Environmental cadmium inhibits testicular testosterone synthesis via Parkin-dependent MFN1 degradation.

Low testosterone (T) levels are associated with many common diseases, such as obesity, male infertility, depression, and cardiovascular disease. It is well known that environmental cadmium (Cd) exposure can induce T decline, but the exact mechanism remains unclear. We established a murine model in which Cd exposure induced testicular T decline. Based on the model, we found Cd caused mitochondrial fusion disorder and Parkin mitochondrial translocation in mouse testes. MFN1 overexpression confirmed that MFN1-dependent mitochondrial fusion disorder mediated the Cd-induced T synthesis suppression in Leydig cells. Further data confirmed Cd induced the decrease of MFN1 protein by increasing ubiquitin degradation. Testicular specific Parkin knockdown confirmed Cd induced the ubiquitin-dependent degradation of MFN1 protein through promoting Parkin mitochondrial translocation in mouse testes. Expectedly, testicular specific Parkin knockdown also mitigated testicular T decline. Mito-TEMPO, a targeted inhibitor for mitochondrial reactive oxygen species (mtROS), alleviated Cd-caused Parkin mitochondrial translocation and mitochondrial fusion disorder. As above, Parkin mitochondrial translocation induced mitochondrial fusion disorder and the following T synthesis repression in Cd-exposed Leydig cells. Collectively, our study elucidates a novel mechanism through which Cd induces T decline and provides a new treatment strategy for patients with androgen disorders.

Exploring the human chorionic gonadotropin induced steroid secretion profile of mouse Leydig tumor cell line 1 by a 20 steroid LC-MS/MS panel.

The canonical androgen synthesis in Leydig cells involves Δ5 and Δ4 steroids. Besides, the backdoor pathway, eompassing 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 500 µL supernatants from unstimulated or 100 pM hCG-exposed mLTC1 cells, separated on a Luna C8 100 × 3 mm, 3 µm column, with 100 µM 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.

Tetramethyl bisphenol a inhibits leydig cell function in late puberty by inducing ferroptosis.

Tetramethyl bisphenol A (TMBPA) is a commonly used bisphenol analog, used as a fire retardant. However, whether it inhibits the function of Leydig cells in late puberty remains unclear. In this study, 35-day-old male Sprague-Dawley rats were gavaged with 0, 10, 100, and 200 mg/kg body weight TMBPA for 21 days. TMBPA significantly reduced serum testosterone levels at 10 mg/kg and higher doses without altering serum luteinizing hormone and follicle-stimulating hormone levels. TMBPA significantly increased serum iron concentraion while reducing the ratio of serum glutathione (GSH) and GSH/GSSG (oxidized glutathione disulfide). In addition, TMBPA significantly increased testicular iron amount at 10 mg/kg and higher doses and malondialdehyde level at 200 mg/kg. TMBPA down-regulated the expression of Leydig cell genes, including Nr5a1, Star, Scarb1, Insl3, Cyp11a1, Cyp17a1, Hsd17b3, and Hsd11b1, and their proteins. In addition, TMBPA markedly down-regulated the expression of genes in the ferroptosis pathway (Tp53, Slc7a11, Sod1, Sod2, Cat, Sqstm1, Keap1, and Hmox1). TMBPA significantly reduced the levels of ferroptosis pathway proteins (TP53, SLC7A11, GPX4, SQSTM1, KEAP1, NRF2, and HMOX1) in Leydig cells in vivo. Immature and adult Leydig cell culture in vitro also showed that TMBPA significantly reduced testosterone concentrations in the medium, which can be reversed by a ferroptosis inhibitor. After 24 h of culture in primary Leydig cells at 10 and 50 µM, TMBPA significantly induced reactive oxygen species and lowered the mitochondrial membrane potential. TMBPA also altered protein levels in the ferroptosis pathway in Leydig cells in vitro. In conclusion, TMBPA directly inhibits the activity of rat Leydig cell steroidogenic enzymes and induces the ferroptosis of Leydig cells, thereby inhibiting the testosterone synthesis of Leydig cells in the late puberty.

Cordycepin mitigates spermatogenic and redox related expression in H2O2-exposed Leydig cells and regulates testicular oxidative apoptotic signalling in aged rats.

