Mechanisms of MEHP Inhibitory Action and Analysis of Potential Replacement Plasticizers on Leydig Cell Steroidogenesis

Steroid production in Leydig cells is stimulated mainly by the pituitary luteinizing hormone, which leads to increased expression of genes involved in steroidogenesis, including the gene encoding the steroidogenic acute regulatory (STAR) protein. Mono(2-ethylhexyl)phthalate (MEHP), the active metabolite of the widely used plasticizer DEHP, is known to disrupt Leydig steroidogenesis but its mechanisms of action remain poorly understood. We found that MEHP caused a significant reduction in hormone-induced steroid hormone production in two Leydig cell lines, MA-10 and MLTC-1. Consistent with disrupted cholesterol transport, we found that MEHP represses cAMP-induced Star promoter activity. MEHP responsiveness was mapped to the proximal Star promoter, which contains multiple binding sites for several transcription factors. In addition to STAR, we found that MEHP also reduced the levels of ferredoxin reductase, a protein essential for electron transport during steroidogenesis. Finally, we tested new plasticizers as alternatives to phthalates. Two plasticizers, dioctyl succinate and 1,6-hexanediol dibenzoate, had no significant effect on hormone-induced steroidogenesis. Our current findings reveal that MEHP represses steroidogenesis by affecting cholesterol transport and its conversion into pregnenolone. We also found that two novel molecules with desirable plasticizer properties have no impact on Leydig cell steroidogenesis and could be suitable phthalate replacements.

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A review of drug-induced Leydig cell hyperplasia and neoplasia in the rat and some comparisons with man

Human & Experimental Toxicology ◽

10.1177/096032719501400703 ◽

1995 ◽

Vol 14 (7) ◽

pp. 562-572 ◽

Cited By ~ 65

Author(s):

DE Prentice ◽

AW Meikle

Keyword(s):

Leydig Cell ◽

Leydig Cells ◽

Safety Assessment ◽

Hormonal Status ◽

Control Mechanisms ◽

Hormone Production ◽

Drug Induced ◽

Cell Hyperplasia ◽

Etiological Factors ◽

Func Tion

This paper describes control of normal Leydig cell func tion and testosterone production. The macroscopic and histopathological appearances of spontaneous Leydig cell hyperplasias and tumors (LCT) in the rat are reviewed together with their incidence and hormonal status. Drugs which induce LCTs in chronic studies are discussed and include busereline, carbamazepine, cimetidine, finas teride, flutamide, gemfibrozil, histrelin, hydralazine, indomethacin, isradipine, lactitol, leuprolide, metronida zole, mesulergine, nafarelin, norprolac and vidarabine. The known mechanisms of LCT induction in the rat are reviewed together with other possible etiological factors. The incidence, clinical picture and etiological factors of LCTs in man are also surveyed. Hormone production in Leydig cells and LCTs in rats and man are compared. Differences between the two species are considered, par ticularly with regard to Leydig cell control mechanisms. The paper concludes that drug-induced LCTs in rats are most probably not predictive for man and their occurrence has little relevance in human safety assessment.

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Interleukin-1alpha stimulates steroidogenic acute regulatory protein expression via p38 MAP kinase in immature rat Leydig cells

Journal of Molecular Endocrinology ◽

10.1677/jme.0.0300059 ◽

2003 ◽

Vol 30 (1) ◽

pp. 59-67 ◽

Cited By ~ 21

Author(s):

K Svechnikov ◽

DM Stocco ◽

O Soder

Keyword(s):

Protein Expression ◽

Map Kinase ◽

Leydig Cell ◽

Leydig Cells ◽

Regulatory Protein ◽

P38 Map Kinase ◽

Dependent Manner ◽

Star Protein ◽

Adult Leydig Cells ◽

Steroidogenic Acute Regulatory

We have investigated the involvement of the steroidogenic acute regulatory (StAR) protein in interleukin-1alpha (IL-1alpha)-induced steroidogenesis in immature (40-day-old) and adult Leydig cells in vitro. Further, IL-1alpha-mediated signaling pathway(s) controlling StAR expression in immature Leydig cells were also studied. IL-1alpha stimulated both androgen production and StAR protein expression in a dose- and time-dependent manner in immature but not adult Leydig cells. These effects of IL-1alpha were prevented by pretreatment of the cells with the specific inhibitors of the p38 MAP kinase, SB203580 and PD169316, suggesting that this kinase is an important part of IL-1alpha signaling in the immature Leydig cell. The present results suggest that IL-1alpha, which is constitutively produced by the rat testis from postnatal day 25, is an important paracrine regulator of postnatal Leydig cell maturation. Regulation of StAR protein expression is one of the possible mechanisms by which IL-1alpha contributes to the differentiation of immature Leydig cells into adult cells.

