Cell Steroidogenesis Research Articles

Link between cell cycle, steroidogenesis and PKA in adrenocortical tumors cells

Link between cell cycle, steroidogenesis and PKA in adrenocortical tumors cells

MCL1 is a key regulator of steroidogenesis in mouse Leydig cells.

Myeloid cell leukemia-1 (MCL1), an anti-apoptotic member of the BCL2 family, is expressed abundantly in the testis. Previous characterization revealed that MCL1 is expressed exclusively in the Leydig cells in the mouse testis, yet what it does in these cells remains unknown. We therefore analyzed testosterone biosynthesis in isolated primary Leydig cells and the MA-10 cell line, in which MCL1 was knocked down using an siRNA strategy. The mRNA abundance of the steroidogenic genes Star, Cyp11a1, Cyp17a1, Hsd3b1, Srd5a, and the luteinizing hormone/choriogonadotropin receptor Lhcgr were significantly reduced following MCL1 knockdown. Of the two enzymes required for testosterone biosynthesis, STAR and P450 SCC (encoded by Cyp11a1) enzyme abundance was also reduced following Mcl1 siRNA treatment, possibly leading to the reduced production of sex steroid precursors, and testosterone in these knockdown cells. Despite its classification as an anti-apoptosis protein, Mcl1 siRNA treatment did not affect cell survival. Collectively, our findings indicate that MCL1 plays a pivotal role in Leydig-cell steroidogenesis, and might provide novel insights into metabolic regulation in this cell. Mol. Reprod. Dev. 83: 226-235, 2016. © 2016 Wiley Periodicals, Inc.

Autocrine/Paracrine Insulin-like Growth Factor Binding Protein-3 Acts as Pro-apoptotic Factor for Leydig cells in the Rat Testis

The secretory insulin-like growth factor-binding protein-3 (IGFBP-3) induces apoptosis via both insulin-like growth factor-I (IGF-I)-dependent and -independent mechanisms. Here, we have examined the effects of IGFBP-3 on Leydig cell apoptosis, proliferation and steroidogenesis. Immunohistochemical analysis of testes of rats at different ages revealed that IGFBP-3 is expressed first after 20 days of postnatal life and is present at a high level in the adult testis. In addition, Western blotting showed that the expression of IGFBP-3 in Leydig cells isolated from 60-day-old rats is higher than in 40-day-old animals. The rate of DNA synthesis (as assessed by incorporation of 3H-thymidine in vitro) in Leydig cells from 40-day-old rats is reduced by IGFBP-3, which also blocks the promotion of cell survival by IGF-I. Moreover, IGFBP-3 induces apoptosis in Leydig cells and, at the same time, attenuates the anti-apoptotic action of IGF-I. Furthermore, IGF-I stimulates secretion of IGFBP-3, -4, and -2 by Leydig cells. The pro-inflammatory cytokine tumor necrosis factor- α induces apoptosis in these same cells and increases their secretion of IGFBP-3 and IGFBP-4. These findings provide the first evidence that IGFBP-3 acts as a pro-apoptotic effector of Leydig cells and can also block the positive effect of IGF-I on cell survival. In addition, IGFBPs appear to modulate interactions between IGF-I and pro-inflammatory cytokines in the testis, suggesting possible participation of these proteins in processes such as testicular inflammation and cancer.

