Ameliorative effects of elderberry (Sambucus nigra L.) extract and extract-derived monosaccharide-amino acid on H2O2-induced decrease in testosterone-deficiency syndrome in a TM3 Leydig cell.

With aging, men develop testosterone-deficiency syndrome (TDS). The development is closely associated with age-related mitochondrial dysfunction of Leydig cell and oxidative stress-induced reactive oxygen species (ROS). Testosterone-replacement therapy (TRT) is used to improve the symptoms of TDS. However, due to its various side effects, research on functional ingredients derived from natural products that do not have side effects is urgently needed. In this study, using the mitochondrial dysfunction TM3 (mouse Leydig) cells, in which testosterone biosynthesis is reduced by H2O2, we evaluated the effects of elderberry extract and monosaccharide-amino acid (fructose-leucine; FL) on mRNA and protein levels related to steroidogenesis-related enzymes steroidogenic acute regulatory protein (StAR), cytochrome P450 11A1(CYP11A1, cytochrome P450 17A1(CYP17A1), cytochrome P450 19A1(CYP19A1, aromatase), 3ß-hydroxysteroid dehydrogenase (3ß-HSD), and 17ß-hydroxysteroid dehydrogenase(17ß-HSD). We analyzed elderberry extract and extract-derived FL for changes in ROS scavenging activity and testosterone secretion. Elderberry extract and FL significantly reduced H2O2-induced intracellular ROS levels, improved testosterone secretion, and increased the mRNA and protein expression levels of steroidogenesis-related enzymes (StAR, 3b-HSD, 17b-HSD, CYP11A1, CYp17A1). However, the conversion of testosterone to estradiol was inhibited by elderberry extract and extract-derived FL, which reduced the mRNA and protein expression of CYP19A1. In conclusion, elderberry extract and FL are predicted to have value as novel functional ingredients that may contribute to the prevention of TDS by ameliorating reduced steroidogenesis.

Antagonistic effect of selenium on programmed necrosis of testicular Leydig cells caused by cadmium through endoplasmic reticulum stress in chicken.

Cadmium (Cd) is a widely distributed environmental contaminant that is highly toxic to animals and humans. However, detailed reports on Cd-induced programmed necrosis have not been seen in chicken testicular Leydig cells. Selenium (Se) is a trace element in the human body that has cytoprotective effects in a variety of pathological damages caused by heavy metals. This study investigated the potential mechanisms of Cd-induced programmed cell necrosis and the antagonistic effect of Se on Cd toxicity. Chicken testis Leydig cells were divided into six groups, namely, control, Se (5 µmol/L Na2SeO3), Cd (20 µmol/L CdCl2), Se + Cd (5 µmol/L Na2SeO3 and 20 µmol/L CdCl2), 4-phenylbutyric acid (4-PBA) + Cd (10 mmol/L 4-phenylbutyric acid and 20 µmol/L CdCl2), and Necrostatin-1 (Nec-1) + Cd (60 µmol/L Necrostatin-1 and 20 µmol/L CdCl2). The results showed that Cd exposure decreased the activity of CAT, GSH-Px, and SOD and the concentration of GSH, and increased the concentration of MDA and the content of ROS. Relative mRNA and protein expression of GRP78, PERK, ATF6, IRE1, CHOP, and JNK increased in the Cd group, and mRNA and protein expression of TNF-α, TNFR1, RIP1, RIP3, MLKL, and PARP1 significantly increased in the Cd group, while Caspase-8 mRNA and protein expression significantly decreased. The abnormal expression of endoplasmic reticulum stress-related proteins was significantly reduced by 4-PBA pretreatment; the increased expression of TNF-α, TNFR1, RIP1, RIP3, MLKL, and PARP1 caused by Cd toxicity was alleviated; and the expression of caspase-8 was upregulated. Conversely, the increased mRNA and protein expression of endoplasmic reticulum stress marker genes (GRP78, ATF6, PERK, IRE1, CHOP, JNK) caused by Cd was not affected after pretreatment with Nec-1. We also found that these Cd-induced changes were significantly attenuated in the Se + Cd group. We clarified that Cd can cause programmed necrosis of chicken testicular Leydig cells through endoplasmic reticulum stress, and Se can antagonize Cd-induced programmed necrosis of chicken testicular Leydig cells.

