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Time-dependent metabolism of androstenedione and formation of androsterone quantified by UPLC-MS/MS. BLTK1 cells were incubated in serum-free medium supplemented with 250 nM AD for different time (0, 30, 90, and 270 min), and the formation of ADT, T and DHT was quantified by UPLC-MS/MS in the cell culture supernatants. Data are shown as mean ± SD of technical triplicates.
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Androgen biosynthesis in males occurs to a large extent in testicular Leydig cells. This study focused on the evaluation of three murine Leydig cell lines as potential screening tool to test xenobiotics interfering with gonadal androgen synthesis. The final step of testosterone (T) production in Leydig cells is catalyzed by the enzyme 17β-hydroxyst...
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... supernatants of BLTK1 cells incubated with 250 nM AD for different time periods were analyzed by UPLC-MS/MS. The main metabolite produced by BLTK1 cells was identified as ADT. The time-dependent loss of AD over time was proportional to the increase of ADT in the culture supernatants (Fig. 2). DHT was below the detection limit and only very low amounts of T could be ...
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Citations
... The use of primary testicular in vitro models, like the current 3D co-culture, can advance our understanding of Leydig cell testosterone production and regulation. This model can simulate testicular physiology, which is not possible with the widely used mouse tumor Leydig cell line MA-10, as it does not express CYP17 and mostly produces progesterone instead of testosterone (Engeli et al. 2018;Zirkin and Papadopoulos 2018). ...
To improve the mechanistic screening of reproductive toxicants in chemical-risk assessment and drug development, we have developed a three-dimensional (3D) heterogenous testicular co-culture model from neonatal mice. Di-n-butyl phthalate (DBP), an environmental contaminant that can affect reproductive health negatively, was used as a model compound to illustrate the utility of the in vitro model. The cells were treated with DBP (1 nM to 100 µM) for 7 days. Automated high-content imaging confirmed the presence of cell-specific markers of Leydig cells (CYP11A1 +), Sertoli cells (SOX9 +), and germ cells (DAZL +). Steroidogenic activity of Leydig cells was demonstrated by analyzing testosterone levels in the culture medium. DBP induced a concentration-dependent reduction in testosterone levels and decreased the number of Leydig cells compared to vehicle control. The levels of steroidogenic regulator StAR and the steroidogenic enzyme CYP11A1 were decreased already at the lowest DBP concentration (1 nM), demonstrating upstream effects in the testosterone biosynthesis pathway. Furthermore, exposure to 10 nM DBP decreased the levels of the germ cell-specific RNA binding protein DAZL, central for the spermatogenesis. The 3D model also captured the development of the Sertoli cell junction proteins, N-cadherin and Zonula occludens protein 1 (ZO-1), critical for the blood–testis barrier. However, DBP exposure did not significantly alter the cadherin and ZO-1 levels. Altogether, this 3D in vitro system models testicular cellular signaling and function, making it a powerful tool for mechanistic screening of developmental testicular toxicity. This can open a new avenue for high throughput screening of chemically-induced reproductive toxicity during sensitive developmental phases.
... Therefore, the absent expression of Lhcgr, Cyp11a1, Hsd3b1, Cyp17a1, Hsd17b3, and Star confirmed that the testosterone synthesis pathway was not functional, which aligns with our findings that testosterone secretion was scarcely detectable in the present in vitro setting. In good agreement with our results, the TM3 cell line was recently reported to be unsuitable for studying gonadal androgen biosynthesis, because it lacks the last critical steroidogenic enzyme, 17β-hydroxysteroid dehydrogenase 3 (17β-hsd3), to synthesize testosterone (Engeli et al. 2018). In summary, the TM3 cell line may not be a great model for investigating testosterone production and studying the regulation of the testosterone synthesis pathway in vitro, as this pathway is non-functional in TM3 cells. ...