CONTEXT: Cordycepin (COR), from Cordyceps militaris L., (Cordycipitaceae), is a valuable agent with immense health benefits. OBJECTIVE: The protective effects of COR in ageing-associated oxidative and apoptosis events in vivo and hydrogen peroxide (H2O2)-exposed spermatogenesis gene alterations in TM3 Leydig cells was investigated. MATERIALS AND METHODS: Male Sprague-Dawley rats were divided into young control (YC), aged control (AC) and COR treated (COR-20) aged groups. COR-20 group received daily doses of COR (20 mg/kg) for 6 months. Cell viability and hormone levels were analysed by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] and enzyme immunoassay kits with COR treated at 1, 5, and 10 µg/mL. Oxidative enzymes, spermatogenic, and apoptotic expression in testis tissues were evaluated by Western blotting and real-time RT-PCR. RESULTS: COR treatment (1, 5, and 10 µg/mL) significantly (p < 0.05 ∼ p < 0.001) inhibited the H2O2-induced decrease in the percentage of viable cells (from 63.27% to 71.25%, 85.67% and 93.97%, respectively), and reduced the malondialdehyde (MDA) content (from 4.28 to 3.98, 3.14 and 1.78 nM MDA/mg protein, respectively). Further, the decreased antioxidant enzymes (glutathione-S-transferase mu5, glutathione peroxidase 4 and peroxiredoxin 3), spermatogenesis-related factors (nectin-2 and inhibin-α) and testosterone levels in H2O2-exposed TM3 cells were significantly (p < 0.05 ∼ p < 0.001) ameliorated by COR. In aged rats, COR (20 mg/kg) restored the altered enzymatic and non-enzymatic antioxidative status and attenuated the apoptotic p53 and Bax/Bcl-2 expression significantly (p < 0.05). CONCLUSION: COR might be developed as a potential agent against ageing-associated and oxidative stress-induced male infertility.

Potential Role of Individual and Combined Effects of T-2 Toxin, HT-2 Toxin and Neosolaniol on the Apoptosis of Porcine Leydig Cells.

T-2 toxin usually co-occurs with HT-2 toxin and neosolaniol (NEO) in the grains and feed. Our previous studies found that T-2 toxin and its metabolites' binary or ternary combination exposure to porcine Leydig cells (LCs) displayed synergism in certain range of dosage and cannot be predicted based on individual toxicity. However, the possible mechanism of these mycotoxins' combined exposure to cell lesions remains unknown. Based on 50% cell viability, the mechanism of apoptosis in porcine Leydig cells was investigated after exposure to T-2, HT-2, NEO individual and binary or ternary combinations. Compared with control, the adenosine triphosphate (ATP) content decreased, reactive oxygen species (ROS) level increased, and mitochondrial membrane potential (MMP) decreased in all treated groups. Additionally, the cell apoptosis rates were significantly increased in test groups (p < 0.05), and the B-cell lymphoma 2 (Bcl-2) Associated X (Bax)/Bcl-2 ratio and the expression of caspase 3, caspase 8, cytochrome c (Cytc) in the treated group are all significantly higher than the control group. Moreover, the expression of Cytc and caspase 8 gene in NEO and T-2+NEO groups was significantly higher than that in other individual and combined groups. It can be concluded that the toxicities of T-2, HT-2, and NEO individually and in combination can induce apoptosis related to the oxidative stress and mitochondrial damage, and the synergistic effect between toxins may be greater than a single toxin effect, which is beneficial for assessing the possible risk of the co-occurrences in foodstuffs to human and animal health.

Comparative Cytotoxic Effects and Possible Mechanisms of Deoxynivalenol, Zearalenone and T-2 Toxin Exposure to Porcine Leydig Cells In Vitro.

Mycotoxins such as zearalenone (ZEN), deoxynivalenol (DON) and T-2 toxin (T-2) are the most poisonous biological toxins in food pollution. Mycotoxin contaminations are a global health issue. The aim of the current study was to use porcine Leydig cells as a model to explore the toxic effects and underlying mechanisms of ZEN, DON and T-2. The 50% inhibitory concentration (IC50) of ZEN was 49.71 µM, and the IC50 values of DON and T-2 were 2.49 µM and 97.18 nM, respectively. Based on the values of IC50, ZEN, DON and T-2 exposure resulted in increased cell apoptosis, as well as disrupted mitochondria membrane potential and cell cycle distribution. The results also showed that ZEN and DON significantly reduced testosterone and progesterone secretion in Leydig cells, but T-2 only reduced testosterone secretion. Furthermore, the expression of steroidogenic acute regulatory (StAR) protein and 3ß-hydroxysteroid dehydrogenase (3ß-HSD) were significantly decreased by ZEN, DON and T-2; whereas the protein expression of cholesterol side-chain cleavage enzyme (CYP11A1) was only significantly decreased by ZEN. Altogether, these data suggest that the ZEN, DON and T-2 toxins resulted in reproductive toxicity involving the inhibition of steroidogenesis and cell proliferation, which contributes to the cellular apoptosis induced by mitochondrial injury in porcine Leydig cells.