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 3ß-hydroxysteroid dehydrogenase (3ß-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 3ß-HSD mRNA expression in the p38 inhibitor group did not change; StAR, P450scc, and 3ß-HSD mRNA expressions were significantly decreased (P < 0.05), and P450c17 mRNA expression in ERK inhibitor group did not change; StAR, P450scc, 3ß-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, 3ß-HSD, and P450c17 expression in primary rooster Leydig cells.

Status of estrogen receptor expression and epigenetic methylation in Leydig cells after exposure to metalloestrogen - selenium.

The trace element selenium (Se) is essential for the maintenance of spermatogenesis and fertility. A growing volume of evidence shows that Se is necessary for testosterone synthesis, and Se can stimulate Leydig cell proliferation. However, Se can also act as a metalloestrogen, which can mimic estrogen and activate the estrogen receptors. This study aimed to investigate Se effect on estrogen signaling and the epigenetic status of Leydig cells. Mouse Leydig cells (MA-10) were cultured in a medium supplemented with different Se concentrations (4, 8 µM) for 24 h. Next, cells were assessed for morphological and molecular (qRT PCR, western blot, immunofluorescence) analyses. Immunofluorescence revealed strong immunosignal for 5-methylcytosine in both control and treated cells, with a stronger signal in the 8 µM treated group. qRT-PCR confirmed an increased expression of methyltransferase 3 beta (Dnmt3b) in 8 µM cells. Analysis of the expression of γH2AX (a marker for double-stranded DNA breaks) revealed an increase in the DNA breaks in cells exposed to 8 µM Se. Selenium exposure did not affect the expression of canonical estrogen receptors (ERα and ERß), however, an increase in membrane estrogen receptor G-protein coupled (GPER) protein expression was observed.To sum up, in a high concentration (8 µM) Se affects GPER expression (non-genomic estrogen signaling) in Leydig cells possibly via acting on receptor protein and/or its binding. This causes DNA breaks and induces changes in Leydig cell methylation status, especially in de novo methylation which is mediated by Dnmt3b.

Nano-selenium Alleviates Cadmium-Induced Mouse Leydig Cell Injury, via the Inhibition of Reactive Oxygen Species and the Restoration of Autophagic Flux.

Cadmium (Cd) is a well-known environmental pollutant that can contribute to male reproductive toxicity through oxidative stress. Nano-selenium (Nano-se) is an active single body of selenium with strong antioxidant properties and low toxicity. Some studies have addressed the potential ameliorative effect of Nano-se against Cd-induced testicular toxicity; however, the underlying mechanisms remain to be investigated. This study aimed to explore the protective effect of Nano-se on Cd-induced mouse testicular TM3 cell toxicity by regulating autophagy process. We showed that cadmium exposure to TM3 cells inhibited cell viability and elevated the level of reactive oxygen species (ROS) generation. Morphology observation by transmission electron microscope and the presence of mRFP-GFP-LC3 fluorescence puncta demonstrated that cadmium increased autophagosome formation and accumulation in TM3 cells, resulting in blocking the autophagic flux of TM3 cells. Meanwhile, cadmium remarkably increased the ratio of LC3-II to LC3-I protein expression (2.07 ± 0.31) and the Beclin-1 protein expression (1.97 ± 0.40) in TM3 cells (P < 0.01). Pretreatment with Nano-se significantly reduced Cd-induced TM3 cell toxicity (P < 0.01). Furthermore, Nano-se treatment reversed Cd-induced ROS production and autophagosome accumulation, and autophagy as evidenced by the ratio of LC3-II to LC3-I and Beclin-1 expression. In addition, ROS scavenger, N-acetyl-L-cysteine (NAC) or autophagy inhibitor, 3-methyladenine (3-MA) reversed cadmium-induced ROS generation, autophagosome accumulation, and autophagy-related protein expression levels, which confirmed that cadmium induced TM3 cell injury via ROS signal pathway and blockage of autophagic flux. Collectively, our results reveal that Nano-se attenuates Cd-induced TM3 cell toxicity through the inhibition of ROS production and the amelioration of autophagy disruption.

Omega-6 highly unsaturated fatty acids in Leydig cells facilitate male sex hormone production.