Cannabidiol (CBD), one of the major components extracted from the plant Cannabis sativa L., has been used as a prescription drug to treat seizures in many countries. CBD-induced male reproductive toxicity has been reported in animal models; however, the underlying mechanisms remain unclear. We previously reported that CBD induced apoptosis in primary human Leydig cells, which constitute the primary steroidogenic cell population in the testicular interstitium. In this study, we investigated the effects of CBD and its metabolites on TM3 mouse Leydig cells. CBD, at concentrations below 30 µM, reduced cell viability, induced G1 cell cycle arrest, and inhibited DNA synthesis. CBD induced apoptosis after exposure to high concentrations (≥ 50 µM) for 24 h or a low concentration (20 µM) for 6 days. 7-Hydroxy-CBD and 7-carboxy-CBD, the main CBD metabolites of CBD, exhibited the similar toxic effects as CBD. In addition, we conducted a time-course mRNA-sequencing analysis in both primary human Leydig cells and TM3 mouse Leydig cells to understand and compare the mechanisms underlying CBD-induced cytotoxicity. mRNA-sequencing analysis of CBD-treated human and mouse Leydig cells over a 5-day time-course indicated similar responses in both cell types. Mitochondria and lysosome dysfunction, oxidative stress, and autophagy were the major enriched pathways in both cell types. Taken together, these findings demonstrate comparable toxic effects and underlying mechanisms in CBD-treated mouse and primary human Leydig cells.
... In 2018, it was revealed that this low testosterone production is altered steroidogenic pathways, in which 17β-HSD is barely expressed. Thu way to accurately study the production of testosterone by LCs is to use huma primary LCs [37]. However, LCs can be used in studies of the endocrine, par autocrine regulation of LCs and how these can be disrupted by EDCs [36,3 more, it might be interesting to access steroidogenesis by measuring intermed process, such as androstenedione. ...
... Since the BLTK1 cells, as happens with other LC lines, do not functionally express 17β-HSD 3 , it is not possible to directly measure the testosterone production, since these produce little to no testosterone under basal conditions [37]. Therefore, to assess steroidogenesis, a competitive ELISA kit was used to detect androstenedione levels, the intermediate androgen that is reduced to testosterone by 17β-HSD 3 . ...
... Since the main function of LCs is to produce testosterone via steroidogenesis [46], any study regarding the effect of a substance in these cells should focus on this primary function. Hence, we used an ELISA kit to measure basal androstenedione production, as it is the final product of steroidogenesis in these cells, as previously mentioned [37]. The group of LCs exposed to 10 µM CrPic 3 and PA (0.96 ± 0.10 ng/mL) showed significantly higher androstenedione production than the LCs from the control group (0.65 ± 0.07 ng/mL) and from the groups of cells exposed to 0.1 µM of CrPic 3 (0.54 ± 0.03 ng/mL), 10 µM of CrPic 3 (0.62 ± 0.09 ng/mL), and 250 µM of PA (0.58 ± 0.08 ng/mL) (Figure 8). ...
Leydig cells (LCs) play a pivotal role in male fertility, producing testosterone. Chromium (III) picolinate (CrPic 3), a contentious supplement with antidiabetic and antioxidant properties, raises concerns regarding male fertility. Using a rodent LC line, we investigated the cytotoxicity of increasing CrPic 3 doses. An insulin resistance (IR) model was established using palmitate (PA), and LCs were further exposed to CrPic 3 to assess its antioxidant/antidiabetic activities. An exometabolome analysis was performed using 1 H-NMR. Mitochondrial function and oxidative stress were evaluated via immunoblot. Steroidogenesis was assessed by quantifying androstenedione through ELISA. Our results uncover the toxic effects of CrPic 3 on LCs even at low doses under IR conditions. Furthermore, even under these IR conditions, CrPic 3 fails to enhance glucose consumption but restores the expression of mitochondrial complexes CII and CIII, alleviating oxidative stress in LCs. While baseline androgen production remained unaffected, CrPic 3 promoted androstenedione production in LCs in the presence of PA, suggesting that it promotes cholesterol conversion into androgenic intermediates in this context. This study highlights the need for caution with CrPic 3 even at lower doses. It provides valuable insights into the intricate factors influencing LCs metabolism and antioxidant defenses, shedding light on potential benefits and risks of CrPic 3 , particularly in IR conditions.
... Our attempts in explaining how these enzymes may regulate androgen production in the mLTC1 cell must be reconciliated with differences with the human Leydig steroidogenesis. Limitations due to different gene expression levels must be considered before making comparative analyses [17]. ...
