TM3 Leydig Cells Research Articles

Prenatal exposure to environmentally relevant levels of PBDE-99 leads to testicular dysgenesis with steroidogenesis disorders

Polybrominated diphenyl ethers (PBDEs) are a widely used class of brominated flame retardants. Exposure to PBDEs could induce testicular damage in mammals, but the effects and potential mechanism of action of prenatal exposure to environmentally relevant PBDEs on testicular development remain unclear. For the in vivo study, pregnant ICR mice were exposed to environmentally relevant levels of 2,2′,4,4′,5-pentabromodiphenyl ether (PBDE-99), a major component of commercial PBDE mixtures. We found that the anogenital index and testicular organ coefficient were significantly decreased, the incidence of cryptorchidism was increased, and testicular histology was disturbed in male offspring. Transcriptomic profiling showed that steroidogenesis disorders were significant in all PBDE-99 exposure groups. The testosterone levels, expressions of testosterone regulators, and the number of CYP11A1-positive and 11β-HSD1-positive Leydig cells were significantly decreased after PBDE-99 exposure. For the in vitro study, TM3 Leydig cells were exposed to PBDE-99 at gradient concentrations. Transcriptomic profiling and validation experiments showed that PBDE-99 upregulated reactive oxygen species, activated the ERK1/2 pathway, inhibited the ubiquitination degradation pathway, and finally induced Leydig cell apoptosis. Cumulatively, these findings revealed that prenatal exposure to environmentally relevant levels of PBDE-99 leads to steroidogenesis disorders by inducing the apoptosis of Leydig cells, causing testicular dysgenesis.

Nanosecond Pulsed Electric Field Only Transiently Affects the Cellular and Molecular Processes of Leydig Cells.

The purpose of this study was to verify whether the nanosecond pulsed electric field, not eliciting thermal effects, permanently changes the molecular processes and gene expression of Leydig TM3 cells. The cells were exposed to a moderate electric field (80 quasi-rectangular shape pulses, 60 ns pulse width, and an electric field of 14 kV/cm). The putative disturbances were recorded over 24 h. After exposure to the nanosecond pulsed electric field, a 19% increase in cell diameter, a loss of microvilli, and a 70% reduction in cell adhesion were observed. Some cells showed the nonapoptotic externalization of phosphatidylserine through the pores in the plasma membrane. The cell proportion in the subG1 phase increased by 8% at the expense of the S and G2/M phases, and the DNA was fragmented in a small proportion of the cells. The membrane mitochondrial potential and superoxide content decreased by 37% and 23%, respectively. Microarray’s transcriptome analysis demonstrated a negative transient effect on the expression of genes involved in oxidative phosphorylation, DNA repair, cell proliferation, and the overexpression of plasma membrane proteins. We conclude that nanosecond pulsed electric field affected the physiology and gene expression of TM3 cells transiently, with a noticeable heterogeneity of cellular responses.

Cyclin-dependent kinase inhibitor 1B acts as a novel molecule to mediate testosterone synthesis and secretion in mouse Leydig cells by luteinizing hormone (LH) signaling pathway.

Cyclin-dependent kinase inhibitor 1B (Cdkn1b, p27) plays important regulatory roles in many cellular processes. p27 is highly expressed in the mouse testis, but its roles and underlying mechanisms for testosterone synthesis and secretion remain not well understood. In the current study, we found that p27 located in Leydig cells and Sertoli cells of adult mouse testis. To explore the function of p27 in Leydig cells, p27 inhibitor and activator were injected into the adult mice, primary Leydig cells and TM3 cells. Our in vivo and in vitro results showed that change in the expression of p27 significantly alters the testosterone in both globe serum and culture medium. Meanwhile, the steroidogenesis-related gene expression was significantly regulated too. Moreover, our in vitro study showed that luteinizing hormone (LH) significantly increased p27 mRNA levels. Furthermore, our results proved that altering the mRNA expression of p27 leads to the synchronized changes of Lhcgr, Star, Cyp11a1, Hsd3b6, Cyp11a1, and Hsd17b3. Alterations of p27 also result in synchronously changes of RAF1 and ERK1/2 phosphorylation. These findings indicate that p27 plays vital roles in LH-induced testosterone production, providing a novel mechanism that p27 acts as an upstream molecule to elevate ERK1/2 phosphorylation to promote the expression of StAR and other cholesterol-metabolizing enzymes.