Anti-steroidogenic effects of cholesterol hydroperoxide trafficking in MA-10 Leydig cells: Role of mitochondrial lipid peroxidation and inhibition thereof by selenoperoxidase GPx4.

Steroid hormone synthesis in steroidogenic cells requires cholesterol (Ch) delivery to/into mitochondria via StAR family trafficking proteins. In previous work, we discovered that 7-OOH, an oxidative stress-induced cholesterol hydroperoxide, can be co-trafficked with Ch, thereby causing mitochondrial redox damage/dysfunction. We now report that exposing MA-10 Leydig cells to Ch/7-OOH-containing liposomes (SUVs) results in (i) a progressive increase in fluorescence probe-detected lipid peroxidation in mitochondrial membranes, (ii) a reciprocal decrease in immunoassay-detected progesterone generation, and ultimately (iii) loss of cell viability with increasing 7-OOH concentration. No significant effects were observed with a phospholipid hydroperoxide over the same concentration range. Glutathione peroxidase GPx4, which can catalyze lipid hydroperoxide detoxification, was detected in mitochondria of MA-10 cells. Mitochondrial lipid peroxidation and progesterone shortfall were exacerbated when MA-10 cells were treated with Ch/7-OOH in the presence of RSL3, a GPx4 inhibitor. However, Ebselen, a selenoperoxidase mimetic, substantially reduced RSL3's negative effects, thereby partially rescuing the cells from peroxidative damage. These findings demonstrate that co-trafficking of oxidative stress-induced 7-OOH can disable steroidogenesis, and that GPx4 can significantly protect against this.

Beneficial effects of roots of Argyreia nervosa (Brum.f.) Bojer on testosterone biosynthesis in testis and spermatogenesis in Wistar rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Roots of Argyreia nervosa (Burm.f.) Bojer is used traditionally as an aphrodisiac and mentioned in the indigenous system of medicine as spermatogenic. The roots of the plant are also used as bitter, tonic, and alternative. AIM OF THE STUDY: To study the effect of n-butanol fraction (BTF) and ethyl acetate fraction (ETF) of methanol extract prepared from the roots of Argyreia nervosa and scopoletin isolated from ETF on testosterone biosynthesis in testis and spermatogenesis using rats. MATERIALS AND METHODS: The effect of BTF, ETF, and scopoletin on the testosterone biosynthesis was evaluated by determining the alteration in expression of mRNA corresponding to steroidogenic enzymes and concentration of testosterone using TM-3 cell line. The ability of BTF and ETF in altering the level of testicular cholesterol and testosterone along with mRNA expression corresponding to 3ß-Hydroxy-Δ5-steroid dehydrogenase (3ß-HSD) and Acute Steroid Regulatory Protein (StAR) was evaluated using rats as experimental animals. The sperm concentration in the seminal fluid was determined, and histological studies of testicular tissues were also carried out. RESULTS: Test solutions containing BTF, ETF, and scopoletin showed a dose-dependent and statistically significant increase in the testosterone content when incubated with TM-3 cells. The test solutions also increased the fold expression of mRNA corresponding to StAR and 3ß-HSD enzymes from TM-3 cells. BTF and ETF elevated testicular testosterone levels by 3.57 and 3.84-fold as compared to control animals, while the fractions showed 9.04 and 10.41-fold alteration in expression of mRNA corresponding to StAR, respectively. BTF and ETF altered the expression of mRNA corresponding to 3ß-HSD by 13.43 and 15.04-fold in testicular tissues; moreover, they elevated the activity of 3ß-HSD by 7.11 and 7.73 fold, respectively. The animals treated with BTF and ETF showed increased sperm concentration. Histological observations showed that the lumen of seminiferous tubules was densely populated with spermatozoa and Leydig cells were intensely stained. Extract prepared from fruits of Tribulus terrestris Linn and testosterone served as positive controls. CONCLUSION: BTF, ETF, and scopoletin could promote testosterone biosynthesis by elevating mRNA expression corresponding to StAR, 3ß-HSD, and by increasing 3ß-HSD activity in the testicular tissues. Elevated testosterone concentration in testis promoted spermatogenesis. The studies provided the probable mechanism through which the roots of A. nervosa act as spermatogenic.