Androgens are produced by adrenal and gonadal cells thanks to the action of specific enzymes. We investigated the role of protein kinase B (Akt) in the modulation of Δ4 steroidogenic enzymes' activity, in the mouse Leydig tumor cell line mLTC1. Cells were treated for 0-24 h with the 3 × 50% effective concentration of human luteinizing hormone (LH) and choriogonadotropin (hCG), in the presence and in the absence of the specific Akt inhibitor 3CAI. Cell signaling analysis was performed by bioluminescence resonance energy transfer (BRET) and Western blotting, while the expression of key target genes was investigated by real-time PCR. The synthesis of progesterone, 17α-hydroxy (OH)-progesterone and testosterone was measured by immunoassay. Control experiments for cell viability and caspase 3 activation were performed as well. We found that both hormones activated cAMP and downstream effectors, such as extracellularly-regulated kinase 1/2 (Erk1/2) and cAMP response element-binding protein (Creb), as well as Akt, and the transcription of Stard1, Hsd3b1, Cyp17a1 and Hsd17b3 genes, boosting the Δ4 steroidogenic pathway. Interestingly, Akt blockade decreased selectively Cyp17a1 expression levels, inhibiting its 17,20-lyase, but not the 17-hydroxylase activity. This effect is consistent with lower Cyp17a1 affinity to 17α-OH-progesterone than progesterone. As a result, cell treatment with 3CAI resulted in 17α-OH-progesterone accumulation at 16-24 h and decreased testosterone levels after 24 h. In conclusion, in the mouse Leydig cell line mLTC1, we found substantial Akt dependence of the 17,20-lyase activity and testosterone synthesis. Our results indicate that different intracellular pathways modulate selectively the dual activity of Cyp17a1.
... Actual hormone synthesis (in vivo) is regulated not only by complex systems at the physiology level but also at the cellular level by organelle environments [76,77]. Although TM3 is not a suitable cell line for scrutinization of direct interference between biomolecules of Leydig cell steroidogenesis [78], PFOA-triggered abnormal status of organelles such as mitochondrial dysfunction which was demonstrated in the present study, might explain the decline in Leydig cell testosterone production. ...
Perfluorooctanoic acid (PFOA) is an environmentally ubiquitous synthetic chemical highly persistent in organisms. PFOA exposure is pernicious to reproductive health as indicated by reports of male infertility. However, the PFOA toxicity mechanism to Leydig cells remains poorly understood. Therefore, this study aimed to investigate the toxicological events occurring in TM3 Leydig cells treated with PFOA (250, 500, 750µM) for 24h. PFOA was shown to significantly decrease cell viability resulting from inhibition of proliferation and elevation of apoptotic ratio in a dose dependent manner. Upregulation of pro-apoptotic gene expressions such as Bax, Bad, and p53, was observed in combination with an increase in the apoptosis-related protein levels of Bax, cleaved caspase-3, cleaved caspase-8, and phosphorylated p53. Furthermore, exposure of PFOA lead to mitochondrial damage involving mitochondrial membrane permeabilization. A release of cytochrome c and collapse of the mitochondrial membrane potential (∆Ψm) were observed compared to the untreated control. Additionally, PFOA stimulated unfolded protein response (UPR) upregulating ER stress marker, Bip/GRP78, and upregulated protein levels of UPR signal molecules IRE1, p-JNK, p-ERK1/2, p-p53, CHOP, and ERO1. Overall, the present study elucidated the ER stress-mitochondrial apoptosis pathway-related molecular mechanisms involved in PFOA-induced cell death in TM3 Leydig cells.
... Most importantly, human ligands trigger steroidogenic signals mainly activating the synthesis of Δ4 hormones [34], although Δ5 steroid production was described as well [35]. However, comparative analyses between mLTC1 and human Leydig steroidogenesis must be considered carefully, as the two models differ for their enzymatic milieu [36]. ...
... AN, 5α-dione and A4 were the most abundant steroids secreted by mLTC1 both in unstimulated and hCG-stimulated conditions, although in different relative abundance, i.e. 35:6:1 and 1:2:2, respectively. The accumulation of these intermediates could be explained by the fact that they all are substrates of the 17β-HSD3, which was found to be minimally expressed in murine Leydig cells [36]. On the opposite hand, levels of Δ5 steroids were undetectable. ...