Taurine ameliorates cytotoxic effects of Di(2-ethylhexyl) phthalate on Leydig cells.

It has been revealed that di(2-ethylhexyl)phthalate (DEHP) has toxic impacts on the male reproductive system. Taurine (TAU) is an amino acid with antioxidant property and beneficial impacts on the male reproductive system. In this study, protective impacts of Taurine (TAU) on DEHP-induced Leydig TM3 cell toxicity were investigated. The cells exposed to DEHP (0.8µmol) or TAU (100mg/ml) for 24hr. Cell viability (MTT assay), apoptosis, oxidative stress and testosterone level were examined. DEHP could significantly decrease the cell viability percentage, reduce testosterone level, increase apoptosis, elevate Bax/ Bcl-2 ratio and enhance caspase-3 and -9 activity in the TM3 cells. Additionally, DEHP significantly elevated malondialdehyde contents and reactive oxygen species levels. It also augmented superoxide dismutase and catalase activity in the Leydig cells. Co-treatment of DEHP with TAU increased viability and testosterone level, while oxidative stress and apoptosis significantly reduced. TAU could decrease Bax/Bcl-2 ratio and caspase-3 and -9 activity in the DEHP-intoxicated cells. Our results have clearly shown that TAU protects TM3 cells against oxidative stress and apoptosis induced by DEHP.

Impact of Toxoplasma gondii infection on TM3 Leydig cells: Alterations in testosterone and cytokines levels

Leydig cells play pivotal roles in eliciting male characteristics by producing testosterone and any damage to these cells can compromise male fertility Toxoplasma gondii (T. gondii) is an intracellular parasite capable to invade any nucleated cell, including cells from male reproductive system. Herein, we evaluated the capacity of RH strain of T. gondii to infect TM3 Leydig cells and the impact of this infection on testosterone and inflammatory mediators production. We first, by performing adherence, infection, and intracellular proliferation assays, we found a significant increase in the number of infected Leydig cells, peaking 48 h after the infection with T. gondii. Supernatants of TM3 infected cells exhibited, in a time-dependent manner, increased levels of testosterone as well as monocyte chemoattractant protein-1 (MCP-1) and interferon-γ (IFN-γ), which is correlated with the robust T. gondii infection. In conclusion, our study provides new insights regarding the harmful effects of T. gondii infection on male reproductive system.

Zinc Protects against Heat Stress-Induced Apoptosis via the Inhibition of Endoplasmic Reticulum Stress in TM3 Leydig Cells.

Heat stress (HS)-induced apoptosis in Leydig cells is mediated by various molecular mechanisms, including endoplasmic reticulum (ER) stress. Zinc, an inorganic mineral element, exhibits several cytoprotective properties, but its potential protective action against Leydig cell apoptosis and the related molecular mechanisms has not been fully elucidated. In this study, we evaluated the effects of zinc sulfate, a predominant chemical form of zinc, exerted on cell viability, apoptosis, and testosterone production in HS-treated TM3 Leydig cells and investigated the underlying signaling pathways. HS treatment inhibited cell viability and induced apoptosis, which was accompanied by the induction of the activity of caspase 3, an executioner of apoptosis, involved in the expression of pro-apoptotic protein B cell lymphoma 2-associated X protein (Bax), and in the reduction of the expression of anti-apoptotic protein B cell lymphoma 2 (Bcl-2), thereby activating ER stress marker protein expression (glucose-regulated protein 78 (GRP78) and CCAAT/enhancer-binding protein homologous protein (CHOP)). However, zinc sulfate led to the attenuation of deleterious effects, including increases in apoptosis, caspase-3 activity, Bax, GRP78, and CHOP expression, and decreases in cell viability and Bcl-2 protein expression in cells treated with HS or thapsigargin (an ER stress activator). Furthermore, 4-phenylbutyric acid (an ER stress inhibitor) treatment markedly alleviated the HS-induced adverse effects in cells exposed to HS, which was similar to zinc sulfate. Additionally, zinc sulfate supplementation in the culture medium effectively restored the HS-induced decrease in testosterone levels in HS-treated cells. In summary, these findings indicate that HS triggers apoptosis in TM3 Leydig cells via the ER stress pathway and that zinc confers protection against these detrimental effects. This study provides new insights into the benefits of using zinc against HS-induced apoptosis and cell injury.

