Diametric Effects of Bacterial Endotoxin Lipopolysaccharide on
Adrenal and Leydig Cell Steroidogenic Acute Regulatory Protein

Abstract Immune activation results in the activation of adrenal steroidogenesis and inhibition of gonadal steroidogenesis. Previous studies indicated that these effects were caused primarily by activation and suppression of the secretion of ACTH and LH, respectively. However, other evidence indicated a direct effect of the immune system on the gonads. In this study, serum testosterone, quantitated by RIA after lipopolysaccharide injection, showed a significant decrease within 2 h. Parallel measurement of serum LH showed no change. There were no differences in LH receptor or cAMP produced in Leydig cells between vehicle- and lipopolysaccharide-injected mice. The 30-kDa form of the steroidogenic acute regulatory (StAR) protein was quantitated, by Western blot, in Leydig cells and was found to decrease in a time-dependent manner. No change in StAR protein messenger RNA (mRNA) was detected by Northern analysis during this time, nor were any changes found in the levels of mRNA for the steroidogenic enzymes P450scc, 3β-hydroxysteroid dehydrogenaseΔ 4-Δ5-isomerase, or P450c17. In the adrenal, StAR protein was increased, as was StAR protein mRNA. No changes were observed in the levels of mRNA for P450scc, 3β-hydroxysteroid dehydrogenaseΔ 4-Δ5-isomerase, or P450c21. Thus, although the mechanisms of regulation differ, changes in the levels of StAR protein are a sensitive indicator of the steroidogenic capacity of these two tissues.

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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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Dibutyryl Cyclic Adenosine Monophosphate Restores the Ability of Aged Leydig Cells to Produce Testosterone at the High Levels Characteristic of Young Cells

Endocrinology ◽

10.1210/en.2004-0639 ◽

2004 ◽

Vol 145 (10) ◽

pp. 4441-4446 ◽

Cited By ~ 29

Author(s):

Haolin Chen ◽

June Liu ◽

Lindi Luo ◽

Barry R. Zirkin

Keyword(s):

Signal Transduction ◽

Leydig Cells ◽

Regulatory Protein ◽

Cyclic Adenosine Monophosphate ◽

Serum Testosterone ◽

Adenosine Monophosphate ◽

Brown Norway ◽

Norway Rat ◽

Brown Norway Rat ◽

Steroidogenic Acute Regulatory

Abstract The wealth of knowledge about the function and regulation of adult Leydig cells, the cells within the mammalian testis that produce testosterone, make these cells ideal for studying principles and mechanisms of aging. A hallmark of mammalian aging is decreased serum testosterone concentration. In the Brown Norway rat, this has been shown to be associated with the reduced ability of aged Leydig cells to produce testosterone in response to LH. Herein, we demonstrate that culturing the aged cells with dibutyryl cAMP, a membrane-permeable cAMP agonist that bypasses the LH receptor-adenlyly cyclase cascade, restores testosterone production to levels comparable to those of young cells and also restores steroidogenic acute regulatory protein and P450scc, the proteins involved in the rate-limiting steps of steroidogenesis. These results strongly suggest that signal transduction deficits are responsible for reduced steroidogenesis by aged Leydig cells and that bypassing signal transduction reverses the steroidogenic decline by the aged cells.

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DAPL1 is a novel regulator of testosterone production in Leydig cells of mouse testis

Scientific Reports ◽

10.1038/s41598-021-97961-6 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Hong-bin Chen ◽

Jorge Carlos Pineda Garcia ◽

Shinako Arizono ◽

Tomoki Takeda ◽

Ren-shi Li ◽

...

Keyword(s):

Leydig Cells ◽

Regulatory Protein ◽

Serum Testosterone ◽

Mouse Testis ◽

Testosterone Levels ◽

Steroidogenic Acute Regulatory ◽

Sexually Mature ◽

In Vitro Data

AbstractLeydig cells in the testes produce testosterone in the presence of gonadotropins. Therefore, male testosterone levels must oscillate within a healthy spectrum, given that elevated testosterone levels augment the risk of cardiovascular disorders. We observed that the expression of death-associated protein-like 1 (DAPL1), which is involved in the early stages of epithelial differentiation and apoptosis, is considerably higher in the testes of sexually mature mice than in other tissues. Accordingly, Dapl1-null mice were constructed to evaluate this variation. Notably, in these mice, the testicular levels of steroidogenic acute regulatory protein (StAR) and serum testosterone levels were significantly elevated on postnatal day 49. The findings were confirmed in vitro using I-10 mouse testis-derived tumor cells. The in vivo and in vitro data revealed the DAPL1-regulated the expression of StAR involving altered transcription of critical proteins in the protein kinase A and CREB/CREM pathways in Leydig cells. The collective findings implicate DAPL1 as an important factor for steroidogenesis regulation, and DAPL1 deregulation may be related to high endogenous levels of testosterone.