The canonical androgen synthesis in Leydig cells involves Δ5 and Δ4 steroids. Besides, the backdoor pathway, encompassing 5α and 5α,3α steroids, is gaining interest in fetal and adult pathophysiology. Moreover, the role of androgen epimers and progesterone metabolites is still unknown. We developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for measuring 20 steroids and used it to investigate the steroid secretion induced by human chorionic gonadotropin (hCG) in the mouse Leydig tumor cell line 1 (mLTC1). Steroids were extracted from 500µL supernatants from unstimulated or 100 pM hCG-exposed mLTC1 cells, separated on a Luna C8 100×3mm, 3µm column, with 100µM NH4F and methanol as mobile phases, and analyzed by positive electrospray ionization and multiple reaction monitoring. Sensitivity ranged within 0.012-38.0 nmol/L. Intra-assay and inter-assay imprecision were <9.1 and 10.0%, respectively. Trueness, recovery and matrix factor were within 93.4-122.0, 55.6-104.1 and 76.4-106.3%, respectively. Levels of 16OH-progesterone, 11-deoxycortisol, androstenedione, 11-deoxycorticosterone, testosterone, 17OH-progesterone, androstenedione, epitestosterone, dihydrotestosterone, progesterone, androsterone and 17OH-allopregnanolone were effectively measured. Traces of 17OH-dihydroprogesterone, androstanediol and dihydroprogesterone were found, whereas androstenediol, 17OH-pregnenolone, dehydroepiandrosterone, pregnenolone and allopregnanolone showed no peak. hCG induced an increase of 80.2-102.5 folds in 16OH-progesterone, androstenedione and testosterone, 16.6 in dihydrotestosterone, 12.2-27.5 in epitestosterone, progesterone and metabolites, 8.1 in 17OH-allopregnanolone and ≤3.3 in 5α and 5α,3α steroids. In conclusion, our LC-MS/MS method allows exploring the Leydig steroidogenesis flow according to multiple pathways. Beside the expected stimulation of the canonical pathway, hCG increased progesterone metabolism and, to a low extent, the backdoor route.
... Contrary to primary Leydig cells, MA-10 cells proliferate and contain an aberrant chromosome number [32]. Despite these issues, MA-10 cells nonetheless respond to hormonal stimulation, like primary Leydig cells, with an increase in steroid hormone production [30,[33][34][35][36] The grey box represents the 35-bp sequence responsible for 70% of Insl3 promoter activity. Results are shown as Fold Activation over control ± SEM. ...
... Contrary to primary Leydig cells, MA-10 cells proliferate and contain an aberrant chromosome number [32]. Despite these issues, MA-10 cells nonetheless respond to hormonal stimulation, like primary Leydig cells, with an increase in steroid hormone production [30,[33][34][35][36], indicating that the signalling cascades, kinases, and transcription factors required for this response are present in MA-10 Leydig cells. More relevant to our current work, MA-10 Leydig cells constitutively express Insl3 [13,19,22,23,27,[37][38][39] and have been validated as a suitable model to study Insl3 gene transcription [27]. ...
The peptide hormone insulin-like 3 (INSL3) is produced almost exclusively by Leydig cells of the male gonad. INSL3 has several functions such as fetal testis descent and bone metabolism in adults. Insl3 gene expression in Leydig cells is not hormonally regulated but rather is constitutively expressed. The regulatory region of the Insl3 gene has been described in various species; moreover, functional studies have revealed that the Insl3 promoter is regulated by various transcription factors that include the nuclear receptors AR, NUR77, COUP-TFII, LRH1, and SF1, as well as the Krüppel-like factor KLF6. However, these transcription factors are also found in several tissues that do not express Insl3, indicating that other, yet unidentified factors, must be involved to drive Insl3 expression specifically in Leydig cells. Through a fine functional promoter analysis, we have identified a 35-bp region that is responsible for conferring 70% of the activity of the mouse Insl3 promoter in Leydig cells. All tri- and dinucleotide mutations introduced dramatically reduced Insl3 promoter activity, indicating that the entire 35-bp sequence is required. Nuclear proteins from MA-10 Leydig cells bound specifically to the 35-bp region. The 35-bp sequence contains GC- and GA-rich motifs as well as potential binding elements for members of the CREB, C/EBP, AP1, AP2, and NF-κB families. The Insl3 promoter was indeed activated 2-fold by NF-κB p50 but not by other transcription factors tested. These results help to further define the regulation of Insl3 gene transcription in Leydig cells.
... MA-10 cells mainly produce progesterone because of a mutation in the Cyp17a1 coding sequence [58]. MA-10 Leydig cells are, therefore, suitable for the study of hormoneinduced steroidogenesis and, more specifically, the early steps of steroidogenesis [62]. Since our current work focuses on the modulation of protein phosphorylation that occurs early following hormone treatment, the MA-10 Leydig cell line is a suitable and convenient model to use. ...