Aphrodisiac potential of Polyalthia bullata (Tongkat Ali) in fowl

Objective: To study the aphrodisiac potential of Polyalthia (P.) bullata in fowl. Methods: In this study, testosterone, as an indicator of the aphrodisiac potential of P. bullata, was investigated for its release from TM3 Leydig cells grown in vitro and in 4 fowls given capsules containing P. bullata at a dose of 10 mg in each capsule twice a day, for 50 days. In the latter in vivo evaluation, mating behaviours were additionally determined after the treated fowls were released to the individual hens, and their testes and liver were dissected for histological examinations. Blood drawn from the fowls was assessed for any changes in diagnostic parameters. Results: In the in vitro test (TM3 Leydig cells), P. bullata was able to increase testosterone to 0.48 nmol/L within 72 h of incubation, compared to the untreated control with only 0.18 nmol/L, i.e., an increase of 170%. In the in vivo test, outcomes in the fowls dosed with P. bullata showed similar positive elevations of testosterone to (9.72±1.10) nmol/L in comparison to the controls that showed a level of only (4.05±0.84) nmol/L. Total frequencies of mating behaviours were observed (wing flapping, body shakes, crowing and beak pecking) to be 23 counts for the test compared to only 15 for the control fowls. Histological examination of the male reproductive organs provided evidence of testosterone boosting based on an observable increase in the activity at the seminiferous tubules of testis tissues without any damaging effects, compared to the controls. In the nine diagnostic blood parameters assessed, including alanine aminotransferase, aspartate aminotransferase, and gamma glutamyltransferase, none was remarkably elevated compared to the controls. The histological changes in the liver were not severe and mainly consisted of only localized moderate but recoverable obstructions and swellings of the vessels and tubules. Conclusions: P. bullata is able to boost testosterone both in vitro and in vivo, with no acute toxicities.

Isolation and characterization of flavonoids from fraction of Blepharis persica (Burm. f.) O. Kuntze upregulated testosterone biosynthesis in vitro using TM3 leydig cells

Background: Seeds of Blepharis persica (Burm. f.) O. Kuntze are mentioned in Ayurveda to treat male reproductive functions debilities. Objectives: The present study aimed to evaluate the effect of extract and fraction prepared from seeds of B. persica, in vitro using TM3 Leydig cells and isolation of constituents from bioactive fraction. Materials and Methods: Methanol extract and ethyl acetate fraction prepared that were tested in vitro, using TM3 cell line for the testosterone production. Cell viability was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Ethyl acetate fraction of methanol extract was further taken up for detailed chemical investigations, considering biological potential. The fraction was subjected to column chromatography. The column was eluted with a step gradient of the proportion of methanol in chloroform for the separation of compounds. The isolated compounds were characterized using Fourier-transform infrared, nuclear magnetic resonance (1H and 13C), and mass spectroscopy for identification. Results: MTT assay performed using TM3 cells revealed that extract and fraction had a half-maximal inhibitory concentration value of more than 300 μg/ml. The methanol extract showed more than half-fold and ethyl acetate fraction of methanolic extract showed a one-fold increase in testosterone at a concentration of 150 μg/ml as compared to nontreated cells. Two constituents isolated from ethyl acetate fraction of methanolic extract were identified as apigenin 7-O-neohesperidoside and naringin using comparative spectral studies. Conclusion: The studies suggested that the plant extract and fraction could upregulate testosterone biosynthesis in Leydig cells. Two compounds isolated from the bioactive fraction might serve as a marker for assessment of the quality of plant material.

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 3β-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 3β-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.

A Protein from Dioscorea polystachya (Chinese Yam) Improves Hydrocortisone-Induced Testicular Dysfunction by Alleviating Leydig Cell Injury via Upregulation of the Nrf2 Pathway.

