Jagged1 intracellular domain modulates steroidogenesis in testicular Leydig cells

Leydig cells represent the steroidogenic lineage of mammalian testis, which produces testosterone. Genetic evidence indicates the requirement of Notch signaling in maintaining a balance between differentiated Leydig cells and their progenitors during fetal development. In primary Leydig cells, Notch1 expression decreases with testicular development, while the expression of its ligand, Jagged1, remains relatively unchanged, suggesting that the roles of Jagged1 extend beyond Notch signaling. In addition, Jagged1 is known to be processed into its intracellular domain, which then translocate to the nucleus. In this study, we investigated the effect of Jagged1 intracellular domain (JICD) on steroidogenesis in Leydig cells. The independent overexpression of JICD in MA-10 Leydig cells was found to inhibit the activity of cAMP-induced Nur77 promoter. In addition, JICD suppressed Nur77 transactivation of the promoter of steroidogenic genes such as P450scc, P450c17, StAR, and 3β-HSD. Further, adenovirus-mediated overexpression of JICD in primary Leydig cells repressed the expression of steroidogenic genes, consequently lowering testosterone production. These results collectively suggest that steroidogenesis in testicular Leydig cells, which is regulated by LH/cAMP signaling, is fine-tuned by Jagged1 during testis development.

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FOXA3, a Negative Regulator of Nur77 Expression and Activity in Testicular Steroidogenesis

International Journal of Endocrinology ◽

10.1155/2021/6619447 ◽

2021 ◽

Vol 2021 ◽

pp. 1-8

Author(s):

Hansle Kim ◽

Sudeep Kumar ◽

Keesook Lee

Keyword(s):

Leydig Cells ◽

Adenosine Monophosphate ◽

Negative Regulator ◽

Fine Tuning ◽

Camp Signaling ◽

Orphan Nuclear Receptor ◽

Repressive Effect ◽

Testicular Steroidogenesis ◽

Steroidogenic Genes

Biosynthesis of testosterone occurs mainly in the testicular Leydig cells. Nur77, an orphan nuclear receptor that is expressed in response to the luteinizing hormone/cyclic adenosine monophosphate (LH/cAMP) signaling pathway, is one of the key factors that regulate steroidogenesis in Leydig cells. The function of Nur77 is modulated through interaction with other proteins. FOXA3, a transcription factor that is crucial for male fertility, is also expressed in Leydig cells. Here, we sought to elucidate the role of FOXA3 in testicular steroidogenesis by focusing on its interaction with Nur77. LH/cAMP signaling induces the onset of steroidogenesis in Leydig cells but has a repressive effect on the expression of FOXA3. Overexpression of FOXA3 in MA-10 Leydig cells repressed cAMP-induced expression of Nur77 and its target steroidogenic genes (StAR, P450c17, and Hsd3β). Furthermore, FOXA3 suppressed Nur77 transactivation of the promoter of steroidogenic genes. In mouse primary Leydig cells, adenovirus-mediated overexpression of FOXA3 had similar effects and resulted in decreased production of testosterone. Taken together, these results suggest the role of FOXA3 in the regulation of steroidogenic genes in Leydig cells and fine-tuning steroidogenesis in the testis.

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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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Glucocorticoids antagonize cAMP-induced Star transcription in Leydig cells through the orphan nuclear receptor NR4A1

Journal of Molecular Endocrinology ◽

10.1677/jme-07-0145 ◽

2008 ◽

Vol 41 (3) ◽

pp. 165-175 ◽

Cited By ~ 44

Author(s):

Luc J Martin ◽

Jacques J Tremblay

Keyword(s):

Leydig Cells ◽

Dependent Manner ◽

Orphan Nuclear Receptor ◽

Western Blots ◽

Testosterone Production ◽

Repressive Effect ◽

Testosterone Biosynthesis ◽

Underlying Mechanisms ◽

Steroidogenic Acute Regulatory ◽

Steroidogenic Genes

It is well established that stress, either physical or psychosocial, causes a decrease in testosterone production by Leydig cells. Glucocorticoids (Gc) are the main mediators of stress response and they convey their repressive effect on Leydig cells through the glucocorticoid receptor (GR). So far, various mechanisms have been proposed to explain the mechanism of action of Gc on Leydig cell steroidogenesis including repression of genes involved in testosterone biosynthesis. Several steroidogenic genes, including steroidogenic acute regulatory (STAR) protein, have been shown to be repressed by Gc in a GR-dependent manner but the underlying mechanisms remain to be fully elucidated. Here, we found that dexamethasone (Dex), a potent synthetic Gc, partly antagonizes the cAMP-dependent stimulation of the mouse Star promoter in MA-10 Leydig cells as revealed by transient transfection assays. This repression requires an element located at −95 bp previously implicated in the activation of the Star promoter by the nuclear receptors, NR4A1 and NR5A1. Dex was found to inhibit NR4A1-dependent transactivation of the Star promoter in Leydig cells by decreasing NR4A1, but not NR5A1, recruitment to the proximal Star promoter as determined by chromatin immunoprecipitation assay. Western blots revealed that Dex did not affect NR4A1 or NR5A1 expression in response to cAMP. These data suggest that NR4A1 would be associated with the GR in a transcriptionally inactive complex as previously demonstrated in pituitary corticotrope cells. Thus, our data provide new molecular insights into the stress-mediated suppression of testosterone production in testicular Leydig cells.