Highly unsaturated fatty acids (HUFAs) are fatty acids with more than three double bonds in the molecule. Mammalian testes contain very high levels of omega-6 HUFAs compared with other tissues. However, the metabolic and biological significance of these HUFAs in the mammalian testis is poorly understood. Here we show that Leydig cells vigorously synthesize omega-6 HUFAs to facilitate male sex hormone production. In the testis, FADS2 (Fatty acid desaturase 2), the rate-limiting enzyme for HUFA biosynthesis, is highly expressed in Leydig cells. In this study, pharmacological and genetic inhibition of FADS2 drastically reduces the production of omega-6 HUFAs and male steroid hormones in Leydig cells; this reduction is significantly rescued by supplementation with omega-6 HUFAs. Mechanistically, hormone-sensitive lipase (HSL; also called LIPE), a lipase that supplies free cholesterol for steroid hormone production, preferentially hydrolyzes HUFA-containing cholesteryl esters as substrates. Taken together, our results demonstrate that Leydig cells highly express FADS2 to facilitate male steroid hormone production by accumulating omega-6 HUFA-containing cholesteryl esters, which serve as preferred substrates for HSL. These findings unveil a previously unrecognized importance of omega-6 HUFAs in the mammalian male reproductive system.

Exceptional Properties of Lepidium sativum L. Extract and Its Impact on Cell Viability, Ros Production, Steroidogenesis, and Intracellular Communication in Mice Leydig Cells In Vitro.

The prevalence of reproductive dysfunction in males has risen in the last few years, and alternative therapies are gradually gaining in popularity. Our in vitro study aimed to evaluate the potential impact of Lepidium sativum L. on mice TM3 Leydig cells, concerning basal parameters such as cell viability, cell membrane integrity, and lysosomal activity, after 24 h and 48 h exposure. Moreover, reactive oxygens species generation, sex-steroid hormone secretion, and intercellular communication were quantified. In the present study, the microgreen extract from Lepidium was rich in ferulic acid, 4-OH benzoic acid, and resveratrol, with a significant antioxidant activity. The results showed that lower experimental doses (62.5-250 µg/mL) could positively affect the observed parameters, with significant differences at 250 µg/mL after 24 h and 48 h, respectively. Potential risks could be associated with higher concentrations, starting at 500 µg/mL, 1000 µg/mL, and 2000 µg/mL of Lepidium. Nevertheless, biochemical quantification indicated a significant antioxidant potential and a rich content of biologically active molecules at the applied doses, and time determined the intracellular response of the cultured model.

Vitamin E and selenium partially prevent cytotoxicity, oxidative stress and DNA damage induced by T-2 toxin in bovine Leydig cells.

The objective of this study was to explore the protective mechanism of Vitamin E (VE) and selenium (Se) against T-2 toxin-induced oxidative damage of bovine Leydig cells. Leydig cells were isolated, cultured and divided into five treatment groups such as: control, T-2, Se + T-2, VE + T-2 and VE + Se + T-2. After treatment for 24 h, the cells and supernatants were harvested to examine the cell viability, the activities and mRNA expression of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and catalase (CAT), the content of malondialdehyde (MDA) and DNA damage. Results showed that T-2 toxin exposure significantly reduced the cell viability, increased the MDA level, reduced GSH-Px, SOD and CAT activities and increased DNA damage (P < 0.05). Meanwhile, T-2 toxin was attributed to the down-regulation of the mRNA expression of GSH-Px, SOD and CAT (P < 0.05). However, VE and Se reduced T-2 toxin-induced oxidative damage and tended to maintain normal levels (P < 0.05). Furthermore, VE and Se substantially up-regulated the activities and mRNA expressions of the GSH-Px, SOD and CAT. In conclusion, VE and Se, due to its anti-oxidative ability, could ameliorate T-2 toxin-induced cytotoxicities by regulating oxidative stress in bovine Leydig cells.

Antagonistic effects of selenium on lead-induced oxidative stress and apoptosis of Leydig cells in sheep.