Leydig cell injury has been described as a primary driver of testicular dysfunction and is affected by oxidative stress. Dioscorea polystachya (Chinese yam) is used to improve testicular dysfunction in clinical and pharmacological research via its antioxidative activity, but the mechanisms underlying the beneficial effect of Chinese yam on testicular dysfunction and its suppression of Leydig cell oxidative damage remain unclear. In this study, we obtained a Chinese yam protein (DP1) and explored its effectiveness and possible mechanism in improving testicular dysfunction in vivo and in vitro. We established a testicular dysfunction model in rats using hydrocortisone (HCT). DP1 increased body weight and organ index, improved the deterioration in testicular morphology (including increasing the diameter of seminiferous tubules and thickness of germinal cell layers, inhibiting testicular cell apoptosis by increasing the Bcl-2/Bax ratio, and impeding collagen leakage by downregulating TGF-β1 and p-SMAD2/3 expression), and restored the testosterone content. In addition, DP1 enhanced the number of Leydig cells in rats and H2O2-induced TM3 Leydig cells, and the effect of DP1 on the apoptosis, fibrosis, and testosterone content of TM3 cells was similar to that observed in vivo. These changes were dependent on the regulation of oxidative stress, including significantly reduced intracellular 8-hydroxy-2-deoxyguanosine levels, enhanced superoxide dismutase activities, and decreased superoxide anion levels, which were confirmed via a superoxide overexpression system. Furthermore, we observed that DP1 promoted Nrf2 nuclear import and upregulated antioxidant factor expression in vivo and in vitro. However, Nrf2 silencing eliminated the ability of DP1 to increase the Bcl-2/Bax ratio, reduce the expression levels of TGF-β1 and p-SMAD2/3, and increase testosterone contents in H2O2-induced TM3 cells. In conclusion, DP1 reversed the HCT-induced testicular apoptosis and fibrosis and decreased testosterone contents by alleviating Leydig cell oxidative damage via upregulation of the Nrf2 pathway.

LncRNAs activate longevity regulation pathway due to aging of Leydig cells caused by DEHP exposure: A transcriptome-based study

Di-(2-ethylhexyl) phthalate (DEHP), one of the most commonly used endocrine-disrupting chemicals, has been shown to cause reproductive dysfunction in humans and animal models. However, very few studies have investigated the impact of DEHP at the post-transcriptional level in mouse testes, and the underlying mechanisms remain unclear. In the present research, TM3 Leydig cells were treated with 200 µM phthalic acid mono-2-ethylhexyl ester (MEHP, bio-metabolite of DEHP), and then the mRNA and lncRNA sequencing of TM3 Leydig cells was performed. Mice were exposed prepubertally to 0 or 500 mg DEHP/kg/day. RNA sequencing of mouse testes was performed to verify the RNA-seq results in vitro. The expression patterns of relevant genes and proteins were verified using real-time quantitative polymerase chain reaction (RT-qPCR) and western blotting. DEHP and MEHP exposure led to testicular damage and accelerated cell aging via ROS accumulation. RNA sequencing analyses indicated that FOXO signaling and longevity regulation pathways were activated in resistance to ROS accumulation. FOXO signaling and longevity regulation pathway-related genes and proteins were also activated. By constructing a competing endogenous RNA (ceRNA) network, we observed that the ceRNA network might play a role in regulating FOXO signaling and longevity regulation pathways in response to excessive ROS accumulation and cell aging. In summary, our data here suggests that the ceRNA network may play a role in regulating FOXO signaling and longevity pathways in response to DEHP exposure in mouse testes.

The Effects of Gold Nanoparticles on Leydig Cells and Male Reproductive Function in Mice.

