Primary Leydig Cells Research Articles

α-Linolenic acid promotes testosterone synthesis by improving mitochondrial function in primary rooster Leydig cells

The present study aimed to investigate the direct effects of α-Linolenic acid (ALA) on the in vitro production of testosterone and the expression of key enzymes and proteins related to steroidogenesis in Leydig cells of roosters. MethodsPurified primary Leydig cells isolated from 65-week-old roosters were purified and treated with different concentrations of ALA treatments: (0 μm/L [control], solvent control group (DMSO), 20 μM/L, 40 μM/L, and 80 μM/L) and cell counting-8 (CCK-8) for cell viability assay, Enzyme-linked immunosorbent assay (ELISA) kit for the determination of testosterone in cell supernatants, quantitative (real-time) PCR, and analysis of activities of antioxidants catalase (CAT), superoxide dismutase (SOD) and malondialdehyde (MDA), evaluation of mitochondrial membrane potential, pro- and anti-apoptotic proteins/genes Bcl-2, Bcl-2-associated X protein (Bax), apoptosis-inducing factor (AIF) were done respectively. ResultsOur results showed that ALA significantly increased testosterone secretion in primary rooster Leydig cells (P < 0.05), and 40 μM/L is the optimal dose. Leydig cells supplemented with ALA (20, 40, 80 μM) increased the expression of key enzymes and proteins 3β-hydroxysteroid dehydrogenase (3β-HSD), steroidogenic acute regulatory protein (StAR), cholesterol side-chain cleavage enzyme (P450scc) concerning steroidogenesis, enhanced antioxidant capability, improved mitochondrial biogenesis, and markedly improved the mitochondrial membrane potential (P < 0.05). Furthermore, the expression of the apoptosis-suppressive gene Bcl-2 was significantly increased, but Bax and AIF expression was decreased in the ALA group compared to that in the control group (P < 0.05). ConclusionALA promoted testosterone production, enhanced steroidogenic enzyme expression, improved mitochondrial function, and antioxidant capacity, and reduced apoptosis in primary rooster Leydig cells, with 40 μM/L identified as the optimal concentration.

Molecular characteristics and regulatory role of insulin-like growth factor 1 gene in testicular Leydig cells of Tibetan sheep.

This study aimed to analyze the molecular characteristics of insulin-like growth factor 1 (IGF1) gene in the testes of Tibetan sheep and its role in the testosterone synthesis and cell development. First, we cloned IGF1 gene for bioinformatics analysis, and the primary Leydig cells (LCs) of Tibetan sheep were isolated to explore its effect on the proliferation, apoptosis and function of LCs. Finally, the specific regulatory mechanism of IGF1 on LCs was analyzed by transcriptome sequencing. Results showed that overexpression of IGF1 increased the proliferation rate and decreased apoptosis of LCs. In addition, overexpression of IGF1 altered expression of genes related to testosterone synthesis and transformation and significantly increased amount of the final product testosterone. Mechanistically, IGF1 stimulated the expression of the proliferating cell nuclear antigen and IGF1R and promoted the proliferation of LCs via the PI3K/Akt signaling pathway. Collectively, what should be clear from the results reported here is that IGF1 might play roles in the proliferation or differentiation and testosterone synthesis of LCs. These findings add to our understanding on the regulation of testosterone synthesis in sheep and other mammals.

Bisphenol H exposure disrupts Leydig cell function in adult rats via oxidative stress-mediated m6A modifications: Implications for reproductive toxicity