Multi- and transgenerational biochemical effects of low-dose exposure to bisphenol A and 4-nonylphenol on testicular interstitial (Leydig) cells.

Bisphenol A (BPA) and 4-nonylphenol (NP) are well-known endocrine-disrupting chemicals (EDCs) that have been proven to affect Leydig cell (LC) functions and testosterone production, but whether BPA and NP have multi- and transgenerational biochemical effects on Leydig cells (LCs) is unknown. Fourier transform infrared (FTIR) spectroscopy is a powerful analytical technique that enables label-free and non-destructive analysis of the tissue specimen. Herein we employed FTIR coupled with chemometrics analysis to identify biomolecular changes in testicular interstitial (Leydig) cells of rats after chronic exposure to low doses of BPA and NP. Cluster segregations between exposed and control groups were observed based on the fingerprint region of 1800-900 cm-1 in all generations. The main biochemical alterations for segregation were amide I, amide II and nucleic acids. BPA and NP single and co-exposure induced significant differences in the ratio of amide I to amide II compared to the corresponding control group in all generations. BPA exposure resulted in remarkable changes of cellular gene transcription and DNA oxidative damage across all generations. Direct exposure to BPA, NP, and BPA&NP of F0 and F1 generations could significantly decrease lipid accumulation in LCs in the F2 and F3 generations. The overall findings revealed that single or co-exposure to BPA and NP at environmental concentrations affects the biochemical structures and properties of LCs.

Bisphenol S exposure induces cytotoxicity in mouse Leydig cells.

Bisphenol S (BPS), an increasingly used alternative to bisphenol A, has been linked to testosterone deficiency and male reproductive dysfunction in laboratory animals. This study aimed to examine the cytotoxicity of BPS exposure to Leydig cells and to investigate its possible mechanisms. After treatment with BPS (100, 200 and 400 µM) for 48 h in vitro, TM3 mouse Leydig cells exhibited a dose-dependent decrease in the viability. Furthermore, BPS challenge triggered oxidative stress manifested by compromised activities of superoxide dismutase and catalase with exaggerated formation of reactive oxygen species. Especially, BPS exposure resulted in augmented mitochondrial permeability transition pore opening, dissipated mitochondrial membrane potential and reduced ATP generation, along with an altered energy metabolism. Moreover, BPS stimulation enhanced BAX expression and caspase-3 activity and inhibited BCL-2 expression. In addition, BPS-treated TM3 cells showed an accumulation of autophagic vacuoles, together with increased Beclin1 and P62 expression and elevated LC3B-II/LC3B-I ratio. These results demonstrated that in vitro exposure to BPS exerted cytotoxicity to TM3 Leydig cells through inducing oxidative stress, mitochondrial impairment, autophagic disturbance and apoptosis.

Chlorocholine chloride induced testosterone secretion inhibition mediated by endoplasmic reticulum stress in primary rat Leydig cells.

Chlorocholine chloride (CCC) is well acknowledged as a plant growth regulator and may be considered as a potential environmental endocrine disrupting chemical. In our previous studies, it was found that CCC exposure at a pubertal stage reduced the serum and testicular levels of testosterone, decreased the sperm motility and delayed the puberty onset. However, the molecular mechanisms of CCC-induced testosterone secretion disorders remain unclear. In this study, we found that CCC exposure above 20 µg/mL inhibited the secretion of testosterone in Sprague-Dawley rats Leydig cells. Proteomic and pathway enrichment analysis indicated that CCC might induce endoplasmic reticulum (ER) stress. Western blot detection showed CCC exposure at 100, 200 µg/mL increased the protein level of glucose-regulated protein 78 (GPR78), C/EBP-homologous protein (CHOP), the ubiquitin-conjugating enzyme E2 D1 (UBE2D1) and the ring finger protein (RNF185) in the Leydig cells. The Leydig cells treated with 4-phenyl butyric acid (4-PBA), an ER stress inhibitor, rescued the testosterone secretion disorders and alleviated CCC-induced increase in the ER stress related protein levels at 200 µg/mL CCC treatment. Overall, CCC in vitro exposure might disturb testosterone production of Leydig cells and endoplasmic reticulum stress was involved in it.