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SUN-719 The Impact of FOXA3 on Testicular Steroidogenesis

Journal of the Endocrine Society ◽

10.1210/jendso/bvaa046.473 ◽

2020 ◽

Vol 4 (Supplement_1) ◽

Author(s):

Hansle Kim ◽

Sudeep Kumar ◽

Keesook Lee

Keyword(s):

Transcription Factors ◽

Leydig Cells ◽

Target Genes ◽

Adult Life ◽

Tumor Cell Line ◽

Testicular Development ◽

Gene Promoters ◽

Testicular Steroidogenesis ◽

The Impact ◽

Steroidogenic Genes

Abstract The Forkhead box(Fox) transcription factors are evolutionarily conserved in organisms and regulate diverse biological processes during development as well as adult life. Among the Fox family, FoxA subfamily members Foxa1-3 have been termed `pioneer’ transcription factors as they bind both nucleosome-bound and nucleosome-free DNA targets with the same recognition site. Foxa3 is the only member of FoxA subfamily that is expressed in both male and female gonads. In the testis, Foxa3 is expressed in spermatids and interstitial Leydig cells. We focused our study to elucidate the role of FOXA3 in Leydig cells and its impact on testicular steroidogenesis. Expression of FOXA3 dramatically decreased in mouse Leydig cells during testicular development. In addition, the time-dependent expression of FOXA3 showed an opposite pattern to that of steroidogenic genes in cAMP-induced primary Leydig cells. Meanwhile, Nur77 is among the prime regulators of steroidogenesis in the testicular Leydig cells. Overexpression of FOXA3 in MA-10 cells (mouse Leydig tumor cell line) repressed the cAMP-induced Nur77 promoter activity, which further resulted in the reduced activity of Nur77-target steroidogenic gene promoters (StAR, CYP17A1and 3β-HSD). Similar to above results, the expression of Nur77 and its target genes,StAR, 3β-HSD and CYP11A1, were repressed by adenovirus-mediated overexpression of FOXA3 in mouse primary Leydig cells, although the expression of CYP17A1, another steroidogenic gene, was differentially affected. These results suggest that FOXA3 locally regulates the expression of steroidogenic genes through Nur77 during testicular development.

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Notch signaling represses GATA4-induced expression of genes involved in steroid biosynthesis

Reproduction ◽

10.1530/rep-15-0226 ◽

2015 ◽

Vol 150 (4) ◽

pp. 383-394 ◽

Cited By ~ 16

Author(s):

Rajani M George ◽

Katherine L Hahn ◽

Alan Rawls ◽

Robert S Viger ◽

Jeanne Wilson-Rawls

Keyword(s):

Notch Signaling ◽

Leydig Cells ◽

Downstream Effector ◽

Effector Genes ◽

Notch Receptors ◽

Expression Of Genes ◽

Downstream Effectors ◽

Proliferation Response ◽

Complex Regulation ◽

Steroidogenic Genes

Notch2 and Notch3 and genes of the Notch signaling network are dynamically expressed in developing follicles, where they are essential for granulosa cell proliferation and meiotic maturation. Notch receptors, ligands, and downstream effector genes are also expressed in testicular Leydig cells, predicting a potential role in regulating steroidogenesis. In this study, we sought to determine if Notch signaling in small follicles regulates the proliferation response of granulosa cells to FSH and represses the up-regulation steroidogenic gene expression that occurs in response to FSH as the follicle grows. Inhibition of Notch signaling in small preantral follicles led to the up-regulation of the expression of genes in the steroid biosynthetic pathway. Similarly, progesterone secretion by MA-10 Leydig cells was significantly inhibited by constitutively active Notch. Together, these data indicated that Notch signaling inhibits steroidogenesis. GATA4 has been shown to be a positive regulator of steroidogenic genes, including STAR protein, P450 aromatase, and 3B-hydroxysteroid dehydrogenase. We observed that Notch downstream effectors HEY1, HEY2, and HEYL are able to differentially regulate these GATA4-dependent promoters. These data are supported by the presence of HEY/HES binding sites in these promoters. These studies indicate that Notch signaling has a role in the complex regulation of the steroidogenic pathway.