Bisphenol H (BPH) has emerged as a potential alternative to bisphenol A (BPA), which has been curtailed for use due to concerns over its reproductive and endocrine toxicity. This study investigates whether BPH exerts antiandrogenic effects by impairing Leydig cell function, a critical component in testosterone production. We administered orally BPH to adult male rats at doses of 0, 1, 10, and 100 mg/kg/day for 7 days. Notably, BPH treatment resulted in a dose-dependent reduction in testicular testosterone levels, with significant decreases observed at ≥ 1 mg/kg/day. Additionally, BPH affected the expression of key genes involved in steroidogenesis and cholesterol metabolism, including Nr5a1, Nr3c4, Lhcgr, Scarb1, and Star, at higher doses (10 and/or 100 mg/kg/day). The study also revealed alterations in antioxidant gene expression (Sod2 and Cat) and modulation of m6A-related genes (Ythdf1–3 and Foxo3) and their proteins. Through MeRIP-qPCR analysis, we identified increased m6A modifications in Scarb1 and Star genes following BPH exposure. In vitro experiments with primary Leydig cells confirmed that BPH enhanced oxidative stress and diminished testosterone production, which were partially mitigated by antioxidant vitamin E supplementation and Ythdf3 knockdown. Meanwhile, simultaneous administration of BPH and vitamin E to primary Leydig cells partially counteracted BPH-induced alterations in the Ythdf3 expression. Our findings underscore a novel mechanism by which BPH disrupts Leydig cell function through the oxidative stress-m6A modification-autophagy pathway, raising concerns about its potential reproductive toxicity.

Fine particulate matter (PM2.5) induces testosterone disruption by triggering ferroptosis through SIRT1/HIF-1α signaling pathway in male mice

Fine particulate matter (PM2.5), a significant component of air pollution particulate matter, is inevitable and closely associated with increasing male reproductive disorder. However, the testicular targets of PM2.5 and its toxicity related molecular mechanisms are still not fully understood. In this study, the conditional knockout (cKO) mice and primary Leydig cells were used to explore the testicular targets of PM2.5 and the related underlying mechanisms. First, apparent the structure impairment of seminiferous tubules, Leydig cells vacuolization, decline of serum testosterone and sperm quality reduction were found in male wild-type (WT) and Sirt1 knockout mice after exposure to PM2.5. Enrichment analyses revealed that differentially expressed genes (DEGs) were enriched in steroid hormone biosynthesis, ferroptosis, and HIF-1 signaling pathway in the mice testes after exposure to PM2.5, which were subsequently verified by the molecular biological analyses. Notably, similar enrichment analyses results were also observed in primary Leydig cells after treatment with PM2.5. In addition, Knockdown of Sirt1 significantly increased PM2.5-induced expression and activation of HIF-1α, which was in parallel to the changes of cellular iron levels, oxidative stress indicators and the ferroptosis markers. In conclusion, this highlights that PM2.5 triggers ferroptosis via SIRT1/HIF-1α signaling pathway to inhibit testosterone synthesis in males. These findings provide a novel research support for the study that PM2.5 causes male reproductive injury.

Androgen synthesis cell-specific CREBZF deficiency alters adrenal cortex steroid secretion and develops behavioral abnormalities in adult male mice.

The global challenge of male infertility is escalating, notably due to the decreased testosterone (T) synthesis in testicular Leydig cells under stress, underscoring the critical need for a more profound understanding of its regulatory mechanisms. CREBZF, a novel basic region-leucine zipper transcription factor, regulates testosterone synthesis in mouse Leydig cells invitro; however, further validation through invivo experiments is essential. Our study utilized Cyp17a1-Cre to knock out CREBZF in androgen-synthesis cells and explored the physiological roles of CREBZF in fertility, steroid hormone synthesis, and behaviors in adult male mice. Conditional knockout (cKO) CREBZF did not affect fertility and serum testosterone level in male mice. Primary Leydig cells isolated from CREBZF-cKO mice showed impaired testosterone secretion and decreased mRNA levels of Star, Cyp17a1, and Hsd3b1. Loss of CREBZF resulted in thickening of the adrenal cortex, especially X-zone, with elevated serum corticosterone and dehydroepiandrosterone levels and decreased serum dehydroepiandrosterone sulfate levels. Immunohistochemical staining revealed increased expression of StAR, Cyp11a1, and 17β-Hsd3 in the adrenal cortex of CREBZF-cKO mice, while the expression of AR was significantly reduced. Along with the histological changes and abnormal steroid levels in the adrenal gland, CREBZF-cKO mice showed higher anxiety-like behavior and impaired memory in the elevated plus maze and Barnes maze, respectively. In summary, CREBZF is dispensable for fertility, and CREBZF deficiency in Leydig cells promotes adrenal function in adult male mice. These results shed light on the requirement of CREBZF for fertility, adrenal steroid synthesis, and stress response in adult male mice, and contribute to understanding the crosstalk between testes and adrenal glands.