Leydig cells produce testosterone, a hormone essential for male sex differentiation and spermatogenesis. The pituitary hormone, LH, stimulates testosterone production in Leydig cells by increasing the intracellular cAMP levels, which leads to the activation of various kinases and transcription factors, ultimately stimulating the expression of the genes involved in steroidogenesis. The second messenger, cAMP, is subsequently degraded to AMP, and the increase in the intracellular AMP levels activates AMP-dependent protein kinase (AMPK). Activated AMPK potently represses steroidogenesis. Despite the key roles played by the various stimulatory and inhibitory kinases, the proteins phosphorylated by these kinases during steroidogenesis remain poorly characterized. In the present study, we have used a quantitative LC-MS/MS approach, using total and phosphopeptide-enriched proteins to identify the global changes that occur in the proteome and phosphoproteome of MA-10 Leydig cells during both the stimulatory phase (Fsk/cAMP treatment) and inhibitory phase (AICAR-mediated activation of AMPK) of steroidogenesis. The phosphorylation levels of several proteins, including some never before described in Leydig cells, were significantly altered during the stimulation and inhibition of steroidogenesis. Our data also provide new key insights into the finely tuned and dynamic processes that ensure adequate steroid hormone production.
... It is known that murine Leydig cell lines can be applied to research early steps of steroidogenesis but not testosterone synthesis. It has been reported that the three murine Leydig cell lines MA-10, BLTK1 and TM3 produce no or only very low testosterone under basal conditions [31]. Because compared to the siControl transfected cells. ...
Background
In recent studies, it was shown that Endoplasmic reticulum-associated degradation (ERAD) is regulated by androgens and small VCP-interacting protein (SVIP) is an ERAD inhibitor. There is no data available about the interactions of ERAD proteins with proteins involved in steroidogenesis. The aim of the study was to investigate the expressions of SVIP, p97/VCP, StAR, CYP17A1 and 3β-HSD in human and mouse.
Methods and results
HLC, TM3 and MA-10 Leydig cell lines were used to determine roles of ERAD proteins in steroidogenesis based on immunofluorescence, Western blot, qRT-PCR, ELISA. Findings showed that StAR, CYP17A1 and 3β-HSD were colocalized with SVIP and p97/VCP in Leydig cells. A decrease in CYP17A1, 3β-HSD and StAR expressions was observed as a result of suppression of SVIP siRNAs and p97/VCP siRNAs expressions in MA10, TM3 and HLC. When siSVIP transfected cells were compared with siSVIP transfected with hCG-exposed cells, SVIP protein expression was significantly increased as compared to the SVIP transfected group in human Leydig cells.
Conclusion
We suggest that the suppression of protein expressions by p97/VCP and SVIP siRNAs in Leydig cells, the effects of proteins involved in steroidogenesis (StAR, CYP17A1 and 3β-HSD) have proven to be originating from p97/VCP and SVIP which were playing a role in the steroidogenesis process. Additionally, it was demonstrated that testosterone levels decreased after transfection with p97/VCP siRNA and SVIP siRNA, p97/VCP and SVIP created an effect on testosterone synthesis while taking place in the steps of testosterone synthesis. Further, it was determined in the study that the SVIP was affected by hCG stimulations.
... Leydig TM3 cells represent a suitable and relevant model of prepubertal Leydig cells for studying GJIC between Leydig cells and early steps of steroidogenesis (Engeli et al., 2018;Kubincová et al., 2019;Mather, 1980;Mather et al., 1982;Yawer et al., 2020). Leydig TM3 cells in the newly established 3D model still express immature markers such as Arl13b, Dlk1, or Wt1 (Nygaard et al., 2015;Uhlén et al., 2015;Supplementary Table S2) even after 14 days in culture, which makes this in vitro system particularly interesting for studying effects of (sub)chronic exposures on prepubertal cells. ...
The testis is a priority organ for developing alternative models to assess male reproductive health hazards of chemicals. This study characterized a 3D in vitro model of murine prepubertal Leydig TM3 cells with improved expression of steroidogenesis markers suitable for image-based screening of testicular toxicity. This 3D scaffold-free spheroid model was applied to explore the impact of prototypical endocrine-disrupting chemicals (EDCs) and environmental reprotoxicants (benzo[a]pyrene, 2- and 9-methylanthracenes, fluoranthene, triclosan, triclocarban, methoxychlor) on male reproductive health. The results were compared to the male reprotoxicity potential of EDCs assessed in a traditional monolayer (2D) culture. The testicular toxicity was dependent not only on the type of culture (2D vs. 3D models) but also on the duration of exposure. Benzo[a]pyrene and triclocarban were the most active compounds, eliciting cytotoxic effects in prepubertal Leydig cells at low micromolar concentrations, which might be a mechanism contributing to their male reprotoxicity.