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Chrysin, a natural flavonoid enhances steroidogenesis and steroidogenic acute regulatory protein gene expression in mouse Leydig cells

Journal of Endocrinology ◽

10.1677/joe-07-0282 ◽

2008 ◽

Vol 197 (2) ◽

pp. 315-323 ◽

Cited By ~ 35

Author(s):

Kuladip Jana ◽

Xiangling Yin ◽

Randolph B Schiffer ◽

Jau-Jiin Chen ◽

Akhilesh K Pandey ◽

...

Keyword(s):

Gene Expression ◽

Leydig Cells ◽

Steroid Hormone ◽

Mrna Level ◽

Regulatory Protein ◽

Threshold Level ◽

Star Protein ◽

Chain Cleavage ◽

Steroidogenic Acute Regulatory ◽

Star Gene

During the aging process of males, testosterone biosynthesis declines in testicular Leydig cells resulting in decreases in various physiological functions. To explore the possibility of delaying the decline using food supplements, we have studied steroidogenic effects of a natural flavonoid, chrysin, in mouse Leydig cells. Chrysin dramatically increased cyclic AMP (cAMP)-induced steroidogenesis in MA-10 mouse Leydig tumor cells. This result was confirmed using Leydig cells isolated from mouse testes. The steroidogenic effect of chrysin is not associated with an increase in expression of the P450 side-chain cleavage enzyme, required for the conversion of cholesterol to pregnenolone. In addition, when 22(R)hydroxylcholesterol was used as a substrate, chrysin induced a non-significant increase in steroid hormone, suggesting that the majority of the observed increase in steroidogenesis was due to the increased supply of substrate cholesterol. These observations were corroborated by showing that chrysin induced a marked increase in the expression of steroidogenic acute regulatory (StAR) protein, the factor that controls mitochondrial cholesterol transfer. Also, chrysin significantly increased StAR promoter activity and StAR mRNA level. Further studies indicated that this compound depressed expression of DAX-1, a repressor in StAR gene transcription. In the absence of cAMP, chrysin did not increase steroidogenesis. However, when a sub-threshold level of cAMP was used, StAR protein and steroid hormone were increased by chrysin to the levels seen with maximal stimulation of cAMP. These results suggest that while chrysin itself is unable to induce StAR gene expression and steroidogenesis, it appears to function by increasing the sensitivity of Leydig cells to cAMP stimulation.

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MAPK Phosphatase-1 (MKP-1) Expression Is Up-Regulated by hCG/cAMP and Modulates Steroidogenesis in MA-10 Leydig Cells

Endocrinology ◽

10.1210/en.2011-0021 ◽

2011 ◽

Vol 152 (7) ◽

pp. 2665-2677 ◽

Cited By ~ 31

Author(s):

Laura Brion ◽

Paula M. Maloberti ◽

Natalia V. Gomez ◽

Cecilia Poderoso ◽

Alejandra B. Gorostizaga ◽

...

Keyword(s):

Hormonal Regulation ◽

Leydig Cells ◽

Map Kinases ◽

Regulatory Gene ◽

Regulatory Protein ◽

Protein Accumulation ◽

Mrna Levels ◽

Dependent Manner ◽

Mapk Phosphatase ◽

Steroidogenic Acute Regulatory

MAP kinases (MAPKs), such as ERK1/2, exert profound effects on a variety of physiological processes. In steroidogenic cells, ERK1/2 are involved in the expression and activation of steroidogenic acute regulatory protein, which plays a central role in the regulation of steroidogenesis. In MA-10 Leydig cells, LH and chorionic gonadotropin (CG) trigger transient ERK1/2 activation via protein kinase A, although the events that lead to ERK1/2 inactivation are not fully described. Here, we describe the hormonal regulation of MAPK phosphatase-1 (MKP-1), an enzyme that inactivates MAPKs, in MA-10 cells. In our experiments, human CG (hCG)/cAMP stimulation rapidly and transiently increased MKP-1 mRNA levels by a transcriptional action. This effect was accompanied by an increase in protein levels in both nuclear and mitochondrial compartments. In cells transiently expressing flag-MKP-1 protein, hCG/cAMP promoted the accumulation of the recombinant protein in a time-dependent manner (10-fold at 1 h). Moreover, hCG/cAMP triggered ERK1/2-dependent MKP-1 phosphorylation. The blockade of cAMP-induced MAPK kinase/ERK activation abated MKP-1 phosphorylation but only partially reduced flag-MKP-1 protein accumulation. Together, these results suggest that hCG regulates MKP-1 at transcriptional and posttranslational level, protein phosphorylation being one of the mechanisms involved in this regulation. Our study also demonstrates that MKP-1 overexpression reduces the effects of cAMP on ERK1/2 phosphorylation, steroidogenic acute regulatory gene promoter activity, mRNA levels, and steroidogenesis, whereas MKP-1 down-regulation by small interfering RNA produces opposite effects. In summary, our data demonstrate that hCG regulates MKP-1 expression at multiple stages as a negative feedback regulatory mechanism to modulate the hormonal action on ERK1/2 activity and steroidogenesis.