Toxicity of cannabidiol and its metabolites in TM3 mouse Leydig cells: a comparison with primary human Leydig 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.

Cell-type specific and differential expression of LINC-RSAS long noncoding RNA declines in the testes during ageing of the rat.

Long noncoding RNAs (lncRNAs) have emerged as major regulators of gene expression, chromatin structure, epigenetic changes, post-transcriptional processing of RNAs, translation of mRNAs into proteins as well as contributing to the process of ageing. Ageing is a universal, slow, progressive change in almost all physiological processes of organisms after attaining reproductive maturity and often associated with age-related diseases. Mammalian testes contain various cell-types, vast reservoir of transcriptome complexity, produce haploid male gametes for reproduction and testosterone for development and maintenance of male sexual characters as well as contribute genetic variation to the species. We report age-related decline in expression and cellular localization of Long intergenic noncoding repeat-rich sense-antisense (LINC-RSAS) RNA in the testes and its major cell-types such as primary spermatocytes, Leydig cells and Sertoli cells during ageing of the rat. LINC-RSAS expression in testes increased from immature (4-weeks) to adult (16- and 44-weeks) and declined from adult (44-weeks) to nearly-old (70-weeks) rats. Genomic DNA methylation in the testes showed a similar pattern. Cell-type specific higher expression of LINC-RSAS was observed in primary spermatocytes (pachytene cells), Leydig cells and Sertoli cells of testes of adult rats. Over-expression of LINC-RSAS in cultured human cell lines revealed its possible role in cell-cycle control and apoptosis. We propose that LINC-RSAS expression is involved in molecular physiology of primary spermatocytes, Leydig cells and Sertoli cells of adult testes and its decline is associated with diminishing function of testes during ageing of the rat.

Multi-omics reveals the testosterone promotion effect mechanism of Cordyceps Sobolifera on Leydig cells

Ethnopharmacological relevanceCordyceps sobolifera (CS) has been traditionally utilized as an ethnic remedy for various health conditions, including chronic kidney diseases, anti-fatigue interventions, and management of chronic inflammation. Notably, CS is recognized for its substantial content of bioactive compounds, among which nucleosides prominently feature as constituents with diverse therapeutic advantages.Aim of the study: This study aims to investigate the effects of CS on testosterone secretion in Leydig cells and explore the underlying mechanism. Materials and methodsLeydig cells were isolated from rat testes to establish a primary rat Leydig cells model. Cell proliferation and testosterone secretion were assessed via the methyl-piperidino-pyrazole (MTT) assay and enzyme-linked immunosorbent assay (ELISA), respectively. Samples earmarked for RNA sequencing (RNA-Seq) analysis facilitated the identification of significantly differentially expressed genes (DEGs), and we conducted Gene Ontology (GO)/Kyoto Encyclopedia of Genes and Genomes (KEGG) functional annotation and enrichment analyses. The veracity of our findings was validated through quantitative real time polymerase chain reaction (qRT-PCR) and western blotting. ResultsThe results showed that CS and guanosine could promote Leydig cell proliferation and bolster testosterone secretion. Our integrative analysis of metabolomics and transcriptomics has unveiled the potential mechanisms governing testosterone synthesis. Specifically, metabolomics has illuminated striking correlations within cholesterol metabolism, and bile secretion. Concurrently, transcriptomics has underscored the pivotal roles played by the cyclic adenosine monophosphate (cAMP) signaling pathway and steroid hormone biosynthesis. Furthermore, our investigation has demonstrated CS's aptitude in elevating the expression of proteins and genes. Notably, our findings have elucidated that these effects can be mitigated by protein kinase A (PKA) and adenylate cyclase (AC) specific inhibitors. ConclusionThis study delineates the cAMP-PKA pathways as plausible mechanisms underpinning the testosterone-enhancing properties of CS, with guanosine emerging as a fundamental bioactive constituent.