Multifaceted epigenetic regulation of porcine testicular aromatase.

Testicular aromatase catalyzes the synthesis of estradiol, which contributes to regulation of porcine Sertoli cell proliferation and postpubertal maintenance of Sertoli cell numbers. Although aromatase enzymatic activity decreases with age and is persistently reprogrammed by prepubertal treatment with the aromatase inhibitor letrozole, the molecular bases for regulation have not been identified. DNA methylation was examined as a potential regulatory mechanism using DNA from Leydig cells isolated from 16-, 40-, and 68-week-old boars and from 68- week-old littermates treated with the aromatase inhibitor, letrozole. Methylation levels of individual CpG dinucleotides located in the distal untranslated exon 1 of the relevant aromatase encoding gene, CYP19A3, were quite high in Leydig cell DNA, and increased further with maturity of boar (P < 0.05), while aromatase activity and transcript abundance decreased more than two-fold. However, reduced aromatase activity following letrozole treatment was not accompanied by altered DNA methylation. Testicular expression of miR378 was altered by prepubertal treatment with letrozole. The data provide evidence for two different epigenetic mechanisms that regulate aromatase expression and enzymatic activity in the boar testis.

Oncostatin M stimulates immature Leydig cell proliferation but inhibits its maturation and function in rats through JAK1/STAT3 signaling and induction of oxidative stress in vitro.

BACKGROUND: Oncostatin M (OSM) is a member of the interleukin-6 group of cytokines, which can regulate cell proliferation, growth, and function. Immature Leydig cells have the ability to proliferate and differentiate, and adult Leydig cells have the function of testosterone synthesis. However, the role and underlying mechanisms of OSM on the proliferation and function of Leydig cells remain unclear. METHODS: The effects of OSM on the proliferation, apoptosis, and function of immature Leydig cells isolated from 35-day-old rats and the function of adult Leydig cells isolated from 63-day-old rats in vitro. RESULTS: OSM stimulated immature Leydig cell proliferation after up-regulating the expression of Ccnd1 and Cdk4 to drive the transition of G1 phase to M2 phase in the cell cycle at 10 and 100 ng/ml. OSM did not affect the apoptosis of immature Leydig cells up to 100 ng/ml. OSM inhibited testosterone production in immature and adult Leydig cells by down-regulating the expression of Lhcgr, Star, Cyp11a1, Hsd3b1, and Cyp17a1 at 1-100 ng/ml. OSM induced reactive oxygen species and down-regulated the expression of antioxidant genes and lowered mitochondrial membrane potential at 10 and 100 ng/ml in both Leydig cells. Janus kinase 1 (JAK1) antagonist filgotinib and signal transducer and activator of transcription 3 (STAT3) antagonist S3I-201 reversed the effect of OSM, indicating that it acts on JAK1/STAT3 signaling. CONCLUSION: Oncostatin M stimulates immature Leydig cell proliferation while inhibiting the function of immature and adult Leydig cells.

Effects of Midazolam on the Development of Adult Leydig Cells From Stem Cells In Vitro.

Background: Midazolam is a neurological drug with diverse functions, including sedation, hypnosis, decreased anxiety, anterograde amnesia, brain-mediated muscle relaxation, and anticonvulsant activity. Since it is frequently used in children and adolescents for extended periods of time, there is a risk that it may affect their pubertal development. Here, we report a potential effect of the drug on the development of Leydig cells (LCs), the testosterone (T)-producing cells in the testis. Methods: Stem LCs (SLCs), isolated from adult rat testes by a magnetic-activated cell sorting technique, were induced to differentiate into LCs in vitro for 3 weeks. Midazolam (0.1-30 µM) was added to the culture medium, and the effects on LC development were assayed. Results: Midazolam has dose-dependent effects on SLC differentiation. At low concentrations (0.1-5 µM), the drug can mildly increase SLC differentiation (increased T production), while at higher concentrations (15-30 µM), it inhibits LC development (decreased T production). T increases at lower levels may be due to upregulations of scavenger receptor class b Member 1 (SCARB1) and cytochrome P450 17A1 (CYP17A1), while T reductions at higher levels of midazolam could be due to changes in multiple steroidogenic proteins. The uneven changes in steroidogenic pathway proteins, especially reductions in CYP17A1 at high midazolam levels, also result in an accumulation of progesterone. In addition to changes in T, increases in progesterone could have additional impacts on male reproduction. The loss in steroidogenic proteins at high midazolam levels may be mediated in part by the inactivation of protein kinase B/cAMP response element-binding protein (AKT/CREB) signaling pathway. Conclusion: Midazolam has the potential to affect adult Leydig cell (ALC) development at concentrations comparable with the blood serum levels in human patients. Further studies are needed to test the effects on human cells.