128 THE ROLE OF ENDOTHELINS IN REGULATING BOVINE GRANULOSA CELLS STEROIDOGENESIS

Endothelins are a group of vasoactive 21 amino acid peptides reported to play roles in steroidogenesis, folliculogenesis, and ovulation (Bridges et al. 2012 Life Sci. 91, 501–506). Nevertheless, the role of endothelins in regulating steroidogenesis in the bovine species requires further investigation. Thus, the objective of this study was to investigate the effects of endothelin 1 (ET-1) and endothelin 2 (ET-2) on bovine granulosa cell (GC) steroidogenesis. Bovine ovaries were obtained from a local abattoir. Follicular fluid was aspirated from small (1–5 mm) follicles and GC were isolated and exposed to various treatments (ET-1, ET-2, or ET-1 plus ET-2 with FSH and with or without insulin-like growth factor-1). In replicated experiments, culture medium was removed and analysed for steroid production via radioimmunoassay. Granulosa cells were either harvested with trypsin and counted using a Coulter Counter or collected with Trizol for RNA extraction and quantification via real-time PCR (18S rRNA was used as a housekeeping gene). Steroid production was expressed as nanograms (in the case of progesterone) and picograms (in the case of oestradiol) per 105 cells per 24 h. Relative quantity of target gene mRNA was expressed as 2–ΔΔCt using the relative comparative threshold cycle (Ct) method. Data were analysed via ANOVA and the general linear models (GLM) procedure of SAS for Windows (SAS Institute Inc., Cary, NC). If a significant main effect was identified, differences among means were determined by Fisher’s protected least significant differences test. The values were reported as least squares means ± standard error of the mean. In the presence of insulin-like growth factor-1, ET-1 significantly inhibited oestradiol production at 300 ng mL–1 (100.30 ± 11.05; P < 0.05), but not at 30 ng mL–1 (114.47 ± 11.05; P > 0.05) in comparison to the control (141.21 ± 11.05), whereas no differences were observed for progesterone production at 300 ng mL–1 (60.11 ± 7.11; P > 0.05) or at 30 ng mL–1 (64.02 ± 7.11; P > 0.05) in comparison to control (76.75 ± 7.11). ET-2 also significantly inhibited oestradiol production at 300 ng mL–1 (91.08 ± 11.87; P < 0.01), but not at 30 ng mL–1 (112.77 ± 11.87; P > 0.05) in comparison to the control in the presence of insulin-like growth factor-1. No significant effect of ET-1 and ET-2 was observed on steroidogenesis of granulosa cells cultured without insulin-like growth factor-1. Consistent with steroids production data, real-time PCR results indicated that, in the presence of IGF-1, ET-1 (5.66 ± 1.05) and ET-2 (5.65 ± 1.05) inhibited (P < 0.05) aromatase gene expression compared to controls (11.33 + 1.05), and ET-1 plus ET-2 (2.42 ± 1.05) reduced (P < 0.05) expression below that observed with either alone. No effect of ET-1 (4.38 ± 0.95; P > 0.05), ET-2 (5.94 ± 0.95; P > 0.05), or ET-1 plus ET-2 (4.57 ± 0.95; P > 0.05) was observed for side-chain cleavage enzyme (CYP11A1) in comparison to controls (4.4 ± 1.07). Altogether, these results indicate that endothelins are involved in the regulation of steroidogenesis of bovine GC.

Nicotinamide nucleotide transhydrogenase (NNT) as a novel molecular target in adrenocortical carcinoma - impact of NNT knockdown on adrenocortical cell proliferation, redox balance and steroidogenesis

Nicotinamide nucleotide transhydrogenase (NNT) as a novel molecular target in adrenocortical carcinoma - impact of NNT knockdown on adrenocortical cell proliferation, redox balance and steroidogenesis

Individual and combined effects of deoxynivalenol and α-zearalenol on cell proliferation and steroidogenesis of granulosa cells in cattle.

This study was conducted to evaluate the impact of deoxynivalenol (DON) and zearalenone (ZEA) metabolite, α-zearalenol (α-Zol), on cell proliferation and steroidogenesis of bovine large (LG) follicle granulosa cells (GC). LGGC were obtained from bovine ovarian follicles (8-22 mm) and were cultured for 2 days in medium containing 10% fetal bovine serum followed by 1 or 2 days in serum-free medium without (control) or with treatments. Three different experiments were performed using different dosages of DON and α-Zol and in different combinations and a fourth experiment evaluated estradiol effects on granulosa cell proliferation. DON inhibited progesterone (P4) and estradiol (E2) production at high dose. α-Zol alone and in combination with DON increased cell growth. Estradiol inhibited cell growth indicating α-Zol is not acting as an estrogen agonist. This study demonstrates that α-Zol and DON can impact in vitro GC function, however further studies will be required to better understand the mechanism of action and reproductive effects of Fusarium mycotoxins.