BackgroundGold nanoparticles (AuNPs) have shown great promise in various biomedical applications, but their effects on male reproductive function remain to be ascertained. The aim of this study was to investigate the uptake, cytotoxicity and testosterone production inhibition of AuNPs in mouse Leydig cells, as well as their accumulation in the testes of male mice and their effects on male reproductive function.ResultsAuNPs (5 nm) were able to be internalized into the endosomes/lysosomes of TM3 Leydig cells, induce the formation of autophagosomes, increase the production of reactive oxygen species (ROS), and disrupt the cell cycle in S phase, resulting in concentration-dependent cytotoxicity and DNA damage. Interestingly, AuNPs significantly reduced testosterone production in TM3 cells by inhibiting the expression of 17α-hydroxylase, an important enzyme in androgen synthesis. After repeated intravenous injection, AuNPs gradually accumulated and retained in the testes of male BALB/c mice in a dose-dependent manner. One week after withdrawal, the level of plasma testosterone in the 0.5 mg/kg AuNPs group was significantly reduced compared to that in the PBS control group, accompanied by the decreased expression of 17α-hydroxylase in the testes. In addition, AuNPs treatment significantly increased the rate of epididymal sperm malformation, but without affecting fertility.ConclusionOur results suggest that AuNPs can accumulate in the testes and reduce testosterone production in Leydig cells by down-regulating the expression of 17α-hydroxylase, thus affecting the quality of epididymal sperm.

Multiple transcriptomic profiling: p53 signaling pathway is involved in DEHP-induced prepubertal testicular injury via promoting cell apoptosis and inhibiting cell proliferation of Leydig cells

Di(2-ethylhexyl) phthalate (DEHP) is a widely-used plasticizer and has long been recognized as an endocrine-disrupting chemical with male reproductive toxicities. DEHP exposure at the prepubertal stage may lead to extensive testicular injury. However, the underlying mechanisms remain to be elucidated. In the present study, we gavaged male C57BL/6 mice with different concentrations of DEHP (0, 250, and 500 mg/kg-bw·d) from postnatal day 22–35, and exposed TM3 Leydig cells with 0, 100, 200, 300, and 400 μM of MEHP (bioactive metabolite of DEHP) for 12–48 h. RNA sequencing was performed both in testicular tissue and TM3 cells. The results showed that DEHP disrupts testicular development and reduces serum testosterone levels in male prepubertal mice. Bioinformatic analysis and experimental verification have revealed that DEHP/MEHP induces cell cycle arrest in TM3 cells and increases apoptosis both in vivo and in vitro. Furthermore, the p53 signaling pathway was found to be activated upon DEHP/MEHP treatment. The inhibition of p53 by pifithrin-α significantly reduced MEHP-induced injuries in TM3 cells. Cumulatively, these findings revealed the involvement of the p53 signaling pathway in DEHP-induced prepubertal testicular injury by promoting cell apoptosis and inhibiting cell proliferation of Leydig cells.

Increased m6A modification of RNA methylation related to the inhibition of demethylase FTO contributes to MEHP-induced Leydig cell injury☆

N6-methyladenosine (m6A) modification, the most prevalent form of RNA methylation, modulates gene expression post-transcriptionally. Di-(2-ethylhexyl) phthalate (DEHP) is a common environmental endocrine disrupting chemical that induces testicular injury due to the inhibition of the demethylase fat mass and obesity-associated protein (FTO) and increases the m6A modification. How FTO-mediated m6A modification in testicular Leydig cell injury induced by DEHP remains unclear. Here, the TM3 Leydig cell line was treated with mono-(2-ethylhexyl) phthalate (MEHP), the main metabolite of DEHP in the body, as well as FB23-2, an inhibitor of FTO. Decreased levels of testosterone in the culture supernatant, significantly increased apoptosis, and a remarkable upregulation of global m6A modification were found in both TM3 cells treated with MEHP and FB23-2. Transcriptome sequencing showed that both treatments significantly induced apoptosis-associated gene expression. Methylated RNA immunoprecipitation sequencing showed that the Leydig cell injury induced by upregulated m6A modification could be associated with multiple physiological disorders, including histone acetylation, reactive oxygen species biosynthesis, MAPK signaling pathway, hormone secretion regulation, autophagy regulation, and male gonadal development. Overall, the inhibition of FTO-mediated up-regulation of m6A could be involved in MEHP-induced Leydig cell apoptosis.