The objective of this study was to investigate the antagonistic effects of selenium (Se) on lead (Pb)-induced oxidative stress and apoptosis of sheep Leydig cells and its underlying mechanism. Leydig cells collected from 8-month-old sheep were treated with Pb (40 µmol/L) and/or Se (2 µmol/L), respectively. CCK-8 assay was used to detect cell proliferation and apoptosis after cultured for 48 h. The abundances of pro-apoptosis (BAX, CASPASE 3 and CASPASE 8) and NRF2-related (NRF2, HO-1, NQO1 and γ-GCS) genes were detected by real-time PCR and western blot analysis, respectively. The results showed that the highest cell viability was observed in the Se group. Compared with the control group, Pb treatment led to the higher ROS level and greater abundances of BAX, CASPASE 3 and CASPASE 8 mRNA transcripts. Treatment with Pb + Se resulted in an increased (P < 0.05) abundances of NRF2, HO-1, NQO1 and γ-GCS mRNA transcripts and proteins. Compared with the Pb group, the Se + Pb treatment dramatically decreased (P < 0.05) the ROS level and relative abundances of pro-apoptosis genes. The greater (P < 0.05) abundances of pro-apoptosis and NRF2-related mRNA transcripts and proteins were also obtained in the Se + Pb group. The abundances of BAX, CASPASE 3 and CASPASE 8 genes in the SeML385 group were greater (P < 0.05) than in the Se group. Compared with the corresponding groups without ML385, treatment with ML385 decreased (P < 0.05) cell viability and the relative abundances of pro-apoptosis and NRF2-related genes. These results indicate that Pb-induced oxidative stress can inhibit the viability of Leydig cells by modulating pro-apoptosis gene expression. NRF2 pathway could be involved in the antagonistic effect of Se on Pb-induced apoptosis of Leydig cells in sheep. This study is expected to provide some experimental evidences for Se treatment of Pb-induced reproductive disorders.

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.

The effect of Apium graveolens L., Levisticum officinale and Calendula officinalis L. on cell viability, membrane integrity, steroidogenesis, and intercellular communication in mice Leydig cells in vitro.

Several plants have the potential to protect essential reproductive processes such as spermatogenesis or steroidogenesis, however, effective concentrations and main mechanisms of action are still unknown. This in vitro study was aimed to assess the effects of Apium graveolens L., Levisticum officinale, and Calendula officinalis L. extracts on the structural integrity, functional activity and gap junctional intercellular communication (GJIC) in mice Leydig cells. TM3 cells were grown in the presence of experimental extracts (37.5; 75; 150 and 300 µg/ml) for 24 h. For the present study, high-performance liquid chromatography analysis was used to quantify flavonoids or phenolic acids. Subsequently, Leydig cell viability was assessed by alamarBlue assay, while the cell membrane integrity was detected by 5-carboxyfluorescein diacetate-acetoxymethyl ester. The level of steroid hormones production was determined by enzyme-linked immunosorbent assay. Additionally, GJIC was assessed by scalpel loading/dye transfer assay. According to our results, Apium graveolens L. significantly increased the viability and cell membrane integrity at 75 µg/ml (109.0±4.3%) followed by a decline at 300 µg/ml (89.4±2.3%). In case of Levisticum officinale and Calendula officinalis L. was observed significant decrease at 150 µg/ml (88.8±11.66%; 87.4±6.0%) and 300 µg/ml (86.2±9.3%; 84.1±4.6%). Furthermore, Apium graveolens L. significantly increased the progesterone and testosterone production (75 and 150 µg/ml) however, Levisticum officinale and Calendula officinalis L. significantly reduced steroid hormones synthesis at 150 and 300 µg/ml. Finally, the disturbance of GJIC was significantly affected at 300 µg/ml of Levisticum officinale (82.5±7.7%) and Calendula officinalis L. (79.8±7.0%). The balanced concentration ratio may support the Leydig cell function, steroidogenesis as well as all essential parameters that may significantly improve reproductive functions.

S-allyl Cysteine Enhances Testosterone Production in Mice and Mouse Testis-Derived I-10 Cells.

Hypogonadism, associated with low levels of testosterone synthesis, has been implicated in several diseases. Recently, the quest for natural alternatives to prevent and treat hypogonadism has gained increasing research interest. To this end, the present study explored the effect of S-allyl cysteine (SAC), a characteristic organosulfur compound in aged-garlic extract, on testosterone production. SAC was administered at 50 mg/kg body weight intraperitoneally into 7-week-old BALB/c male mice in a single-dose experiment. Plasma levels of testosterone and luteinizing hormone (LH) and testis levels of proteins involved in steroidogenesis were measured by enzymatic immunoassay and Western blot, respectively. In addition, mouse testis-derived I-10 cells were also used to investigate the effect of SAC on steroidogenesis. In the animal experiment, SAC significantly elevated testosterone levels in both the plasma and the testis without changing the LH level in plasma and increased phosphorylated protein kinase A (p-PKA) levels. Similar results were also observed in I-10 cells. The findings demonstrating the increasing effect of SAC on p-PKA and mRNA levels of Cyp11a suggest that SAC increases the testosterone level by activating the PKA pathway and could be a potential target for hypogonadism therapeutics.