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Interleukin-1 inhibits Leydig cell steroidogenesis without affecting steroidogenic acute regulatory protein messenger ribonucleic acid or protein levels

Journal of Endocrinology ◽

10.1677/joe.0.1560461 ◽

1998 ◽

Vol 156 (3) ◽

pp. 461-467 ◽

Cited By ~ 28

Author(s):

T Lin ◽

D Wang ◽

DM Stocco

Keyword(s):

Mrna Expression ◽

Leydig Cell ◽

Leydig Cells ◽

Interleukin 1 ◽

Mrna Level ◽

Primary Cultures ◽

Regulatory Protein ◽

Steroidogenic Acute Regulatory Protein ◽

Protein Levels ◽

Steroidogenic Acute Regulatory

The rate-limiting step of steroidogenesis is the transport of the substrate cholesterol from the outer to the inner mitochondrial membrane which involves a cycloheximide-sensitive newly synthesized protein. A protein believed to carry out this function was recently cloned from MA-10 mouse Leydig tumor cells and named the steroidogenic acute regulatory protein (StAR). In the present study, we evaluated the expression and regulation of StAR in primary cultures of rat Leydig cells. StAR mRNA was expressed in Leydig cells as two major transcripts of 3.8 and 1.7 kb and one minor transcript of 1.2 kb. Induction of StAR mRNA transcripts could be detected as early as 30 min after the addition of human choriogonadotropin (hCG) with peak levels attained between 2 and 4 h. hCG in concentrations of 0.1-10 ng/ml caused a dose-dependent increase in StAR mRNA expression. hCG administered at a dose of 10 ng/ml increased the 3.8 kb StAR mRNA level about 14-fold and the 1.7 kb StAR mRNA level about 13.6-fold. hCG-stimulated StAR mRNA was associated with increased StAR protein levels as determined by immunoblot analysis (a 4.5-fold increase). Murine interleukin-1 alpha (mIL-1 alpha) at a concentration of 100 ng/ml inhibited hCG-induced cytochrome P450 side-chain cleavage (P450 scc) mRNA expression and testosterone formation almost completely. Interestingly, mIL-1 alpha had no effect on hCG-induced StAR mRNA or protein levels. Furthermore, mIL-1 alpha (10 ng/ml) decreased conversion of (22R)-hydroxycholesterol to testosterone while the conversion of pregnenolone, 17-hydroxypregnenolone, dehydroepiandrosterone and androstenedione to testosterone were not affected. These results indicate that the major inhibitory effect of IL-1 on Leydig cell function occurs at the level of P450 scc.

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The increased number of Leydig cells by di(2-ethylhexyl) phthalate comes from the differentiation of stem cells into Leydig cell lineage in the adult rat testis

Toxicology ◽

10.1016/j.tox.2013.01.021 ◽

2013 ◽

Vol 306 ◽

pp. 9-15 ◽

Cited By ~ 18

Author(s):

Jingjing Guo ◽

Xing-Wang Li ◽

Yong Liang ◽

Yufei Ge ◽

Xiaomin Chen ◽

...

Keyword(s):

Stem Cells ◽

Leydig Cell ◽

Leydig Cells ◽

Cell Lineage ◽

Rat Testis ◽

Adult Rat ◽

Ethylhexyl Phthalate

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Wt1 dictates the fate of fetal and adult Leydig cells during development in the mouse testis

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00425.2014 ◽

2014 ◽

Vol 307 (12) ◽

pp. E1131-E1143 ◽

Cited By ~ 33

Author(s):

Qing Wen ◽

Qiao-Song Zheng ◽

Xi-Xia Li ◽

Zhao-Yuan Hu ◽

Fei Gao ◽

...

Keyword(s):

Leydig Cell ◽

Testosterone Level ◽

Leydig Cells ◽

Wilms Tumor ◽

Suppressor Gene ◽

Testicular Development ◽

Gene Encoding ◽

Testis Development ◽

Adult Mice ◽

Serum Lh

Wilms' tumor 1 ( Wt1) is a tumor suppressor gene encoding ∼24 zinc finger transcription factors. In the mammalian testis, Wt1 is expressed mostly by Sertoli cells (SCs) involved in testis development, spermatogenesis, and adult Leydig cell (ALC) steroidogenesis. Global knockout (KO) of Wt1 is lethal in mice due to defects in embryogenesis. Herein, we showed that Wt1 is involved in regulating fetal Leydig cell (FLC) degeneration and ALC differentiation during testicular development. Using Wt1−/flox; Amh-Cre mice that specifically deleted Wt1 in the SC vs. age-matched wild-type (WT) controls, FLC-like-clusters were found in Wt1-deficient testes that remained mitotically active from postnatal day 1 (P1) to P56, and no ALC was detected at these ages. Leydig cells in mutant adult testes displayed morphological features of FLC. Also, FLC-like cells in adult mutant testes had reduced expression in ALC-associated genes Ptgds, Sult1e1, Vcam1, Hsd11b1, Hsd3b6, and Hsd17b3 but high expression of FLC-associated genes Thbs2 and Hsd3b1. Whereas serum LH and testosterone level in mutant mice were not different from controls, intratesticular testosterone level was significantly reduced. Deletion of Wt1 gene also perturbed the expression of steroidogenic enzymes Star, P450c17, Hsd3b6, Hsd3b1, Hsd17b1, and Hsd17b3. FLCs in adult mutant testes failed to convert androstenedione to testosterone due to a lack of Hsd17b3, and this defect was rescued by coculturing with fetal SCs. In summary, FLC-like cells in mutant testes are putative FLCs that remain mitotically active in adult mice, illustrating that Wt1 dictates the fate of FLC and ALC during postnatal testis development.