Cholesterol Hydroperoxide Co-trafficking in Testosterone-generating Leydig Cells: GPx4 Inhibition of Cytotoxic and Anti-steroidogenic Effects

Trafficking of intracellular cholesterol (Ch) to and into mitochondria of steroidogenic cells is required for steroid hormone biosynthesis. This trafficking is typically mediated by one or more proteins of the steroidogenic acute regulatory (StAR) family. Our previous studies revealed that 7-OOH, a redox-active cholesterol hydroperoxide, could be co-trafficked with Ch to/into mitochondria of MA-10 Leydig cells, thereby inducing membrane lipid peroxidation (LPO) which impaired progesterone biosynthesis. These negative effects of 7-OOH were inhibited by endogenous selenoperoxidase GPx4, indicating that this enzyme could protect against 7-OOH-induced oxidative damage/dysfunction. In the present study, we advanced our Leydig focus to cultured murine TM3 cells and then to primary cells from rat testis, both of which produce testosterone. Using a fluorescent probe, we found that extensive free radical-mediated LPO occurred in mitochondria of stimulated primary Leydig cells during treatment with liposomal Ch+7-OOH, resulting in a significant decline in testosterone output relative to that with Ch alone. Strong enhancement of LPO and testosterone shortfall by RSL3 (a GPx4 inhibitor) and reversal thereof by Ebselen (a GPx4 mimetic), suggested that endogenous GPx4 was playing a key antioxidant role. 7-OOH in increasing doses was also cytotoxic to these cells, RSL3 exacerbating this in Ebselen-reversable fashion. Moreover, GPx4 knockdown increased cell sensitivity to LPO with reduced testosterone output. These findings, particularly with primary Leydigs (which best represent cells in intact testis) suggest that GPx4 plays a key protective role against peroxidative damage/dysfunction induced by 7-OOH co-trafficking with Ch.

Induction of apoptosis by cannabidiol and its main metabolites in human Leydig cells.

Cannabidiol (CBD) is one of the most prevalent and abundant cannabinoids extracted from the plant Cannabis sativa. CBD has been reported to induce male reproductive toxicity in animal models. In this study, we examined the effects of CBD and its main metabolites, 7-carboxy-CBD and 7-hydroxy-CBD, on primary human Leydig cells, which play a crucial role in male reproductive health. Our results showed that CBD, at concentrations below the Bayesian benchmark dose (BMD)50, inhibited the growth of human Leydig cells by arresting the cell cycle at G1/S transition, disrupting cell cycle regulators, and decreasing DNA synthesis. Concentration-response transcriptomic profiling identified that apoptosis was one of the top biological processes significantly affected by treatment with CBD for 24h. The occurrence of apoptosis was confirmed by increased activation of caspase-3/7 and an increased proportion of annexin V and propidium iodide (PI)-positive cells. Similar to CBD, both 7-carboxy-CBD and 7-hydroxy-CBD decreased cell viability and induced apoptosis after treatment for 24h. 7-Hydroxy-CBD and 7-carboxy-CBD showed lower cytotoxicity than CBD, and 7-carboxy-CBD had the lowest cytotoxicity among the three compounds. Our findings revealed that CBD and its main metabolites can cause adverse effects on primary human Leydig cells.

Bisphenol S stimulates Leydig cell proliferation but inhibits differentiation in pubertal male rats through multiple mechanisms.

Bisphenol S (BPS) is a novel bisphenol A (BPA) analogue, a ubiquitous environmental pollutant that disrupts male reproductive system. Whether BPS affects Leydig cell maturation in male puberty remains unclear. Male Sprague-Dawley rats (age of 35 days) were daily gavaged to 0, 1, 10, 100, and 200 mg/kg/day from postnatal days 35-56. BPS at 1-10 mg/kg/day and higher doses markedly reduced serum testosterone and progesterone levels but it at 200 mg/kg/day significantly increased estradiol level. BPS at 100 and 200 mg/kg/day significantly elevated serum luteinizing hormone (LH) levels. BPS at 1-10 mg/kg/day and higher doses significantly reduced inhibin A and inhibin B levels. BPS at 100 and 200 mg/kg/day markedly increased CYP11A1+ Leydig cell number, but did not affect HSD11B1+ (a mature Leydig cell marker) cell number. BPS at 10 mg/kg/day and higher doses significantly downregulated the expression of Cyp11a1 and at 100 and 200 mg/kg/d significantly lowered Cyp17a1, Hsd11b1, and Nr5a1 in the testes. BPS at 100 and/or 200 mg/kg/day significantly elevated Lhb in the pituitary. BPS at 100 and 200 mg/kg/day significantly increased the phosphorylation of AKT1, AKT2, and CREB without affecting total AKT1, AKT2, and CREB levels. BPS at 1-100 μM significantly suppressed testosterone production and induced proliferation of primary immature Leydig cells after 24 h of treatment and these actions were reversed by estrogen receptor α antagonist, ICI 182780, and partially reversed by vitamin E. BPS at 0.1-10 μM significantly increased oxidative stress of Leydig cells in vitro. BPS also directly inhibited 17β-hydroxysteroid dehydrogenase 3 activity at 10-100 μM. In conclusion, BPS causes hypergonadotropic androgen deficiency in male rats during pubertal exposure via activating ESR1 and inducing ROS in immature Leydig cells and directly inhibiting 17β-hydroxysteroid dehydrogenase 3 activity.