Endocrine-disrupting chemicals rapidly affect intercellular signaling in Leydig cells

A decline in male fertility possibly caused by environmental contaminants, namely endocrine-disrupting chemicals (EDCs), is a topic of public concern and scientific interest. This study addresses a specific role of testicular gap junctional intercellular communication (GJIC) between adjacent prepubertal Leydig cells in endocrine disruption and male reproductive toxicity. Organochlorine pesticides (lindane, methoxychlor, DDT), industrial chemicals (PCB153, bisphenol A, nonylphenol and octylphenol) as well as personal care product components (triclosan, triclocarban) rapidly dysregulated GJIC in murine Leydig TM3 cells. The selected GJIC-inhibiting EDCs (methoxychlor, triclosan, triclocarban, lindane, DDT) caused the immediate GJIC disruption by the relocation of gap junctional protein connexin 43 (Cx43) from the plasma membrane and the alternation of Cx43 phosphorylation pattern (Ser368, Ser279, Ser282) of its full-length and two N-truncated isoforms. After more prolonged exposure (24 h), EDCs decreased steady-state levels of full-length Cx43 protein and its two N-truncated isoforms, and eventually (triclosan, triclocarban) also tight junction protein Tjp-1. The disturbance of GJIC was accompanied by altered activity of mitogen-activated protein kinases MAPK-Erk1/2 and MAPK-p38, and a decrease in stimulated progesterone production. Our results indicate that EDCs might disrupt testicular homeostasis and development via disruption of testicular GJIC, a dysregulation of junctional and non-junctional functions of Cx43, activation of MAPKs, and disruption of an early stage of steroidogenesis in prepubertal Leydig cells. These critical disturbances of Leydig cell development and functions during a prepubertal period might be contributing to impaired male reproduction health later on.

2-Monokloropropandiol (2-MCPD)’nin Sitotoksik Etkilerinin Fare TM3 Leydig ve TM4 Sertoli Hücreleri Üzerinde in vitro Değerlendirilmesi

Amaç: 2-monokloropropandiol (2-MCPD), 3-monokloropropandiol (3-MCPD) ve esterleri ile glisidil yağ asidi esteri (GE), gıda veya gıda işlem kaynaklı kontaminantlar olarak adlandırılmaktadır. Bu gıda kontaminantlarının başta palmiye yağı olmak üzere bitkisel yağlar, margarinler, pasta ve kek gibi işlem görmüş gıdalarda ve bebek mamalarında tespit edilmesi, güvenlikleri konusundaki ilgiyi arttırmıştır. Avrupa Gıda Güvenliği Otoritesi’nin (European Food Safety Authority, EFSA) Mayıs 2016 yılında yayınlamış olduğu bilimsel görüşe göre bitkisel yağ ve işlenmiş gıdalarda bulunan GE, 3-MCPD, 2-MCPD ve bunların yağ asidi esterlerinin insan sağlığı için zararlı olduğu raporlanmıştır. Ancak 2-MCPD toksisitesi hakkında yeterli veri olmadığı için tam bir değerlendirme yapılamamıştır. Bu amaçla 2-MCPD’nin, TM3 Leydig ve TM4 Sertoli hücre hatları üzerinde in vitro sitotoksik, apoptotik etkileri ve deoksiribonükleik asit (DNA) hasarı yapma potansiyeli değerlendirilmiştir.
 Gereç ve Yöntem: Hücre canlılığı, 3-(4,5-dimetiltiyazol-2-il)-2,5-difeniltetrazolyum bromid (MTT) ve nötral kırmızısı alım (NRU) yöntemleri ile değerlendirilmiştir. Akış sitometrisi ile hücre ölüm tipinin belirlenmesinde aneksin V-FITC/PI ikili boyama kiti kullanılmıştır. Genotoksisite değerlendirmesinde komet yöntemi uygulanmıştır. 
 Bulgular: Hücre canlılık yöntemleri sonucu 2-MCPD’nin test edilen konsantrasyonlarda 1000 µM’a kadar sitotoksik olmadığı tespit edilmiştir. Aynı zamanda 2-MCPD’nin, sub-toksik konsantrasyonlarda (1-1000 µM) her iki hücre hattında da (TM3 Leydig ve TM4 Sertoli hücreleri) DNA hasarını indüklemediği bulunmuştur. 
 Sonuç: 2-MCPD, her ne kadar test edilen konsantrasyonlarda (1-1000 µM) sitotoksik ve apoptotik etki göstermese de daha yüksek konsantrasyonlarda hücre ölümüne ve DNA hasarına neden olabilir. Ancak bu yüksek konsantrasyonlar günlük maruziyet dozlarından çok uzaktır.