Effect of mercury on porcine ovarian granulosa cells in vitro

The objective of this in vitro study was to examine dose–dependent changes in the secretion activity [progesterone (P4) and insulin-like growth factor–I (IGF–I)] of porcine ovarian granulosa cells after experimental mercury (Hg) administration, including its apoptotic potential so as to ascertain the possible involvement of Hg in steroidogenesis. Ovarian granulosa cells were incubated with mercuric chloride [mercury (II) chloride or HgCl2] at the doses 50–250 μg mL−1 for 18 h and compared with control group without Hg addition. Release of P4 and IGF–I by ovarian granulosa cells was assessed by RIA and apoptosis by TUNEL assay. Observations show that P4 release by granulosa cells was significantly (P < 0.05) inhibited at all the doses, while IGF-I release was not affected at any of the doses used, although a decreasing trend in the release of IGF–I was noted in comparison to control. An increasing trend of apoptosis of granulosa cells was noted, the difference being significant (P < 0.05) only at the dose 130 μg mL−1 HgCl2, in comparison to control. Obtained data suggest a direct effect of Hg on the release of steroid hormone progesterone but not growth factor IGF-I, and a dose-dependent effect on apoptosis of porcine ovarian granulosa cells. Results indicate the interference of Hg in the pathways of steroidogenesis and apoptosis of porcine ovarian granulosa cells.

OxeR1 regulates angiotensin II and cAMP-stimulated steroid production in human H295R adrenocortical cells

Hormone-regulated steroidogenesis and StAR protein induction involve the action of lipoxygenated products. The products of 5-lipoxygenase act on inflammation and immunity by stimulation of a membrane receptor called OxeR1. The presence of OxeR1 in other systems has not been described up to date and little is known about its mechanism of action and other functions. In this context, the aim of this study was the identification and characterization of OxeR1 as a mediator of cAMP-dependent and independent pathways. Overexpression of OxeR1 in MA-10 Leydig cells increased cAMP-dependent progesterone production. Angiotensin II and cAMP stimulation of adrenocortical human H295R cells produced an increase in StAR protein induction and steroidogenesis in cells overexpressing OxeR1 as compared to mock-transfected cells. Additionally, activation of OxeR1 caused a time-dependent increase in ERK1/2 phosphorylation. In summary, membrane receptor OxeR1 is involved in StAR protein induction and activation of steroidogenesis triggered by cAMP or angiotensin II, acting, at least in part, through ERK1/2 activation.

Potential benefits of adlay on hyperandrogenism in human chorionic gonadotropin-treated theca cells and a rodent model of polycystic ovary syndrome

This study investigated the therapeutic potential of adlay on polycystic ovary syndrome (PCOS) and its possible underlying mechanism. The active anti-androgenic components in adlay were further analyzed. Rat ovarian theca cells were treated with human chorionic gonadotropin to stimulate androstenedione (AD) secretion. The ethyl acetate subfraction obtained from the ethanolic extract of adlay hull (AHE-EA) exerted superior efficacy against AD production. Four major constituents in AHE-EA, including 5,7-dihydroxychromone, liquiritigenin, isoliquiritigenin, and homoeriodictyol, were identified by HPLC–MS and exhibited strong inhibition against high AD levels. In a DHEA-induced PCOS rat model, administration of AHE-EA significantly decreased serum AD levels, improved hyperglycemia and insulin resistance, and attenuated oxidative stress and inflammatory responses in the ovaries. Histopathological morphology of ovarian tissues confirmed that AHE-EA could restore the estrus cycles and normal ovarian histology. Mechanistic characterization demonstrated that AHE-EA inhibited DHEA-induced hyperandrogenemia through modulate steroidogenic acute regulatory protein-related steroidogenesis in theca cells.