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Translocator Protein/Peripheral Benzodiazepine Receptor Is Not Required for Steroid Hormone Biosynthesis

Endocrinology ◽

10.1210/en.2013-1556 ◽

2014 ◽

Vol 155 (1) ◽

pp. 89-97 ◽

Cited By ~ 150

Author(s):

Kanako Morohaku ◽

Susanne H. Pelton ◽

Daniel J. Daugherty ◽

W. Ronald Butler ◽

Wenbin Deng ◽

...

Keyword(s):

Leydig Cells ◽

Steroid Hormone ◽

Conditional Knockout ◽

Translocator Protein ◽

Cholesterol Transport ◽

Type I ◽

Hormone Production ◽

Steroid Hormone Biosynthesis ◽

Genetic Ablation ◽

Hormone Biosynthesis

Molecular events that regulate cellular biosynthesis of steroid hormones have been a topic of intense research for more than half a century. It has been established that transport of cholesterol into the mitochondria forms the rate-limiting step in steroid hormone production. In current models, both the steroidogenic acute regulatory protein (StAR) and the translocator protein (TSPO) have been implicated to have a concerted and indispensable effort in this cholesterol transport. Deletion of StAR in mice resulted in a critical failure of steroid hormone production, but deletion of TSPO in mice was found to be embryonic lethal. As a result, the role of TSPO in cholesterol transport has been established only using pharmacologic and genetic tools in vitro. To allow us to explore in more detail the function of TSPO in cell type-specific experimental manipulations in vivo, we generated mice carrying TSPO floxed alleles (TSPOfl/fl). In this study we made conditional knockout mice (TSPOcΔ/Δ) with TSPO deletion in testicular Leydig cells by crossing with an anti-Mullerian hormone receptor type II cre/+ mouse line. Genetic ablation of TSPO in steroidogenic Leydig cells in mice did not affect testosterone production, gametogenesis, and reproduction. Expression of StAR, cytochrome P450 side chain cleavage enzyme, 3β-hydroxysteroid dehydrogenase/Δ5-Δ4 isomerase type I, and TSPO2 in TSPOcΔ/Δ testis was unaffected. These results challenge the prevailing dogma that claims an essential role for TSPO in steroid hormone biosynthesis and force reexamination of functional interpretations made for this protein. This is the first study examining conditional TSPO gene deletion in mice. The results show that TSPO function is not essential for steroid hormone biosynthesis.

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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 ◽

10.1210/endo.141.9.7678 ◽

2000 ◽

Vol 141 (9) ◽

pp. 3137-3148 ◽

Cited By ~ 16

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.

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"STEROID-CELL" ANTIBODY IN ENDOCRINE DISEASES

Acta Endocrinologica ◽

10.1530/acta.0.0760729 ◽

1974 ◽

Vol 76 (4) ◽

pp. 729-740 ◽

Cited By ~ 15

Author(s):

Peter Kamp ◽

Per Platz ◽

Jørn Nerup

Keyword(s):

Leydig Cells ◽

Addison’S Disease ◽

Addison's Disease ◽

Hormone Production ◽

Cortical Cells ◽

Endocrine Diseases ◽

Cell Antibody ◽

Indirect Immunofluorescence Technique ◽

Males And Females ◽

Adrenal Cortical Cells

ABSTRACT By means of an indirect immunofluorescence technique, sera from 116 patients with Addison's disease, an equal number of age and sex matched controls and 97 patients with other endocrine diseases were examined for the occurrence of antibody to steroid-producing cells in ovary, testis and adrenal cortex. Fluorescent staining was observed in the theca cells of growing follicles, the theca lutein cells, testicular Leydig cells and adrenal cortical cells, i. e. cells which contain enzyme systems used in steroid hormone production. The "steroid-cell" antibody was present in 24 % of the patients with idiopathic Addison's disease, equally frequent in males and females, and in 17 % of the patients with tuberculous Addison's disease, but was rarely found in controls, including patients with other endocrine diseases. Female hypergonadotrophic hypogonadism made an exception, since the "steroid-cell" antibody was found in about half the cases with this condition.

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