scholarly journals Comparison of the Effects of Dibutyl and Monobutyl Phthalates on the Steroidogenesis of Rat Immature Leydig Cells

2016 ◽  
Vol 2016 ◽  
pp. 1-12 ◽  
Author(s):  
Linxi Li ◽  
Xiaomin Chen ◽  
Guoxin Hu ◽  
Sicong Wang ◽  
Renai Xu ◽  
...  
Keyword(s):  
Leydig Cell ◽  
Leydig Cells ◽  
Dibutyl Phthalate ◽  
Potent Inhibitor ◽  
Endocrine System ◽  
Mrna Levels ◽  
Expression Levels ◽  
Reproductive Endocrine ◽  
The Media ◽  
Monobutyl Phthalate

Dibutyl phthalate (DBP) is a widely used synthetic phthalic diester and monobutyl phthalate (MBP) is its main metabolite. DBP can be released into the environment and potentially disrupting mammalian male reproductive endocrine system. However, the potencies of DBP and MBP to inhibit Leydig cell steroidogenesis and their possible mechanisms are not clear. Immature Leydig cells isolated from rats were cultured with 0.05–50 μM DBP or MBP for 3 h in combination with testosterone synthesis regulator or intermediate. The concentrations of 5α-androstanediol and testosterone in the media were measured, and the mRNA levels of the androgen biosynthetic genes were detected by qPCR. The direct actions of DBP or MBP on CYP11A1, CYP17A1, SRD5A1, and AKR1C14 activities were measured. MBP inhibited androgen production by the immature Leydig cell at as low as 50 nM, while 50 μM was required for DBP to suppress its androgen production. MBP mainly downregulatedCyp11a1andHsd3b1expression levels at 50 nM. However, 50 μM DBP downregulatedStar,Hsd3b1, andHsd17b3expression levels and directly inhibited CYP11A1 and CYP17A1 activities. In conclusion, DBP is metabolized to more potent inhibitor MBP that downregulated the expression levels of some androgen biosynthetic enzymes.

scholarly journals Regulation of cytochrome P450 aromatase gene expression in adult rat Leydig cells: comparison with estradiol production

2001 ◽  
Vol 168 (1) ◽  
pp. 95-105 ◽  
Author(s):  
C Genissel ◽  
J Levallet ◽  
S Carreau
Keyword(s):  
Gene Expression ◽  
Cytochrome P450 ◽  
Cyclic Amp ◽  
Leydig Cell ◽  
Half Life ◽  
Leydig Cells ◽  
Aromatase Activity ◽  
Mrna Levels ◽  
Low Concentrations ◽  
Cytochrome P450 Aromatase

Regulation of aromatase gene expression in purified rat Leydig cells has not yet been investigated. Therefore, using a highly specific quantitative RT-PCR method, we have measured the amount of cytochrome P450 aromatase (P450arom) mRNA and aromatase activity in mature rat Leydig cells submitted to various treatments during 24 h. Estradiol production was enhanced in a dose-related manner in the presence of testosterone, the maximum (28% increase) being obtained with 200 ng/ml. Related to the P450arom mRNA levels, a decrease was observed in the presence of low concentrations (50 and 100 ng/ml) of testosterone, then a 20% increase of the amount of transcripts was recorded for the higher concentrations (200-500 ng/ml). The same result was obtained in the presence of 5alpha-dihydrotestosterone (an androgen resistant to aromatase activity). The addition of ovine LH (oLH; 0.1-50 ng/ml) to the Leydig cell culture medium induced a dose-related augmentation of estradiol output up to 10 ng/ml oLH, although a decrease was observed with 50 ng/ml when compared with maximal values. mRNA levels slightly decreased in the presence of low concentrations (0.1-1 ng/ml) of oLH, an effect that was abolished by the addition of testosterone; mRNA levels were increased by oLH (5-10 ng/ml) 35 and 75% respectively in the absence and presence of testosterone (when compared with Leydig cells incubated without treatment). With 50 ng/ml oLH, a large augmentation (twofold) of the P450arom mRNA level either without or with testosterone was observed. Dibutyryl cyclic AMP (1 mM) mimicked the effect of oLH. The half-life of the P450arom mRNAs was twofold increased in the presence of testosterone and oLH when compared with the half-life in the absence of treatment (5.8+/-0.6 h). Taken together, our data have demonstrated that, in freshly isolated Leydig cells from mature rat testes, the regulation of aromatase expression and enzymatic activity is under LH (through cyclic AMP) and steroid control; moreover seminiferous tubule-secreted factor(s) are also involved. Therefore, rat Leydig cell aromatase is controlled at both transcriptional and post-transcriptional steps by endocrine and/or locally produced modulators.