Angiotensin II-Induced Protein Kinase D Activates the ATF/CREB Family of Transcription Factors and Promotes StAR mRNA Expression

Aldosterone synthesis is initiated upon the transport of cholesterol from the outer to the inner mitochondrial membrane, where the cholesterol is hydrolyzed to pregnenolone. This process is the rate-limiting step in acute aldosterone production and is mediated by the steroidogenic acute regulatory (StAR) protein. We have previously shown that angiotensin II (AngII) activation of the serine/threonine protein kinase D (PKD) promotes acute aldosterone production in bovine adrenal glomerulosa cells, but the mechanism remains unclear. Thus, the purpose of this study was to determine the downstream signaling effectors of AngII-stimulated PKD activity. Our results demonstrate that overexpression of the constitutively active serine-to-glutamate PKD mutant enhances, whereas the dominant-negative serine-to-alanine PKD mutant inhibits, AngII-induced StAR mRNA expression relative to the vector control. PKD has been shown to phosphorylate members of the activating transcription factor (ATF)/cAMP response element binding protein (CREB) family of leucine zipper transcription factors, which have been shown previously to bind the StAR proximal promoter and induce StAR mRNA expression. In primary glomerulosa cells, AngII induces ATF-2 and CREB phosphorylation in a time-dependent manner. Furthermore, overexpression of the constitutively active PKD mutant enhances the AngII-elicited phosphorylation of ATF-2 and CREB, and the dominant-negative mutant inhibits this response. Furthermore, the constitutively active PKD mutant increases the binding of phosphorylated CREB to the StAR promoter. Thus, these data provide insight into the previously reported role of PKD in AngII-induced acute aldosterone production, providing a mechanism by which PKD may be mediating steroidogenesis in primary bovine adrenal glomerulosa cells.

Mouse Leydig Cells with Different Androgen Production Potential Are Resistant to Estrogenic Stimuli but Responsive to Bisphenol A Which Attenuates Testosterone Metabolism

It is well known that estrogens and estrogen-like endocrine disruptors can suppress steroidogenic gene expression, attenuate androgen production and decrease differentiation of adult Leydig cell lineage. However, there is no information about the possible link between the potency of Leydig cells to produce androgens and their sensitivity to estrogenic stimuli. Thus, the present study explored the relationship between androgen production potential of Leydig cells and their responsiveness to estrogenic compounds. To investigate this relationship we selected mouse genotypes contrasting in sex hormone levels and differing in testosterone/estradiol (T/E2) ratio. We found that two mouse genotypes, CBA/Lac and C57BL/6j have the highest and the lowest serum T/E2 ratio associated with increased serum LH level in C57BL/6j compared to CBA/Lac. Analysis of steroidogenic gene expression demonstrated significant upregulation of Cyp19 gene expression but coordinated suppression of LHR, StAR, 3βHSDI and Cyp17a1 in Leydig cells from C57BL/6j that was associated with attenuated androgen production in basal and hCG-stimulated conditions compared to CBA/Lac mice. These genotype-dependent differences in steroidogenesis were not linked to changes in the expression of estrogen receptors ERα and Gpr30, while ERβ expression was attenuated in Leydig cells from C57BL/6j compared to CBA/Lac. No effects of estrogenic agonists on steroidogenesis in Leydig cells from both genotypes were found. In contrast, xenoestrogen bisphenol A significantly potentiated hCG-activated androgen production by Leydig cells from C57BL/6j and CBA/Lac mice by suppressing conversion of testosterone into corresponding metabolite 5α-androstane-3α,17β-diol. All together our data indicate that developing mouse Leydig cells with different androgen production potential are resistant to estrogenic stimuli, while xenoestrogen BPA facilitates hCG-induced steroidogenesis in mouse Leydig cells via attenuation of testosterone metabolism. This cellular event can cause premature maturation of Leydig cells that may create abnormal intratesticular paracrine milieu and disturb proper development of germ cells.

In vitro effects of deoxynivalenol and β-Zearalenol alone and in combination on steroidogenesis in bovine small granulosa cells

In vitro effects of deoxynivalenol and β-Zearalenol alone and in combination on steroidogenesis in bovine small granulosa cells

Effect of ET-1, FGF-2, and HGF on intracellular steroidogenesis in prostate cancer cells.

