biological replicates (4 control and 4 treatment). Male mummichog were exposed to 0, 5, and 50 M-BM-5g/L DHT for 21 days. Individuals from the high concentration, 50 ug/L DHT, were used for the microarray analysis.

Project description:Steroidogenic acute regulatory protein (Star) facilitates cholesterol transfer into the inner mitochondrial membrane in the acute and regulated production of steroid hormones. Mice lacking Star (Star-/-) share the phenotypes with patients with congenital lipoid adrenal hyperplasia such as compromised production of steroid hormones and florid accumulation of cholesterol esters in adrenal glands and gonads. To define specific patterns of molecular changes with disruption of Star, we performed a transcriptome analysis of steroidogenic cells in adrenal glands. We harvested adrenal glands at E17.5 or 18.5 from seven wild-type (Star+/+) mice or four Star-/- mice having the transgene targeting enhanced green fluorescent protein (eGFP) under the control of regulatory sequences of mouse Star gene. Steroidogenic cells were selectively isolated by fluorescent-activated cell sorting. The gene expression profile of the fluorescence-positive cells was obtained with Agilent Whole Mouse Genome Microarray and was confirmed by quantitative real-time PCR. We identified 961 and 622 genes that were significantly up-regulated and down-regulated, respectively, in Star-/- mice compared with Star+/+ mice (fold difference ≥2, p value of Student t test <0.05, and Benjamini-Hochberg false discovery rate of multiple comparison test <0.2). In Star-/- mice, expression levels of genes involved in cholesterol mobilization and efflux or immune response as antigen presenting cells were significantly increased, and transition from fetal to adult adrenocortical cells were significantly decreased, whereas those of genes related to steroid hormone biosynthesis or cholesterol biosynthesis and influx were not significantly changed. The trancriptome analysis revealed hitherto undescribed characteristic changes in adrenocortical cells and expanded our understanding of the pathophysiology of the steroidogenic cells with disruption of Star. Gene expression profiles of isolated adrenal steroidogenic cells were compared between seven wild-type mice and four knockout mice lacking steroidogenic acute regulatory protein at E17.5-18.5.

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:We recently demonstrated that Fsh modifies in vitro the testicular transcriptome of rainbow trout at early stages of spermatogenesis. Some of the regulated genes were found related to pathways highly relevant for the testicular functions i.e. spermatogenesis and steroidogenesis. Unlike in mammals, in fish both gonadotropins are able to efficiently stimulate the steroidogenesis, likely through a direct interaction with their cognate receptors present on the Leydig cells. In this context, we wondered whether the effects of Fsh on the tubular compartment were mediated through the production of steroids. To address this issue we performed in vitro incubations of testis explants in the presence of Fsh alone or in combination with an inhibitor of the steroidogenesis, the trilostane. Trilostane significantly reduced or suppressed the response to Fsh of many genes showing that important aspects of the Fsh action on fish testis is indirect and requires the production of steroids. Interestingly, most of the genes regulated in response to Fsh through the steroid mediation were similarly regulated by Lh and many were also found regulated by androgen treatment. On the other hand, for many other genes the response to Fsh was not affected in the presence of trilostane. A majority of those genes were also preferentially regulated by Fsh, as compared to Lh, suggesting that specific regulatory effects of Fsh were independent of steroid production. Finally antagonistic effects between Fsh and steroids were found, in particular for genes encoding clue factors of steroidogenesis or for genes of the Igf system that tended to be inhibited by androgens. Testes were collected from all-male population rainbow trout at early stages of spermatogenesis.Testes were chopped in small pieces, in sterile conditions and then pooled and mixed. Testis fragments were randomly distributed (60-80 mg per well) on Nunc polycarbonate membrane inserts in 24-well plates filled with 300 ÂµL of modified L15 culture medium supplemented with 2% Ultroser SF. Incubation were performed in six replicates for 96 hours, at 12Â°C, in the absence or presence of purified trout Fsh alone (500 ng/mL) or in combination with 10 Âµg/mL trilostane, an inhibitor of 3 beta-hydroxysteroid dehydrogenase. A pre-incubation with trilostane was done for 1 hour before adding Fsh.

Dataset Information

Effects of 5α-dihydrotestosterone on male mummichog (Fundulus heteroclitus) testes steroid production and gene expression patterns

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