scholarly journals Slit/Robo signaling regulates Leydig cell steroidogenesis

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Emmanuelle Martinot ◽  
Derek Boerboom
Keyword(s):  
Cancer Progression ◽  
Leydig Cell ◽  
Leydig Cells ◽  
Adult Mouse ◽  
Primary Cultures ◽  
Mouse Testis ◽  
Mrna Levels ◽  
Pathologic Processes ◽  
In Vitro Treatment

Abstract Background First identified as a regulator of neuronal axon guidance, Slit/Robo signaling has since been implicated in additional physiologic and pathologic processes, such as angiogenesis, organogenesis and cancer progression. However, its roles in the regulation of testis function have been little explored. Methods Immunohistochemistry and RT-qPCR analyses were performed to detect the expression of Slit/Robo signaling effectors in the adult mouse testis. To identify the roles and mechanisms of Slit/Robo signaling in the regulation of steroidogenesis, RT-qPCR, immunoblotting and hormone measurements were carried out using Leydig cells (primary cultures and the MA10 cell line) treated with exogenous SLIT ligands, and testes from Robo1-null mice. Results Slit1, -2 and -3 and Robo1 and -2 expression was detected in the adult mouse testis, particularly in Leydig cells. In vitro treatment of Leydig cells with exogenous SLIT ligands led to a decrease in the expression of the steroidogenic genes Star, Cyp11a1, and Cyp17a1. SLIT2 treatment decreased the phosphorylation of the key steroidogenic gene regulator CREB, possibly in part by suppressing AKT activity. Furthermore, SLIT2 treatment reduced the responsiveness of MA10 cells to luteinizing hormone by decreasing the expression of Lhcgr. Consistent with these in vitro results, an increase in testicular Star mRNA levels and intra-testicular testosterone concentrations were found in Robo1-null mice. Finally, we showed that the expression of the Slit and Robo genes in Leydig cells is enhanced by testosterone treatment in vitro, by an AR-independent mechanism. Conclusion Taken together, these results suggest that Slit/Robo signaling represents a novel mechanism that regulates Leydig cell steroidogenesis. It may act in an autocrine/paracrine manner to mediate negative feedback by testosterone on its own synthesis.

scholarly journals Comparison of cell types in the rat Leydig cell lineage after ethane dimethanesulfonate treatment

Reproduction ◽  
2013 ◽  
Vol 145 (4) ◽  
pp. 371-380 ◽  
Author(s):  
Jingjing Guo ◽  
Hongyu Zhou ◽  
Zhijian Su ◽  
Bingbing Chen ◽  
Guimin Wang ◽  
...  
Keyword(s):  
Leydig Cell ◽  
Leydig Cells ◽  
Pubertal Development ◽  
Cell Lineage ◽  
Hydroxysteroid Dehydrogenase ◽  
Mrna Levels ◽  
Steroidogenic Enzymes ◽  
Isolated Cells ◽  
Adult Leydig Cells