α-Linolenic acid-regulated testosterone biosynthesis via activation of the JNK-SF-1 signaling pathway in primary rooster Leydig cells

As a functional fatty acid, α-linolenic acid (ALA) is essential in promoting animal testosterone biosynthesis. This study investigated the effects of ALA on testosterone biosynthesis and the possible mechanism underlying the signaling pathway in primary Leydig cells of the rooster. Methods: Primary rooster Leydig cells were treated with ALA (0, 20, 40, or 80 μmol/L) or pretreated with a p38 inhibitor (50 μmol/L), a c-Jun NH2-terminal kinase (JNK) inhibitor (20 μmol/L), or an extracellular signal-regulated kinase (ERK) inhibitor (20 μmol/L) before ALA treatment. Testosterone content in the conditioned culture medium was detected using an enzyme-linked immunosorbent assay (ELISA). The expression of steroidogenic enzymes and JNK-SF-1 signaling pathway factors was detected using real-time fluorescence quantitative PCR (qRT-PCR). Results: Supplementation with ALA significantly increased testosterone secretion within culture media (P < 0.05), and the optimized dose was 40 μmol/L. Compared with the control group, steroidogenic acute regulatory protein (StAR), cholesterol side-chain cleavage enzyme (P450scc), and 3β-hydroxysteroid dehydrogenase (3β-HSD) mRNA expression significantly increased (P < 0.05) in the 40 μmol/L ALA group; 17-hydroxylase/c17-20 lyase (P450c17) and p38 mRNA expressions were not significantly different in the 40 μmol/L ALA group; ERK and JNK mRNA expressions were significantly upregulated (P < 0.05) in 40 μmol/L ALA group. In the inhibitor group, testosterone levels were significantly downregulated (P < 0.05). Compared with the 40 μmol/L ALA group, StAR, P450scc, and P450c17 mRNA expressions were significantly decreased (P < 0.05), and 3β-HSD mRNA expression in the p38 inhibitor group did not change; StAR, P450scc, and 3β-HSD mRNA expressions were significantly decreased (P < 0.05), and P450c17 mRNA expression in ERK inhibitor group did not change; StAR, P450scc, 3β-HSD, and P450c17 mRNA expressions were significantly decreased (P < 0.05) in JNK inhibitor group. Additionally, the increased steroidogenic factor 1 (SF-1) gene expression levels induced by ALA were reversed when the cells were pre-incubated with JNK and ERK inhibitors. The levels in the JNK inhibitor group were significantly lower than those in the control group (P < 0.05). Conclusion: ALA may promote testosterone biosynthesis by activating the JNK-SF-1 signaling pathway to upregulate StAR, P450scc, 3β-HSD, and P450c17 expression in primary rooster Leydig cells.

Primary culture and endocrine functional analysis of Leydig cells in ducks (Anas platyrhynchos).

Testicular Leydig cells (LCs) are the primary known source of testosterone, which is necessary for maintaining spermatogenesis and male fertility. However, the isolation, identification, and functional analysis of testosterone in duck LCs are still ambiguous. The aim of the present study was to establish a feasible method for isolating highly purified primary duck LCs. The highly purified primary duck LCs were isolated from the fresh testes of 2-month-old ducks via the digestion of collagenase IV and Percoll density gradient centrifugation; hematoxylin and eosin (H&E), immunohistochemistry (IHC) staining, ELISA, and radioimmunoassay were performed. Results revealed that the LCs were prominently noticeable in the testicular interstitium of 2-month-old ducks as compared to 6-month-old and 1-year-old ducks. Furthermore, IHC demonstrated that the cultured LCs occupied 90% area of the petri dish and highly expressed 3β-HSD 24h after culture (hac) as compared to 48 and 72 hac. Additionally, ELISA and radioimmunoassay indicate that the testosterone level in cellular supernatant was highly expressed in 24 and 48 hac, whereas the testosterone level gradually decreased in 72 and 96 hac, indicating the primary duck LCs secrete testosterone at an early stage. Based on the above results, the present study has effectively developed a technique for isolating highly purified primary duck LCs and identified its biological function in synthesizing testosterone.