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Cyclin-Dependent Kinase 5 Regulates Steroidogenic Acute Regulatory Protein and Androgen Production in Mouse Leydig Cells

Endocrinology ◽

10.1210/en.2008-0496 ◽

2008 ◽

Vol 150 (1) ◽

pp. 396-403 ◽

Cited By ~ 14

Author(s):

Ho Lin ◽

Mei-Chih Chen ◽

Chien-Te Ku

Keyword(s):

Leydig Cells ◽

Kinase Activity ◽

Regulatory Protein ◽

Protein A ◽

Male Reproduction ◽

Serine Phosphorylation ◽

Cyclin Dependent Kinase ◽

Star Protein ◽

Steroidogenic Acute Regulatory ◽

Cyclin Dependent Kinase 5

The roles of cyclin-dependent kinase 5 (Cdk5) in central nervous system and neurodegenerative diseases have been intensely investigated in recent decades. Because protein expressions of Cdk5 and its regulator, p35, have been identified in Leydig cells, it is informative to further explore the novel function of Cdk5/p35 in male reproduction. Here we show that Cdk5/p35 protein expression and kinase activity in mouse Leydig cells are regulated by human chorionic gonadotrophin (hCG) in both dose- and time-dependent manners. Blocking of Cdk5 by molecular inhibitors or small interfering RNA resulted in reduction of testosterone production by Leydig cells. cAMP, a second messenger in LH signaling, was identified as a factor in hCG-dependent regulation of Cdk5/p35. Importantly, Cdk5 protein and kinase activity could support accumulation of steroidogenic acute regulatory (StAR) protein, a crucial component of steroidogenesis. We additionally addressed the protein interaction between Cdk5/p35 and StAR. The Cdk5-dependent serine phosphorylation of StAR indicated a possible mechanism by which Cdk5 induced accumulation of StAR protein. In conclusion, Cdk5 modulates hCG-induced androgen production in mouse Leydig cells, possibly through regulation of StAR protein levels. These results indicate that Cdk5 may play an important role in male reproductive endocrinology and is a potential therapeutic target in androgen-related diseases. The physiological function of cyclin-dependent kinase 5 (Cdk5) in mouse Leydig cells is to regulate androgen production through stabilizing the steroiodogenic acute regulatory (StAR) protein.

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Kolaviron Biflavanoids of Garcinia Kola Seeds Protect Atrazine-Induced Cytotoxicity in Primary Cultures of Rat Leydig Cells

International Journal of Toxicology ◽

10.1177/1091581812445476 ◽

2012 ◽

Vol 31 (4) ◽

pp. 407-415 ◽

Cited By ~ 17

Author(s):

Sunny O. Abarikwu ◽

Ebenezer O. Farombi ◽

Aditya B. Pant

Keyword(s):

Superoxide Dismutase ◽

Messenger Rna ◽

Leydig Cells ◽

Primary Cultures ◽

Regulatory Protein ◽

Mrna Levels ◽

Garcinia Kola ◽

Chain Cleavage ◽

Cleavage Enzyme ◽

Steroidogenic Acute Regulatory

We sought to explore the mechanism by which kolaviron (Kol) protects against atrazine (ATZ)-induced toxicity of cultured interstitial Leydig cells (ILCs). In our experiments, treatment with Kol improved Leydig cell viability and significantly reduced malondialdehyde (MDA) and reactive oxygen species (ROS) levels. Further investigations revealed a reduction in glutathione peroxidase (GSH-Px), glutathione reductase (GR), glutathione-S-transferase (GST) and elevation of superoxide dismutase 1 (SOD-1) and superoxide dismutase 2 (SOD-2) as measured by messenger RNA (mRNA) expression. Additionally, the ATZ-induced alterations in the mRNA transcript copy numbers of steroidogenesis genes: steroidogenic acute regulatory protein (StAR), cytochrome P450 side-chain cleavage enzyme (CYP11A1), and 3β-hydroxysteroid dehydrogenase (3β-HSD) were shifted toward the control values by Kol. Taken together, these findings indicate that Kol protects ILCs from ATZ-induced toxicity via the reduction in ROS and MDA levels and induce normalization of mRNA levels of all the tested genes.