e16083 Background: In the prostatic epithelium endothelin-1 (ET-1), fibroblast growth factor-2 (FGF2) and hepatocyte growth factor (HGF) are involved in cell proliferation and inhibition of apoptosis. The aim of this study is to investigate whether neuropeptides such as ET-1 and growth factors such as FGF and HGF may constitute an alternate pathway to intracellular steroidogenesis. Methods: PC3 cells, an androgen independent prostate cancer cell line were cultured in RPMI 1640, fully supplemented with FBS 10% and in serum free conditions. The cells were incubated with ET-1(10nM, 3 hrs incubation), FGF-2 (10 ng/ml, 3 hrs incubation) and HGF(33 ng/ml, 3 hrs incubation), and then progesterone, 17-OH progesterone and aldosterone were measured in cell lysates by LC-MS using LTQ Orbitrap XL MS 2.5.5 SP1 (Thermo-Fisher) equipped with Accela AS 2.2.1, Accela pump. The data were analysed by Xcalibur 2.1.0 Software. Results: In baseline conditions PC3 cells produce no progesterone but high amounts of 17-OH progesterone and small amounts of aldosterone. Upon stimulation with endothelin there was a significant increase in progesterone, significant decrease in 17-OH progesterone and a very significant increase in aldosterone. Similarly stimulation with both HGF and FGF resulted in significant decreases in 17-OH progesterone and increases in aldosterone. Conclusions: Our results imply that in androgen deprivation conditions intracellular steroidogenesis can still take place by stimulation via seven membrane and tyrosine kinase receptors, contributing in part to a steroid dependent phenotype in addition to a steroid independent one, thus implying the potential for new therapeutic interventions.

In the Ovary, the Rhox Library Is the Lender and Some Genes Are the Borrowers

In the Ovary, the Rhox Library Is the Lender and Some Genes Are the Borrowers

Swainsonine differentially affects steroidogenesis and viability in caprine luteal cells in vitro

Plants containing swainsonine (SW) have been reported to impair reproductive function and fertility after long-term ingestion by livestock. However, direct effects of SW on luteal cell steroidogenesis remain unclear. In this study, primary and transfected luteal cells were used to investigate the effects of SW on progesterone secretion and cell viability and the mechanisms involved in these processes. After treatment with various concentrations of SW for 24 or 48 hours, progesterone production and the number of living cells were assessed using radioimmunoassay and trypan blue dye exclusion assay, respectively. Lower concentrations of SW enhanced basal, 22R-hydroxycholesterol- or pregnenolone-stimulated progesterone secretion (P < 0.05), whereas higher concentrations of SW inhibited progesterone secretion (P < 0.05). Lower concentrations of SW promoted expression of P450 side-chain cleavage enzyme and 3β-hydroxysteroid dehydrogenase, two key enzymes involved in luteal cell steroidogenesis, at mRNA and protein levels (P < 0.05), but did not affect expression of steroidogenic acute regulatory protein and cell proliferation. In contrast, higher concentrations of SW inhibited luteal cell proliferation by inducing growth phase 1/quiescent state cell cycle arrest and apoptosis (P < 0.05). Taken together, these results demonstrated that lower concentrations of SW induced progesterone production through upregulation of P450 side-chain cleavage enzyme and 3β-hydroxysteroid dehydrogenase without affecting cell viability, whereas higher concentrations of SW induced cell cycle arrest and apoptosis and impaired steroidogenesis. These findings provided new insights into understanding the effect of SW on luteal cell steroidogenesis.

Correlation between cell cycle, steroidogenesis and PKA RIA and RIIB subunits in adrenocortical tumors cells

Correlation between cell cycle, steroidogenesis and PKA RIA and RIIB subunits in adrenocortical tumors cells

The relationship between ceramide cascade and Lox-1 receptor influences steroidogenesis in bovine granulosa cells through cytoplasmatic release of calcium

The relationship between ceramide cascade and Lox-1 receptor influences steroidogenesis in bovine granulosa cells through cytoplasmatic release of calcium

GATA4 Is Required for Steroidogenesis in MA-10 Leydig Cells.

GATA4 Is Required for Steroidogenesis in MA-10 Leydig Cells.