Short-term exposure to perfluorotetradecanoic acid affects the late-stage regeneration of Leydig cells in adult male rats.

Perfluorotetradecanoic acid (PFTeDA) is one of perfluoroalkyl substances widely found in the environment. PFTeDA may cause the dysfunction of male reproductive system. However, whether PFTeDA affects the regeneration of Leydig cells remains unclear. The objective of this study was to examine the effects of short-term exposure of PFTeDA on the late-stage maturation of Leydig cells. Fifty-four adult Sprague-Dawley male rats were daily gavaged with PFTeDA (0, 10, or 20 mg/kg body weight) for 10 days, and then were injected intraperitoneally with ethylene dimethane sulfonate (EDS, 75 mg/kg body weight/once) to ablate Leydig cells to induce their regeneration. On day 21 (early stage) and 56 (late stage) after EDS, hormone levels, gene expression, and protein levels were measured. PFTeDA did not affect the early stage of Leydig cell regeneration, because it had no effect on serum testosterone, luteinizing hormone, and follicle-stimulating hormone levels, Leydig cell number, and its gene and protein expression. PFTeDA significantly reduced serum testosterone level and down-regulated the expression of Leydig cell genes (Cyp11a1, Hsd3b1, Cyp17a1, Hsd17b3, Hsd11b1, and Insl3) and their proteins (CYP11A1, HSD3B1, CYP17A1, HSD17B3, and INSL3), decreased the phosphorylation of AKT1 and ERK1/2, as well as lowered sperm count in the epididymis at 20 mg/kg. In conclusion, short-term exposure to PFTeDA blocks the late-stage maturation of Leydig cells.

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.

Amodiaquine promotes testosterone production and de novo synthesis of cholesterol and triglycerides in Leydig cells.

Testosterone is a hormone essential for male reproductive function. It is produced primarily by Leydig cells in the testicle through activation of steroidogenic acute regulatory protein and a series of steroidogenic enzymes, including a cytochrome P450 side-chain cleavage enzyme (cytochome P450 family 11 subfamily A member 1), 17α-hydroxylase (cytochrome P450 family 17 subfamily A member 1), and 3ß-hydroxysteroid dehydrogenase. These steroidogenic enzymes are mainly regulated at the transcriptional level, and their expression is increased by the nuclear receptor 4A1. However, the effect on Leydig cell function of a small molecule-activating ligand, amodiaquine (AQ), is unknown. We found that AQ effectively and significantly increased testosterone production in TM3 and primary Leydig cells through enhanced expression of steroidogenic acute regulatory protein, cytochome P450 family 11 subfamily A member 1, cytochrome P450 family 17 subfamily A member 1, and 3ß-hydroxysteroid dehydrogenase. Concurrently, AQ dose-dependently increased the expression of 3-hydroxy-3-methylglutaryl-CoA reductase, a key enzyme in the cholesterol synthesis pathway, through induction of the transcriptional and DNA-binding activities of nuclear receptor 4A1, contributing to increased cholesterol synthesis in Leydig cells. Furthermore, AQ increased the expression of fatty acid synthase and diacylglycerol acyltransferase and potentiated de novo synthesis of fatty acids and triglycerides (TGs). Lipidomics profiling further confirmed a significant elevation of intracellular lipid and TG levels by AQ in Leydig cells. These results demonstrated that AQ effectively promotes testosterone production and de novo synthesis of cholesterol and TG in Leydig cells, indicating that AQ may be beneficial for treating patients with Leydig cell dysfunction and subsequent testosterone deficiency.

MEHP induces alteration of mitochondrial function and inhibition of steroid biosynthesis in MA-10 mouse tumor Leydig cells.