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Repeated immobilization stress disturbed steroidogenic machinery and stimulated the expression of cAMP signaling elements and adrenergic receptors in Leydig cells

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00554.2011 ◽

2012 ◽

Vol 302 (10) ◽

pp. E1239-E1251 ◽

Cited By ~ 25

Author(s):

Natasa J. Stojkov ◽

Marija M. Janjic ◽

Maja M. Bjelic ◽

Aleksandar I. Mihajlovic ◽

Tatjana S. Kostic ◽

...

Keyword(s):

Adrenergic Receptors ◽

Leydig Cells ◽

Immobilization Stress ◽

Regulatory Protein ◽

Camp Signaling ◽

Partial Recovery ◽

Transcription Analysis ◽

Releasing Hormone ◽

Testosterone Production ◽

Glucocorticoid Signaling

This study was designed to evaluate the effect of acute (2 h daily) and repeated (2 h daily for 2 or 10 consecutive days) immobilization stress (IMO) on: 1) the steroidogenic machinery homeostasis; 2) cAMP signaling; and the expression of receptors for main markers of 3) adrenergic and 4) glucocorticoid signaling in Leydig cells of adult rats. The results showed that acute IMO inhibited steroidogenic machinery in Leydig cells by downregulation of Scarb1 (scavenger receptor class B), Cyp11a1 (cholesterol side-chain cleavage enzyme), Cyp17a1 (17α-hydroxylase/17,20 lyase), and Hsd17b3 (17β-hydroxysteroid dehydrogenase) expression. In addition to acute IMO effects, repeated IMO increased transcription of Star (steroidogenic acute regulatory protein) and Arr19 (androgen receptor corepressor 19 kDa) in Leydig cells. In the same cells, the transcription of adenylyl cyclases (Adcy7, Adcy9, Adcy10) and cAMP-specific phosphodiesterases ( Pde4a, Pde4b, Pde4d, Pde7a, Pde8a) was stimulated, whereas the expression of the genes encoding protein kinase A subunits were unaffected. Ten times repeated IMO increased the levels of all adrenergic receptors and β-adrenergic receptor kinase ( Adrbk1) in Leydig cells. The transcription analysis was supported by cAMP/testosterone production. In this signaling scenario, partial recovery of testosterone production in medium/content was detected. The physiological significance of the present results was proven by ex vivo application of epinephrine, which increased cAMP/testosterone production by Leydig cells from control rats in greater fashion than from stressed. IMO did not affect the expression of transcripts for Crhr1/Crhr2 (corticotropin releasing hormone receptors), Acthr (adrenocorticotropin releasing hormone receptor), Gr (glucocorticoid receptor), and Hsd11b1 [hydroxysteroid (11-β) dehydrogenase 1], while all types of IMO stimulated the expression of Hsd11b2, the unidirectional oxidase with high affinity to inactivate glucocorticoids. Thus, presented data provide new molecular/transcriptional base for “fight/adaptation” of Leydig cells and new insights into the role of cAMP, epinephrine, and glucocorticoid signaling in recovery of stress-impaired Leydig cell steroidogenesis.

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Gene expression and functional abnormalities in XX/Sry Leydig cells

Scientific Reports ◽

10.1038/s41598-020-80741-z ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Shogo Yanai ◽

Takashi Baba ◽

Kai Inui ◽

Kanako Miyabayashi ◽

Soyun Han ◽

...

Keyword(s):

Gene Expression ◽

Germ Cell ◽

Y Chromosome ◽

Sertoli Cells ◽

Leydig Cells ◽

Gene Expressions ◽

Germ Cell Development ◽

Testis Development ◽

Testosterone Production ◽

Adult Leydig Cells

AbstractThe SRY gene induces testis development even in XX individuals. However, XX/Sry testes fail to produce mature sperm, due to the absence of Y chromosome carrying genes essential for spermatogenesis. XX/Sry Sertoli cells show abnormalities in the production of lactate and cholesterol required for germ cell development. Leydig cells are essential for male functions through testosterone production. However, whether XX/Sry adult Leydig cells (XX/Sry ALCs) function normally remains unclear. In this study, the transcriptomes from XY and XX/Sry ALCs demonstrated that immediate early and cholesterogenic gene expressions differed between these cells. Interestingly, cholesterogenic genes were upregulated in XX/Sry ALCs, although downregulated in XX/Sry Sertoli cells. Among the steroidogenic enzymes, CYP17A1 mediates steroid 17α-hydroxylation and 17,20-lyase reaction, necessary for testosterone production. In XX/Sry ALCs, the latter reaction was selectively decreased. The defects in XX/Sry ALCs, together with those in the germ and Sertoli cells, might explain the infertility of XX/Sry testes.