Nesfatin-1 regulates steroidogenesis in mouse Leydig cells

Nesfatin-1 is a polypeptide hormone known to regulate appetite and energy metabolism and is derived from the precursor protein nucleobindin 2 (NUCB2). Recent studies have shown that nesfatin-1 is expressed in many peripheral tissues in mice, including the reproductive organs. However, its function and regulation in the testis remain unknown. In this study, we investigated the expression of Nucb2 mRNA and nesfatin-1 protein in mouse Leydig cells and the Leydig cell line, TM3 cells. We also examined whether Nucb2 mRNA expression is regulated by gonadotropins and whether exogenous nesfatin-1 affects steroidogenesis in primary Leydig cells isolated from the testis and TM3 cells. We found that Nucb2 mRNA and nesfatin-1 protein were present in primary Leydig cells and TM3 cells, and nesfatin-1 binding sites were also found in both cell types. Nucb2 mRNA expression in testis, primary Leydig cells, and TM3 cells was increased after treatment with pregnant mare’s serum gonadotropin and human chorionic gonadotropin. After nesfatin-1 treatment, the expression of steroidogenesis-related enzyme genes Cyp17a1 and Hsd3b was upregulated in primary Leydig cells and TM3 cells. Our results suggest that NUCB2/nesfatin-1 expression in mouse Leydig cells may be regulated through the hypothalamic-pituitary-gonadal axis and that nesfatin-1 produced by Leydig cells may locally regulate steroidogenesis in an autocrine manner. This study provides insight into the regulation of NUCB2/nesfatin-1 expression in Leydig cells and the effect of nesfatin-1 on steroidogenesis, which may have implications for male reproductive health.

Anti-apoptotic effect of adrenomedullin gene delivery on Leydig cells by suppressing TGF-β1 via the Hippo signaling pathway

This study aims to establish whether adrenomedullin (ADM) is capable to restore the steroidogenic functions of Leydig cells by suppressing transforming growth factor-β1 (TGF-β1) through Hippo signaling. Primary Leydig cells were treated with lipopolysaccharide (LPS), an adeno-associated virus vector that expressed ADM (Ad-ADM) or sh-RNA of TGF-β1 (Ad-sh-TGF-β1). The cell viability and medium concentrations of testosterone were detected. Gene expression and protein levels were determined for steroidogenic enzymes, TGF-β1, RhoA, YAP, TAZ and TEAD1. The role of Ad-ADM in the regulation of TGF-β1 promoter was confirmed by ChIP and Co-IP. Similar to Ad-sh-TGF-β1, Ad-ADM mitigated the decline in the number of Leydig cells and plasma concentrations of testosterone by restoring the gene and protein levels of SF-1, LRH1, NUR77, StAR, P450scc, 3β-HSD, CYP17 and 17β-HSD. Similar to Ad-sh-TGF-β1, Ad-ADM not only inhibited the LPS-induced cytotoxicity and cell apoptosis but also restored the gene and protein levels of SF-1, LRH1, NUR77, StAR, P450scc, 3β-HSD, CYP17 and 17β-HSD, along with the medium concentrations of testosterone in LPS-induced Leydig cells. Like Ad-sh-TGF-β1, Ad-ADM improved LPS-induced TGF-β1 expression. In addition, Ad-ADM suppressed RhoA activation, enhanced the phosphorylation of YAP and TAZ, reduced the expression of TEAD1 which interacted with HDAC5 and then bound to TGF-β1 gene promoter in LPS-exposed Leydig cells. It is thus suspected that ADM can exert anti-apoptotic effect to restore the steroidogenic functions of Leydig cells by suppressing TGF-β1 through Hippo signaling.