A potential mechanism associated with lead-induced spermatogonia and Leydig cell toxicity and mitigative effect of selenium in chicken.

Lead (Pb) is a toxic heavy metal pollutants and can damage male reproductive function. Selenium (Se) possesses an ability of antagonizing Pb toxicity. However, biological events in the process of Pb toxicity and mitigative effect of Se are not well understood. The aim of present research was to investigate potential mechanism of Se against Pb toxicity from the perspective of oxidative stress, heat shock response and autophagy in the spermatogonia and Leydig cell of chicken. The cells from one-day-old male Hyline chickens were treated with Se (0.5 µmol/L) and/or Pb (20 µmol/L) for 24 h, respectively. Cell viability, cell ultrastucture, Pb and Se concentrations, testosterone level, oxidative stress indicators and relative expression of heat shock proteins (HSPs) and autophagy-related genes were measured. The results showed that spermatogonia was more tolerant to Pb than Leydig cell; cell injury was confirmed via histological assessment, cell viability and testosterone level; oxidative stress was further indicated by the decrease of catalase, glutathione peroxidase, glutathione-s-transferase and superoxide dismutase activities and the increase of malondialdehyde and reactive oxygen species contents. Pb increased expression of HSPs (27, 40, 60, 70 and 90). Meanwhile Pb induced autophagy through up-regulation of autophagy-related proteins 5, Beclin 1, Dynein, light chain 3 (LC3)-I and LC3-II and down-regulation of mammalian target of rapamycin in two type cells of chicken. However, Se intervention mitigated the aforementioned alterations caused by Pb. In conclusion, Pb led to oxidative stress, which triggered heat shock response and autophagy; Se administration mitigated reproductive toxicity of Pb through strengthening antioxidant defense in the spermatogonia and Leydig cell of chicken.

[Xiongcan Yishen Prescription up-regulates the expressions of cholesterol transport proteins, steroidogenic enzymes and SF-1 in the Leydig cells of rats with late-onset hypogonadism].

OBJECTIVE: To investigate the effects of Xiongcan Yishen Prescription (XYP) on the expressions of cholesterol transport proteins, steroidogenic enzymes and steroidogenic factor-1 (SF-1) in the Leydig cells of the rats with late-onset hypogonadism (LOH). METHODS: Twenty-five 18-month-old male SD rats were randomly divided into five groups of equal number, LOH model control, testosterone propionate (TP) and low-, medium- and high-dose XYP, and another 5 two-month-old male SD rats included as normal controls. After modeling, the animals in the TP group were treated by intramuscular injection of TP at 5.21 mg/kg qd alt, those in the low-, medium- and high-dose XYP groups intragastrically with XYP at 10.4, 20.8 and 41.6 g/kg qd alt respectively, and those in the LOH model and normal control groups with saline, all for 28 successive days. Then, all the rats were sacrificed for determination of the expressions of the cholesterol transport proteins StAR and TSPO, steroidogenic enzymes CYP11A1, HSD3B7 and HSD17B4, and SF-1 in the Leydig cells by Western blot. RESULTS: The expressions of StAR, TSPO, CYP11A1, HSD3B7, HSD17B4 and SF-1 in the Leydig cells were significantly decreased in the LOH model controls compared with those in the normal controls (P< 0.05), but remarkably increased in the low-, medium- and high-dose XYP groups in comparison with those in the LOH model control group (P< 0.05). CONCLUSIONS: Xiongcan Yishen Prescription can up-regulate the expressions of the cholesterol transport proteins StAR and TSPO, steroidogenic enzymes CYP11A1, HSD3B7 and HSD17B4, and SF-1 in the rat Leydig cells, which might be one of the possible mechanisms of the prescription in the treatment of LOH.

Steroidal Glycosides from Allium tuberosum Seeds and Their Roles in Promoting Testosterone Production of Rat Leydig Cells.