Di-(2-ethylhexyl) phthalate (DEHP) is a plasticizer that is widely used in manufacturing. Previous studies have shown that mono-(2-ethylhexyl) phthalate (MEHP), the active metabolite of DEHP, has inhibitory effects on luteinizing hormone (LH)-stimulated steroid biosynthesis by Leydig cells. The molecular mechanisms underlying its effects, however, remain unclear. In the present study, we examined the effects of MEHP on changes in mitochondrial function in relationship to reduced progesterone formation by MA-10 mouse tumor Leydig cells. Treatment of MA-10 cells with MEHP (0-300 µM for 24 h) resulted in dose-dependent inhibition of LH-stimulated progesterone biosynthesis. Biochemical analysis data revealed that the levels of the mature steroidogenic acute regulatory protein (STAR), a protein that works at the outer mitochondrial membrane to facilitate the translocation of cholesterol for steroid formation, was significantly reduced in response to MEHP exposures. MEHP also caused reductions in MA-10 cell mitochondrial membrane potential (ΔΨm) and mitochondrial respiration as evidenced by decreases in the ability of the mitochondria to consume molecular oxygen. Additionally, significant increases in the generation of mitochondrial superoxide were observed. Taken together, these results indicate that MEHP inhibits steroid formation in MA-10 cells at least in part by its effects on mitochondrial function.

Gene expression profiles of two testicular somatic cell lines respond differently to 4-nitrophenol mediating vary reproductive toxicity.

4-Nitrophenol (PNP) has been extensively used in manufacturing for several decades. Its toxic effects on the male reproductive system have been reported, but the underlying mechanisms remain unclear. In this study, we utilized two testicular somatic cell lines (TM3 and TM4 cells) to explore the possible toxic effects of PNP on the male reproductive system. The activity of the cells after exposure to different doses of PNP (0.01, 0.1, 1, 10 and 100 µM) was evaluated. PNP treatment at 10 µM significantly inhibited cell viability, and 10 µM PNP was thus selected for subsequent experiments. Although PNP (10 µM) inhibited cell proliferation, promoted cell apoptosis, and changed the cell cycle distribution and ultrastructure in both types of cells, these effects were more significant in the TM4 cells. In addition, an Agilent mouse mRNA array was used to identify the gene expression differences between the control and PNP (10 µM) exposed TM3 and TM4 cells. The microarray analysis identified 67 and 1372 differentially expressed genes mainly concentrated in endothelial cell morphogenesis and anatomical structure development in TM3 cells and associated with cardiovascular system development and circulatory system development in TM4 cells. Moreover, a pathway analysis revealed that PNP not only predominately affected meiotic recombination and meiosis in TM3 cells, but also influenced axon guidance and developmental biology in TM4 cells. These results suggest that TM3 and TM4 cells exhibit different responses to PNP, which might mediate different toxic mechanisms.

Valproic acid during pregnancy decrease the number of spermatogenic cells and testicular volume in the offspring of mice: Stereological quantification.

Valproic acid (VPA) is a drug used to treat epilepsy, bipolar disorders and headaches. As a secondary effect, this antiepileptic drug can cause a decrease in androgens and gonadotropins, and dose-dependent testicular defects, such as reduction of testicular weights, sperm motility and degeneration of the seminiferous tubules. In offspring exposed to VPA, its effects have not been evaluated, so the study aimed to determine the morphological effects of the use of VPA along testicular development in mice. 30 adult female BALB/c mice were crossed and divided by age, with embryos of 12.5 days post coitum (dpc), fetuses of 17.5 dpc and male mice 6 weeks postnatal. In each case, the pregnant mouse received 600 mg/kg of VPA, making up the VPA groups, or 0.3 mL of 0.9% physiological solution for the control groups, from the beginning to the end of the pregnancy, orally.t. A morpho-quantitative analysis was carried out on the gonadal development of the male offspring. In the groups treated with VPA, at all ages studied they had lower testicular volume. At 12.5 dpc, they showed less testicular development in the form of sex cords, with fewer gonocytes and somatic cells. At 17.5 dpc, they presented greater interstitial space, fewer spermatogonial, sustentacular Sertoli, peritubular and interstitial Leydig cells. At 6 weeks postnatal, they presented fewer spermatogonia, pachytene spermatocytes, elongated spermatids, sustentacular Sertoli and interstitial Leydig cells, with statistically significant differences. In conclusion, prenatal exposure to VPA causes histopathological alterations in the offspring of mice in testicular development, from the embryonic stage to 6 weeks postnatal.