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Aromatase and 11β-hydroxysteroid dehydrogenase 2 localisation in the testes of pigs from birth to puberty linked to changes of hormone pattern and testicular morphology

Reproduction Fertility and Development ◽

10.1071/rd07136 ◽

2008 ◽

Vol 20 (4) ◽

pp. 505 ◽

Cited By ~ 5

Author(s):

A. Wagner ◽

R. Claus

Keyword(s):

Blood Plasma ◽

Sertoli Cell ◽

Germ Cells ◽

Leydig Cells ◽

Hydroxysteroid Dehydrogenase ◽

Blood Collection ◽

Testicular Development ◽

Post Mortem ◽

Cell Numbers ◽

Testicular Morphology

Oestrogens and glucocorticoids are important for spermatogenesis and are regulated via aromatase for oestradiol synthesis and 11β-hydroxysteroid dehydrogenase 2 (11β-HSD 2) as an inactivator of cortisol. In the present study postnatal changes of these two enzymes were monitored together with testicular development and hormone concentrations. Pigs were assigned to three periods: Weeks 0–5, Weeks 5–11 or Weeks 11–17. In Period 1, groups of four piglets were killed after each week. Blood plasma and testes were sampled immediately post mortem. For Periods 2 and 3, groups of six pigs were fitted with vein catheters for daily blood collection. Testes from all pigs were obtained after killing. Levels of testosterone, oestradiol, LH, FSH and cortisol were determined radioimmunologically. The 11β-HSD 2- and aromatase-expressing cells were stained immunocytochemically. All hormones were maximal 2 weeks after birth. A rise of LH, testosterone and oestradiol occurred again at Week 17. FSH and cortisol remained basal. Parallel to the first postnatal rise, the presence of aromatase and 11β-HSD 2 in Leydig cells increased, together with germ and Sertoli cell numbers. Expression was low from 3 to 5 weeks, was resumed after Week 5 and was maximal at Week 17. The amount of 11β-HSD 2 in germ cells was greatest at birth, decreased thereafter and was absent after Week 3.

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Sustained in vivo blockade of α1-adrenergic receptors prevented some of stress-triggered effects on steroidogenic machinery in Leydig cells

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00100.2013 ◽

2013 ◽

Vol 305 (2) ◽

pp. E194-E204 ◽

Cited By ~ 12

Author(s):

Natasa J. Stojkov ◽

Marija M. Janjic ◽

Aleksandar Z. Baburski ◽

Aleksandar I. Mihajlovic ◽

Dragana M. Drljaca ◽

...

Keyword(s):

Adrenergic Receptors ◽

Leydig Cells ◽

High Affinity ◽

Testosterone Production ◽

Transcription Profiles ◽

Steroidogenic Cells ◽

Main Regulator ◽

Stimulatory Factor

This study was designed to systematically analyze and evaluate the effects of in vivo blockade of α1-adrenergic receptors (α1-ADRs) on the stress-induced disturbance of steroidogenic machinery in Leydig cells. Parameters followed 1) steroidogenic enzymes/proteins, transcription factors, and cAMP/testosterone production; 2) the main hallmarks of stress (epinephrine, glucocorticoids); and 3) transcription profiles of ADRs and oxidases with high affinity to inactivate glucocorticoids. Results showed that sustained blockade of α1-ADRs prevented stress-induced 1) decrease of the transcripts/proteins for main steroidogenic CYPs (CYP11A1, CYP17A1); 2) decrease of Scarb1 and Hsd3b1 transcripts; 3) decrease of transcript for Nur77, one of the main activator of the steroidogenic expression; and 4) increase of Dax1 and Arr19, the main steroidogenic repressors in Leydig cells. In the same cells, the expression of steroidogenic stimulatory factor Creb1, StAR, and androgen receptor increased. In this signaling scenario, stress-induced stimulation of Adra1a/Adra1b/Adrbk1 and Hsd11b2 (the unidirectional oxidase with high affinity to inactivate glucocorticoids) was not changed. Blockade additionally stimulated stress-increased transcription of the most abundantly expressed ADRs Adra1d/Adrb1/Adrb2 in Leydig cells. In the same cells, stress-decreased testosterone production, the main marker of Leydig cells functionality, was completely prevented, while reduction of cAMP, the main regulator of androgenesis, was partially prevented. Accordingly, the presented data provide a new molecular/transcriptional base for “fight/adaptation” of steroidogenic cells and new molecular insights into the role of α1-ADRs in stress-impaired Leydig cell steroidogenesis. The results are important in term of wide use of α1-ADR selective antagonists, alone/in combination, to treat high blood pressure, nightmares associated with posttraumatic stress disorder, and disrupted sexual health.

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