The objective of this study was to purify cells in the Leydig cell lineage following regeneration after ethane dimethanesulfonate (EDS) treatment and compare their steroidogenic capacity. Regenerated progenitor (RPLCs), immature (RILCs), and adult Leydig cells (RALCs) were isolated from testes 21, 28 and 56 days after EDS treatment respectively. Production rates for androgens including androsterone and 5α-androstane-17β, 3α-diol (DIOL), testosterone and androstenedione were measured in RPLCs, RILCs and RALCs in media after 3-h in vitro culture with 100 ng/ml LH. Steady-state mRNA levels of steroidogenic enzymes and their activities were measured in freshly isolated cells. Compared to adult Leydig cells (ALCs) isolated from normal 90-day-old rat testes, which primarily produce testosterone (69.73%), RPLCs and RILCs primarily produced androsterone (70.21%) and DIOL (69.79%) respectively. Leydig cells isolated from testes 56 days post-EDS showed equivalent capacity of steroidogenesis to ALCs and primarily produced testosterone (72.90%). RPLCs had cholesterol side-chain cleavage enzyme, 3β-hydroxysteroid dehydrogenase 1 and 17α-hydroxylase but had almost no detectable 17β-hydroxysteroid dehydrogenase 3 and 11β-hydroxysteroid dehydrogenase 1 activities, while RILCs had increased 17β-hydroxysteroid dehydrogenase 3 and 11β-hydroxysteroid dehydrogenase 1 activities. Because RPLCs and RILCs had higher 5α-reductase 1 and 3α-hydroxysteroid dehydrogenase activities they produced mainly 5α-reduced androgens. Real-time PCR confirmed the similar trends for the expressions of these steroidogenic enzymes. In conclusion, the purified RPLCs, RILCs and RALCs are similar to those of their counterparts during rat pubertal development.

scholarly journals Ghrelin Inhibits the Proliferative Activity of Immature Leydig Cells in Vivo and Regulates Stem Cell Factor Messenger Ribonucleic Acid Expression in Rat Testis

Endocrinology ◽  
2004 ◽  
Vol 145 (11) ◽  
pp. 4825-4834 ◽  
Author(s):  
M. L. Barreiro ◽  
F. Gaytan ◽  
J. M. Castellano ◽  
J. S. Suominen ◽  
J. Roa ◽  
...  
Keyword(s):  
Stem Cell ◽  
Leydig Cell ◽  
Stem Cell Factor ◽  
Proliferative Activity ◽  
Leydig Cells ◽  
Human Testis ◽  
Seminiferous Tubules ◽  
Mrna Levels ◽  
Messenger Ribonucleic Acid

Abstract Ghrelin has emerged as putative regulator of an array of endocrine and nonendocrine functions, including cell proliferation. Recently, we provided evidence for the expression of ghrelin in mature, but not in undifferentiated, Leydig cells of rat and human testis. Yet testicular actions of ghrelin, other than modulation of testosterone secretion, remain unexplored. In the present study we evaluated the effects of ghrelin on proliferation of Leydig cell precursors during puberty and after selective elimination of mature Leydig cells by treatment with ethylene dimethane sulfonate. In these settings, intratesticular injection of ghrelin significantly decreased the proliferative activity of differentiating immature Leydig cells, estimated by 5-bromodeoxyuridine labeling. This response was selective and associated, in ethylene dimethane sulfonate-treated animals, with a decrease in the mRNA levels of stem cell factor (SCF), i.e. a key signal in spermatogenesis and a putative regulator of Leydig cell development. Thus, the effects of ghrelin on SCF gene expression were evaluated. In adult rats, ghrelin induced a significant decrease in SCF mRNA levels in vivo. Such an inhibitory action was also detected in vitro using cultures of staged seminiferous tubules. The inhibitory effect of ghrelin in vivo was dependent on proper FSH input, because it was detected in hypophysectomized rats only after FSH replacement. Overall, it is proposed that acquisition of ghrelin expression by Leydig cell precursors during differentiation may operate as a self-regulatory signal for the inhibition of the proliferative activity of this cell type through direct or indirect (i.e. SCF-mediated) mechanisms. In addition, we present novel evidence for the ability of ghrelin to modulate the expression of the SCF gene, which may have implications for the mode of action of this molecule in the testis as well as in other physiological systems.

scholarly journals MEF2 Is Restricted to the Male Gonad and Regulates Expression of the Orphan Nuclear Receptor NR4A1

2014 ◽  
Vol 28 (6) ◽  
pp. 886-898 ◽  
Author(s):  
Caroline Daems ◽  
Luc J. Martin ◽  
Catherine Brousseau ◽  
Jacques J. Tremblay
Keyword(s):  
Nuclear Receptor ◽  
Leydig Cell ◽  
Leydig Cells ◽  
Sex Differentiation ◽  
Dominant Negative ◽  
Mrna Levels ◽  
Orphan Nuclear Receptor ◽  
Hormonal Stimulation ◽  
Regulatory Cascade ◽  
And Function