A systematic phytochemical study on the components in the seeds of Allium tuberosum was performed, leading to the isolation of 27 steroidal glycosides (SGs 1-27). The structures of SGs were identified mainly by nuclear magnetic resonance and mass spectrometries as well as the necessary chemical evidence. In the SGs, 1-10 and 22-26 are new steroidal saponin analogues. An in vitro bioassay indicates that 1, 2, 7, 8, 10, 13-15, 20, 23, and 26 display promotional roles in testosterone production of rat Leydig cells with the EC50 values of 1.0 to 4.5 µM, respectively.

Differentially expressed genes on the growth of mouse Leydig cells treated with standardised Eurycoma longifolia extract.

Eurycoma (E.) longifolia Jack (Tongkat Ali) is a widely applied medicine that has been reported to boost serum testosterone and increase muscle mass. However, its actual biological targets and effects on an in vitro level remain poorly understood. Therefore, the present study aimed to investigate the effects of a standardised E. longifolia extract (F2) on the growth and its associated gene expression profile in mouse Leydig cells. F2, even at lower doses, was found to induce a high level of testosterone by ELISA. The level was as high as the levels induced by eurycomanone and formestane in Leydig cells. However, Leydig cells treated with F2 demonstrated reduced viability, which was likely due to the diminished cell population at the G0/G1 phase and increased cell population arrested at the S phase in the cell cycle, as assessed by MTT assay and flow cytometry, respectively. Cell viability was revived when the treatment time­point was prolonged to 96 h. Genome­wide gene analysis by reverse transcription­quantitative PCR of F2­treated Leydig cells at 72 h, when the cell growth was not revived, and 96 h, when the cell growth had started to revive, revealed cyclin­dependent kinase­like 2 (CDKL2) to be a potential target in regulating the viability of F2­treated Leydig cells. Functional analysis, as analysed using GeneMANIA Cytoscape program v.3.6.0 (https://genemania.org/), further suggested that CDKL2 could act in concert with Casitas B­lineage lymphoma and sphingosine kinase 1 interactor­A­kinase anchoring protein domain­containing genes to regulate the viability of F2­treated Leydig cells. The findings of the present study provide new insights regarding the potential molecular targets associated with the biological effect of E. longifolia extract on cell growth, particularly on the cell cycle, which could aid in enhancing the bioefficacy and reducing the toxicity of this natural product in the future.

Antioxidant, anti-inflammatory and anti-apoptotic effects of hydro-ethanolic extract of Cyperus esculentus L. (tigernut) on lead acetate-induced testicular dysfunction in Wistar rats.

AIM: Lead acetate impairs testicular function by enhancing testicular oxidative stress and apoptosis. Cyperus esculentus possesses antioxidants and has shown great improvement of testicular function. This study investigated the protective effect of hydro-ethanolic extract of Cyperus esculentus on lead acetate-induced testicular dysfunction in Wistar rats. MATERIALS AND METHODS: Twenty-five male Wistar rats (180-195 g) were randomly divided into 5 groups (n = 5) namely: Normal control (NC), Lead control (PbC) (20 mg/kg b.w. i.p.), C. esculentus-treated (CE) (500 mg/kg b.w p.o.), Pb + CE(500) (20 mg/kg of lead and 500 mg/kg of extract) and Pb + CE(1000) (20 mg/kg of lead and 1000 mg/kg of extract). Administration lasted for 21 days. RESULTS: Sperm count, motility, viability, serum testosterone and follicle stimulating hormone, Johnsen's score, Leydig cell count, Sertoli cell count, testicular testosterone, B-cell lymphoma protein-2 (Bcl-2), steroidogenic acute regulatory protein, cytochrome P450 A1, 3ß-hydroxysteroid dehydrogenase (HSD), 17ß-HSD, enzymatic antioxidant activities and total antioxidant capacity were significantly (p < 0.05) decreased in PbC compared with NC. These parameters however increased significantly (p < 0.05) in Pb + CE(500) and Pb + CE(1000) compared with PbC. Lead acetate upregulated (p < 0.05) testicular malondialdehyde, nitric oxide, glucose, lactate, lactate dehydrogenase, C-reactive protein, tumor necrosis factor-α, interleukin (IL)-6, IL-1ß, Bcl-2 associated X (Bax), Bax/Bcl-2 and cleaved caspase-3 levels. All these parameters were downregulated (p < 0.05) in Pb + CE(500) and Pb + CE(1000) in comparison with PbC. CONCLUSION: C. esculentus exhibited a dose-dependent mitigation of lead acetate-induced testicular dysfunction in Wistar rats via its antioxidant, anti-inflammatory and anti-apoptotic effects.