Novel treatment and insight for irradiation-induced injuries: Dibucaine ameliorates irradiation-induced testicular injury by inhibiting fatty acid oxidation in primary Leydig cells

BackgroundMale infertility is a worldwide problem but few treatments, especially irradiation-induced testicular injury. The aim of this research was to investigate novel drugs for the treatment of irradiation-induced testicular injury. MethodsWe administered dibucaine (0.8 mg/kg) intraperitoneally to male mice (6 mice per group) after five consecutive daily 0.5 Gy whole-body irradiation, and evaluated its ameliorating efficacy by testicular HE staining and morphological measurements. Drug affinity responsive target stability assay (Darts) were used to find target protein and pathway; mouse primary Leydig cells were isolated and to explore the mechanism (Flow cytometry, Western blot, and Seahorse palmitate oxidative stress assays); finally rescue experiments were completed by combining dibucaine with fatty acid oxidative pathway inhibitors and activators. ResultsThe testicular HE staining and morphological measurements in dibucaine treatment group was significantly better than that in irradiation group (P < 0.05); sperm motility and mRNA levels of spermatogenic cell markers were also higher than those in the latter (P < 0.05). Darts and Western blot results showed that dibucaine targets CPT1A and downregulate fatty acid oxidation. Flow cytometry, Western blot, and Palmitate oxidative stress assays of primary Leydig cells demonstrated that dibucaine inhibits fatty acid oxidation in Leydig cells. Dibucaine combined with etomoxir/baicalin confirmed that its inhibition of fatty acid oxidation was beneficial in ameliorating irradiation-induced testicular injury. ConclusionsIn conclusion, our data suggest that dibucaine ameliorates irradiation-induced testicular injury in mice by inhibiting fatty acid oxidation in Leydig cells. This will provide novel ideas for the treatment of irradiation-induced testicular injury.

Selenium nanoparticles improve nickel-induced testosterone synthesis disturbance by down-regulating miR-708-5p/p38 MAPK pathway in Leydig cells.

The present study was designed to investigate the role of miR-708-5p/p38 mitogen-activated protein kinase (MAPK) pathway during the mechanism of selenium nanoparticles (Nano-Se) against nickel (Ni)-induced testosterone synthesis disorder in rat Leydig cells. We conducted all procedures based on in vitro culture of rat primary Leydig cells. After treating Leydig cells with Nano-Se and NiSO4 alone or in combination for 24 h, we determined the cell viability, reactive oxygen species (ROS) levels, testosterone production, and the protein expression of key enzymes involved in testosterone biosynthesis: steroidogenic acute regulatory (StAR) and cytochrome P450 cholesterol side chain cleavage enzyme (CYP11A1). The results indicated that Nano-Se antagonized cytotoxicity and eliminated ROS generation induced by NiSO4 , suppressed p38 MAPK protein phosphorylation and reduced miR-708-5p expression. Importantly, we found that Nano-Se upregulated the expression of testosterone synthase and increased testosterone production in Leydig cells. Furthermore, we investigated the effects of p38 MAPK and miR-708-5p using their specific inhibitor during Nano-Se against Ni-induced testosterone synthesis disorder. The results showed that Ni-inhibited testosterone secretion was alleviated by Nano-Se co-treatment with p38 MAPK specific inhibitor SB203580 and miR-708-5p inhibitor, respectively. In conclusion, these findings suggested Nano-Se could inhibit miR-708-5p/p38 MAPK pathway, and up-regulate the key enzymes protein expression for testosterone synthesis, thereby antagonizing Ni-induced disorder of testosterone synthesis in Leydig cells.