Abstract Leydig cell steroidogenesis is controlled by the pituitary gonadotropin LH that activates several signaling pathways, including the Ca2+/calmodulin kinase I (CAMKI) pathway. In other tissues, CAMKI regulates the activity of the myocyte enhancer factor 2 (MEF2) transcription factors. MEF2 factors are essential regulators of cell differentiation and organogenesis in numerous tissues but their expression and role in the mammalian gonad had not been explored. Here we show that MEF2 factors are expressed in a sexually dimorphic pattern in the mouse gonad. MEF2 factors are present in the testis throughout development and into adulthood but absent from the ovary. In the testis, MEF2 was localized mainly in the nucleus of both somatic lineages, the supporting Sertoli cells and the steroidogenic Leydig cells. In Leydig cells, MEF2 was found to activate the expression of Nr4a1, a nuclear receptor important for hormone-induced steroidogenesis. In these cells MEF2 also cooperates with forskolin and CAMKI to enhance Nr4a1 promoter activity via two MEF2 elements (−318 and −284 bp). EMSA confirmed direct binding of MEF2 to these elements whereas chromatin immunoprecipitation revealed that MEF2 recruitment to the proximal Nr4a1 promoter was increased following hormonal stimulation. Modulation of endogenous MEF2 protein level (small interfering RNA-mediated knockdown) or MEF2 activity (MEF2-Engrailed active dominant negative) led to a significant decrease in Nr4a1 mRNA levels in Leydig cells. All together, our results identify MEF2 as a novel testis-specific transcription factor, supporting a role for this factor in male sex differentiation and function. MEF2 was also positioned upstream of NR4A1 in a regulatory cascade controlling Leydig cell gene expression.

Growth hormone-induced STAT5B regulates Star gene expression through a cooperation with cJUN in mouse MA-10 Leydig cells

Endocrinology ◽  
2021 ◽  
Author(s):  
Pierre-Olivier Hébert-Mercier ◽  
Francis Bergeron ◽  
Nicholas M Robert ◽  
Samir Mehanovic ◽  
Kenley Joule Pierre ◽  
...  
Keyword(s):  
Gene Expression ◽  
Growth Hormone ◽  
Leydig Cell ◽  
Leydig Cells ◽  
Cell Function ◽  
Mrna Levels ◽  
Leydig Cell Function ◽  
Star Gene

Abstract Leydig cells produce androgens that are essential for male sex differentiation and reproductive function. Leydig cell function is regulated by several hormones and signaling molecules, including growth hormone (GH). Although GH is known to upregulate Star gene expression in Leydig cells, its molecular mechanism of action remains unknown. The STAT5B transcription factor is a downstream effector of GH signaling in other systems. While STAT5B is present in both primary and Leydig cell lines, its function in these cells has yet to be ascertained. Here we report that treatment of MA-10 Leydig cells with GH or overexpression of STAT5B induces Star mRNA levels and increases steroid hormone output. The mouse Star promoter contains a consensus STAT5B element (TTCnnnGAA) at -756 bp to which STAT5B binds in vitro (EMSA and supershift) and in vivo (ChIP) in a GH-induced manner. In functional promoter assays, STAT5B was found to activate a -980 bp mouse Star reporter. Mutating the -756 bp element prevented STAT5B binding but did not abrogate STAT5B-responsiveness. STAT5B was found to functionally cooperate with DNA-bound cJUN. The STAT5B/cJUN cooperation was only observed in Leydig cells and not in Sertoli or fibroblast cells, indicating that additional Leydig cell-enriched transcription factors are required. The STAT5B/cJUN cooperation was lost only when both STAT5B and cJUN elements were mutated. In addition to identifying the Star gene as a novel target for STAT5B in Leydig cells, our data provide important new insights into the mechanism of GH and STAT5B action in the regulation of Leydig cell function.

scholarly journals The Peroxisome Proliferator Perfluorodecanoic Acid Inhibits the Peripheral-Type Benzodiazepine Receptor (PBR) Expression and Hormone-Stimulated Mitochondrial Cholesterol Transport and Steroid Formation in Leydig Cells*