Pfaffia glomerata hydroalcoholic extract stimulates penile tissue in adult Swiss mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Roots of Pfaffia glomerata are used in traditional medicine as aphrodisiacs and sexual stimulants. AIM OF THE STUDY: The aim of this study was to evaluate the action of the hydroalcoholic extract from the roots of Pfaffia glomerata on the Leydig cells, cavernous bodies and other penile constituents, as well as on serum testosterone and 17ß-estradiol levels of adult mice. MATERIALS AND METHODS: Mature male Swiss mice were divided into 6 groups: control (water), sildenafil citrate, 3 groups receiving daily doses of P. glomerata extract (100, 200 and 400 mg/kg) and one group receiving intermittent doses of P. glomerata (200 mg/kg/3-3d). RESULTS: The proportions of blood vessels, lymphatic space and estradiol levels were increased. On the other hand, reduction of testosterone levels due to Leydig cells death was observed. As for penile parameters, volumetric proportions of cavernous bodies, collagen and nitric oxide were increased, while smooth muscle content was decreased. CONCLUSIONS: Despite that the long term intake of P. glomerata extract was related to a stimulant action, reduction on Leydig cell viability induced decreased testosterone production.

Investigation of the Properties and Effects of Salvia Officinalis L. on the Viability, Steroidogenesis and Reactive Oxygen Species (ROS) Production in TM3 Leydig Cells in Vitro.

The aim of our study was to reveal the in vitro effects of Salvia officinalis L. (37.5, 75, 150, 200, 250, 300 and 600 µg/ml) extract on the TM3 Leydig cell viability, membrane integrity, steroidogenesis and reactive oxygen species production after 24 h and 48 h cultivation. For the present study, the extract prepared from Salvia officinalis L. leaves was analysed by high performance liquid chromatography (HPLC) for selected flavonoids and phenolic acids followed by a determination of its free radicals scavenging activity (DPPH). Furthermore, Leydig cell viability was assessed by the mitochondrial toxicity assay (MTT), while the membrane integrity was evaluated by 5- carboxyfluorescein diacetate-acetoxymethyl ester (5-CFDA-AM). The level of steroid hormones was performed by enzyme-linked immunosorbent assay (ELISA) from the culture media, while the superoxide radical generation was measured by the nitroblue tetrazolium chloride (NBT) assay. The results show that experimental concentrations did not damage the cell membrane integrity and viability when present at below 300 µg/ml, it was only at 600 µg/ml that a significant (P<0.05) cell viability decline was observed after a 48 h cultivation. A significant (P<0.05) stimulation of testosterone secretion was recorded at 250 µg/ml for 24 h, while the prolonged cultivation time significantly (P<0.05) increased the testosterone and progesterone production at 150, 200, 250 and 300 µg/ml. Furthermore, none of the selected doses exhibited significant ROS-promoting effects however, the highest dose of Salvia initiated the free radical scavenging activity in cultured mice Leydig cells.

GLI3 resides at the intersection of hedgehog and androgen action to promote male sex differentiation.

Urogenital tract abnormalities are among the most common congenital defects in humans. Male urogenital development requires Hedgehog-GLI signaling and testicular hormones, but how these pathways interact is unclear. We found that Gli3XtJ mutant mice exhibit cryptorchidism and hypospadias due to local effects of GLI3 loss and systemic effects of testicular hormone deficiency. Fetal Leydig cells, the sole source of these hormones in developing testis, were reduced in numbers in Gli3XtJ testes, and their functional identity diminished over time. Androgen supplementation partially rescued testicular descent but not hypospadias in Gli3XtJ mutants, decoupling local effects of GLI3 loss from systemic effects of androgen insufficiency. Reintroduction of GLI3 activator (GLI3A) into Gli3XtJ testes restored expression of Hedgehog pathway and steroidogenic genes. Together, our results show a novel function for the activated form of GLI3 that translates Hedgehog signals to reinforce fetal Leydig cell identity and stimulate timely INSL3 and testosterone synthesis in the developing testis. In turn, exquisite timing and concentrations of testosterone are required to work alongside local GLI3 activity to control development of a functionally integrated male urogenital tract.