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Inhibition of Cyclooxygenase-2 Activity Enhances Steroidogenesis and Steroidogenic Acute Regulatory Gene Expression in MA-10 Mouse Leydig Cells

Endocrinology ◽

10.1210/en.2002-0081 ◽

2003 ◽

Vol 144 (8) ◽

pp. 3368-3375 ◽

Cited By ~ 45

Author(s):

XingJia Wang ◽

Matthew T. Dyson ◽

Youngah Jo ◽

Douglas M. Stocco

Keyword(s):

Gene Expression ◽

Protein Expression ◽

Leydig Cells ◽

Promoter Activity ◽

Regulatory Gene ◽

Steroid Production ◽

Star Protein ◽

Cox Inhibitor ◽

Steroidogenic Acute Regulatory ◽

Star Gene

Abstract To study the mechanism for the regulatory effect of arachidonic acid (AA) on steroidogenesis, the role of cyclooxygenase (COX) in steroid production and steroidogenic acute regulatory (StAR) gene expression was investigated. Although stimulation with 0.05 mm dibutyryl cAMP (Bt2cAMP) did not increase StAR protein or progesterone in MA-10 mouse Leydig cells, the addition of 1 μm of the COX inhibitor indomethacin increased StAR protein expression and progesterone production by 5.7-fold and 34.3-fold, respectively. In the presence of indomethacin, the level of Bt2cAMP required for maximal steroidogenesis was reduced from 1.0 mm to 0.25 mm. Similar results were obtained in studies on StAR promoter activity and in Northern blot analyses of StAR mRNA expression, suggesting that inhibition of COX activity enhanced StAR gene transcription. COX2 (an inducible isoform of COX) was constitutively detected in MA-10 cells. Although SC560, a selective COX1 inhibitor, did not affect steroidogenesis, the COX2 inhibitor NS398 significantly enhanced Bt2cAMP-stimulated StAR protein expression and steroid production. Overexpression of the COX2 gene in COS-1 cells significantly inhibited StAR promoter activity. The results of the present study suggest that inhibition of COX2 activity increases the sensitivity of steroidogenesis to cAMP stimulation in MA-10 Leydig cells.

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Involvement of the Thromboxane A2 Receptor in the Regulation of Steroidogenic Acute Regulatory Gene Expression in Murine Leydig Cells

Endocrinology ◽

10.1210/en.2008-1425 ◽

2009 ◽

Vol 150 (7) ◽

pp. 3267-3273 ◽

Cited By ~ 3

Author(s):

Akhilesh K. Pandey ◽

Xiangling Yin ◽

Randolph B. Schiffer ◽

James C. Hutson ◽

Douglas M. Stocco ◽

...

Keyword(s):

Gene Expression ◽

Leydig Cells ◽

Regulatory Gene ◽

Thromboxane A2 ◽

Cyclooxygenase 2 ◽

Thromboxane A2 Receptor ◽

Star Protein ◽

Dependent Inhibition ◽

Steroidogenic Acute Regulatory ◽

Star Gene

Recent studies suggested an involvement of thromboxane A2 in cyclooxygenase-2-dependent inhibition of steroidogenic acute regulatory (StAR) gene expression. The present study further investigated the role of thromboxane A2 receptor in StAR gene expression and steroidogenesis in testicular Leydig cells. The thromboxane A2 receptor was detected in several Leydig cell lines. Blocking thromboxane A2 binding to the receptor using specific antagonist SQ29548 or BM567 resulted in dose-dependent increases in StAR protein and steroid production in MA-10 mouse Leydig cells. The results were confirmed with Leydig cells isolated from rats. StAR promoter activity and StAR mRNA level in the cells were also increased after the treatments, suggesting an involvement of the thromboxane A2 receptor in StAR gene transcription. Furthermore study indicated that blocking the thromboxane A2 receptor reduced dosage sensitive sex reversal-adrenal hypoplasia congenita critical region on the X chromosome, gene 1 protein, a transcriptional repressor of StAR gene expression. Specific binding of the antagonists to the receptors on cellular membrane was demonstrated by binding assays using 3H-SQ29548 and binding competition between 3H-SQ29548 and BM567. Whereas SQ29548 enhanced cAMP-induced StAR gene expression, in the absence of cAMP, it was unable to increase StAR protein and steroidogenesis. However, when the receptor was blocked by the antagonist, subthreshold levels of cAMP were able to induce maximal levels of StAR protein expression, suggesting that blocking the thromboxane A2 receptor increase sensitivity of MA-10 cells to cAMP stimulation. Taken together, the results from the present and previous studies suggest an autocrine loop, involving cyclooxygenase-2, thromboxane A synthase, and thromboxane A2 and its receptor, in cyclooxygenase-2-dependent inhibition of StAR gene expression.

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