Endocrinology ◽  
2000 ◽  
Vol 141 (9) ◽  
pp. 3137-3148 ◽  
Author(s):  
Noureddine Boujrad ◽  
Branislav Vidic ◽  
Maria Gazouli ◽  
Martine Culty ◽  
Vassilios Papadopoulos
Keyword(s):  
Leydig Cell ◽  
Mrna Stability ◽  
Leydig Cells ◽  
Benzodiazepine Receptor ◽  
Model Systems ◽  
Cell Protein ◽  
Cholesterol Transport ◽  
Peroxisome Proliferator ◽  
Mrna Levels ◽  
Peripheral Type

Abstract The peroxisome proliferator perfluordecanoic acid (PFDA) has been shown to exert an antiandrogenic effect in vivo by acting directly on the interstitial Leydig cells of the testis. The objective of this study was to examine the in vitro effects of PFDA and identify its site of action in steroidogenesis using as model systems the mouse tumor MA-10 and isolated rat Leydig cells. PFDA inhibited in a time- and dose-dependent manner the hCG-stimulated Leydig cell steroidogenesis. This effect was localized at the level of cholesterol transport into the mitochondria. PFDA did not affect either the total cell protein synthesis or the mitochondrial integrity. Moreover, it did not induce any DNA damage. Morphological studies indicated that PFDA induced lipid accumulation in the cells, probably due to the fact that cholesterol mobilized by hCG did not enter the mitochondria to be used for steroidogenesis. In search of the target of PFDA, we examined its effect on key regulatory mechanisms of steroidogenesis. PFDA did not affect the hCG-induced steroidogenic acute regulatory protein (StAR) levels. However, it was found to inhibit the mitochondrial peripheral-type benzodiazepine receptor (PBR) ligand binding capacity, 18-kDa protein, and messenger RNA (mRNA) levels. Further studies indicated that PFDA did not affect PBR transcription, but it rather accelerated PBR mRNA decay. Taken together, these data suggest that PFDA inhibits the Leydig cell steroidogenesis by affecting PBR mRNA stability, thus inhibiting PBR expression, cholesterol transport into the mitochondria, and the subsequent steroid formation. Moreover, this action of PFDA on PBR mRNA stability indicates a new mechanism of action of peroxisome proliferators distinct from the classic transcription-mediated regulation of target genes.

Androgen receptor signalling in testicular Leydig cells is essential for Leydig cell maturation and survival

2014 ◽  
Author(s):  
Laura O'Hara ◽  
Kerry McInnes ◽  
Ioannis Simitsidellis ◽  
Steph Morgan ◽  
Laura Milne ◽  
...  
Keyword(s):  
Androgen Receptor ◽  
Leydig Cell ◽  
Leydig Cells ◽  
Cell Maturation ◽  
Receptor Signalling

scholarly journals Methylobacterium oryzae Influences Isoepoxydon Dehydrogenase Gene Expression and Patulin Production by Penicillium expansum

Water ◽  
2021 ◽  
Vol 13 (10) ◽  
pp. 1427
Author(s):  
Tiago Barros Afonso ◽  
Lúcia Chaves Simões ◽  
Nelson Lima
Keyword(s):  
Gene Expression ◽  
Distribution Systems ◽  
Water Distribution ◽  
Penicillium Expansum ◽  
Transcriptional Level ◽  
Positive Trend ◽  
Mrna Levels ◽  
Expression Levels ◽  
Production Values ◽  
Methylobacterium Oryzae

Biofilms can be considered the main source of microorganisms in drinking water distribution systems (DWDS). The ecology of a biofilm is dependent on a variety of factors, including the presence of microbial metabolites excreted by its inhabitants. This study reports the effect of the Gram-negative bacteria Methylobacterium oryzae on the idh gene expression levels and patulin production of Penicillium expansum mature biofilms. For this purpose, a RT-qPCR method to quantify idh mRNA levels was applied. In addition, the idh expression levels were compared with the patulin production. The results obtained revealed that the effect of the bacterium on pre-established P. expansum biofilms is dependent on the time of interaction. More mature P. expansum biofilms appear to be more resistant to the inhibitory effect that M. oryzae causes towards idh gene expression and patulin production. A positive trend was observed between the idh expression and patulin production values. The results indicate that M. oryzae affects patulin production by acting at the transcriptional level of the idh gene.

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