Mechanism of phytoestrogen action in Leydig cells of ganders (Anser anser domesticus): Interaction with estrogen receptors and steroidogenic enzymes

Phytoestrogens (PE) are plant-derived compounds that have an estrogen-like activity and they can influence male and female reproduction. The possible mechanisms of PE action may be including: the binding to estrogen receptors (ER) and the interaction with the key steroidogenic enzymes. The aim of this study was to investigate if PE has effect on steroidogenesis of gander testicular cells by above-described pathways. The Leydig cells were isolated from testes of White Kołuda® ganders at the peak of their reproductive activity (March). These Leydig cells (1×105per mL) were pre-incubated with the ER inhibitor – ICI 182, 780 (100 nM) for 3 h and then these cells were incubated with PE (5 and 50 μM): genistein, daidzein, equol and coumestrol during next 20 h or untreated control and the Leydig cells that were previously treated (20 h) with genistein (5 and 50 μM) were incubated for next 6 h with steroid intermediates (20 μM) as testosterone (T) precursors: hydroxycholesterol, pregnenolone, progesterone and androstenedione. Concentrations of T in the samples of incubation medium were measured using radioimmunoassay. Genistein, daidzein, and equol (5 and 50 μM) decreased (P < 0.05) T secretion by incubated gander Leydig cells and ICI 182, 780 did not eliminate the inhibitory effect of these PE. After genistein (50 μM) treatment, basal and stimulated with 22R-hydroxycholesterol, pregnenolone, progesterone and androstenedione, T production by testicular cells was decreased (P < 0.05). In contrast, genistein at lower dose (5 μM) did not affect the stimulatory effects of testosterone precursors. In conclusion, the inhibition of testosterone secretion by the phytoestrogens in gander Leydig cells did not depend on estrogen receptors. The suppression of steroidogenesis in these cells may be in part conducted by interaction of phytoestrogens with key steroidogenic enzymes. However, further studies are required to elucidate the phytoestrogen mechanism of action in gander testicular cells.

Abstract 808: Endocrine resistance: The influence of stromal-epithelial interaction on intracrine pathways of oestrogen synthesis

Abstract Resistance to endocrine therapy, both de novo and acquired, poses a significant clinical challenge. Two primary pathways, the aromatase and steroid sulphatase (STS) pathways, exist for the production of estrogenic steroids from androstenedione and DHEAS, respectively. Numerous studies have demonstrated an upregulation of these pathways in breast tumour tissue compared to normal breast tissue. Therapies targeting oestrogenic pathways form the mainstay of the pharmacological approach to breast cancer treatment. The SERM, Tamoxifen, and aromatase inhibitors (AI) such as Letrozole, have shown substantial clinical benefit in hormone-dependent breast cancer patients. Additionally, to date one STS inhibitor has entered clinical trials. Despite this success, a proportion of hormone-receptor positive patients do not respond to hormone therapy with 40% of patients who initially show a response to Tamoxifen relapsing. As little is known about the influence of the tumour microenvironment, particularly stromal-fibroblasts, on these pathways in the development of endocrine resistance, we have examined the influence of stromal-epithelial interactions on the expression of enzymes of steroidogenesis in endocrine resistant cells. Wild type and tamoxifen-resistant MCF-7 breast cancer cells (MCF-7wt and MCF-7TamR, respectively) were indirectly co-cultured with primary fibroblasts derived from breast tumour and reduction mammoplasty samples, using 0.4μm 6-well inserts. Expression levels of 17α-hydroxysteroid dehydrogenase types 1 (17α-HSD1) and 2, (17α-HSD2), aromatase, oestrogen receptor ≤ (ERα) and β, progesterone receptor, steroid sulphatase (STS), and steroid sulphotransferase were assessed basally and in co-culture using RT-PCR. Significantly higher basal expression of STS (p&amp;lt;0.001), aromatase (p=0.012) and 17α-HSD2 (p=0.032) was seen in fibroblast versus epithelial lines. On co-culture a 2.5 - 5 fold up-regulation of aromatase expression was seen in MCF-7wt and MCF-7TamR (p=0.009, p=0.046 respectively). The expression of STS (p=0.150, p=0.05) and 17α-HSD1 (p=0.001, p=0.071) was also increased. Epithelial lines showed a significant down-regulation in ERα on co-culture. Both malignant and normal fibroblast lines demonstrated up-regulation of aromatase expression on co-culture (p=0.037). These data suggest that stromal-fibroblasts make a significant contribution to the tumour hormonal environment through both expression and modulation of the pathways associated with intracrine oestrogen production. The differential effect of cell-type combinations implicates these interactions in both oestrogen sensitivity and insensitivity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 808. doi:1538-7445.AM2012-808