Molecular insights underlying the adverse effects of bisphenol A on gonadal somatic cells' steroidogenic activity

Bisphenol A (BPA) exposure may impair gonadal steroidogenesis, although the underlying mechanism is not well known. Hereby, we assessed BPA action on human primary granulosa (hGC) and mouse Leydig cells (BLTK-1) proliferation, cytotoxicity, hormone secretion, and steroidogenic enzyme/receptor gene profile. hGC and BLTK-1 cells were stimulated with increasing concentrations of BPA (10-12 M to 10-4 M for cell proliferation assay, 10-8 M to 10-4 M for LDH-cytotoxicity assay, and 10-9 M to 10-5 M for hormone secretion and genes expression analysis). BPA at low concentrations (pM – nM) did not affect cell proliferation in either cell type, although was toxic at higher (µM) concentrations. BPA stimulation at low nM concentrations decreased the production of estradiol (E2) and testosterone (T) in BLTK-1, E2, and progesterone in hGCs. BPA down-regulated Star, Cyp11a1, and Hsd17b3, but up-regulated Cyp19a1, Esr1, Esr2, and Gpr30 expression in BLTK-1 cells. In hGC, BPA down-regulated STAR, CYP19A1, PGRMC1, and PAQR7 but up-regulated ESR2 expression. Estrogen receptor degrader fulvestrant (FULV) attenuated BPA inhibition of hormone production in both cell lines. FULV also blocked the BPA-induced Gpr30 up-regulation in BLTK-1 cells, whereas in hGC, failed to reverse the down-regulation of PGRMC1, STAR, and CYP19A1. Our findings provide novel mechanistic insights into environmentally-relevant doses of BPA action through both nuclear estrogen receptor-dependent and independent mechanisms affecting cultured granulosa and Leydig cell steroidogenesis.

Ciliary neurotrophic factor stimulates stem/progenitor Leydig cell proliferation but inhibits differentiation into its lineage in rats.

Ciliary neurotrophic factor (CNTF) is a member of the interleukin-6 family of cytokines. CNTF drives many cells for their development. However, its effects on Leydig cells (LCs) development remains unclear. In the current study, we used 3D seminiferous tubule culture system to induce the proliferation and differentiation of tubule-associated stem Leydig cells (SLCs) and primary progenitor Leydig cells (PLCs) culture to address the effects of CNTF. We found that CNTF stimulated the proliferation of SLCs but inhibited its development to the LCs lineage. The CNTF-mediated effects can be reversed by STAT3 inhibitor S3I-201 and PI3K inhibitor wortmannin, indicating that CNTF acts via STAT3-PI3K signaling pathways to increase stem/progenitor Leydig cell proliferation. CNTF at 1 and 10 ng/mL significantly decreased androgen production by PLCs. Microarray analysis for CNTF-treated PLCs showed that CNTF blocked steroidogenic pathways through downregulating Scarb1, Star and Hsd3b1 possibly through the downregulating the transcription factor Nr5a1 expression. CNTF stimulates the proliferation but blocks the differentiation of stem/progenitor Leydig cells. This article is protected by copyright. All rights reserved.

Grim-19 plays a key role in mitochondrial steroidogenic acute regulatory protein stability and ligand-binding properties in Leydig cells

Grim-19 (gene associated with retinoid-IFN-induced mortality 19), the essential component of complex I of mitochondrial respiratory chain, functions as a noncanonical tumor suppressor by controlling apoptosis and energy metabolism. However, additional biological actions of Grim-19 have been recently suggested in male reproduction. We investigated here the expression and functional role of Grim-19 in murine testis. Testicular Grim-19 expression was detected from mouse puberty and increased progressively thereafter, and GRIM-19 protein was observed to be expressed exclusively in interstitial Leydig cells (LCs), with a prominent mitochondrial localization. Invivo lentiviral vector-mediated knockdown of Grim-19 resulted in a significant decrease in testosterone production and triggered aberrant oxidative stress in testis, thus impairing male fertility by inducing germ cell apoptosis and oligozoospermia. The control of testicular steroidogenesis by GRIM-19 was validated using the invivo knockdown model with isolated primary LCs and invitro experiments with MA-10 mouse Leydig tumor cells. Mechanistically, we suggest that the negative regulation exerted by GRIM-19 deficiency-induced oxidative stress on steroidogenesis may be the result of two phenomena: a direct effect through inhibition of phosphorylation of steroidogenic acute regulatory protein (StAR) and subsequent impediment to StAR localization in mitochondria and an indirect pathway that is to facilitate the inhibiting role exerted by the extracellular matrix on the steroidogenic capacity of LCs via promotion of integrin activation. Altogether, our observations suggest that Grim-19 plays a potent role in testicular steroidogenesis and that its alterations may contribute to testosterone deficiency-related disorders linked to metabolic stress and male infertility.