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.

Phytochemical content, antioxidant activities and androgenic properties of four South African medicinal plants

Introduction:This study aimed to investigate the phytochemical contents, antioxidant activities, and androgenic properties ofPeltophorum africanumSond,Trichilia emeticaVahl,Terminalia sambesiaca, andXimenia caffra.Methods:The finely powdered leaves of the selected plants were extracted using acetone, aqueous, and methanol as solvents. The total phenolics and flavonoids contents were determined from gallic acid and quercetin standard curves. The antioxidant activities of these extracts were evaluated using 1, 1-Diphenyl-2-picrylhydrazyl ( DPPH) assay. The effect of plant extracts (100-1000 µg/mL) on TM3 Leydig cells was assessed using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay. Testosterone levels were measured using ELISA kit.Results:The methanol extracts ofT. sambesiacaandX. caffrarevealed higher total phenolic and flavonoid contents (102.13±2.32 mg/g GAE and 1.05±0.04 mg/g QE, respectively) than other plant extracts. The acetone and methanol extracts ofP. africanumrevealed the best IC50value (12.50 ± 0.052 µg/mL) against DPPH than the other plant extracts and ascorbic acid. The MTT assay results showed that all varying concentrations of plant extracts maintained cell viability and were not cytotoxic with IC50values of greater than 20 µg/mL. The methanol extract ofT. sambesiacahad the highest testosterone production at 500 µg/mL (0.399 ng/mL) when compared with the basal control while at the concentration of 500 µg/mL the acetone extracts ofP. africanumandT. sambesiacahad significantly high testosterone production 0.147 and 0.188 ng/mL respectively when compared with basal control.Conclusion:The results reveal that these plants possess antioxidants and androgenic property and suggest the potential use for the treatment of male infertility.

Propofol induces apoptosis by activating caspases and the MAPK pathways, and inhibiting the Akt pathway in TM3 mouse Leydig stem/progenitor cells.

Propofol is an anesthetic agent moderating GABA receptors in the nervous system. A number of studies have demonstrated that propofol exerts a negative effect on neural stem celldevelopment in the neonatal mouse hippocampus. However, to the best of our knowledge, there is no study available to date illustrating whether neonatal exposure to propofol affects Leydig stem/progenitor cell development for normal male reproductive development and functions, and the regulatory mechanism remains elusive. In the present study, TM3 cells, a mouse Leydig stem/progenitor cell line, was treated with propofol. The data illustrated that propofol significantly reduced TM3 cell viability. TM3 subG1 phase cell numbers were significantly increased by propofol assayed by flow cytometric analysis. AnnexinV/PI double staining assay of the TM3 Leydig cells also demonstrated that propofol increased TM3 cell apoptosis. In addition, cleaved caspase‑8, ‑9 and ‑3 and/or poly(ADP‑ribose) polymerase (PARP) were significantly activated by propofol in the TM3 cells. Furthermore, the expression levels of phospho‑JNK, phospho‑ERK1/2 and phospho‑p38 were activated by propofol in the TM3 cells, indicating that propofol induced apoptosis through the mitogen‑activated protein kinase (MAPK) pathway. Additionally, propofol diminished the phosphorylation of Akt to increase the apoptosis of TM3 cells. On the whole, the findings of the present study demonstrate that propofol induces TM3 cell apoptosis by activating caspases and MAPK pathways, as well as by inhibiting the Akt pathway in TM3 cells. These findings illustrate that propofol affects the viability of Leydig stem/progenitor cells possibly related to the development of the male reproductive system.

BMAL1 knockdown promoted apoptosis and reduced testosterone secretion in TM3 Leydig cell line

Brain and muscle Arnt-like protein-1 (BMAL1) is a clock gene that plays an important role in hormone secretion and apoptosis, but its effect on Leydig cells is unidentified. Here the role of BMAL1 in apoptosis and testosterone secretion in TM3 Leydig cell line were investigated by inhibiting its expression using small interfering RNA (siRNA). Results showed that BMAL1 knockdown promoted the apoptosis of Leydig cells and expression of (BCL2 associated X) BAX mRNA and protein, and reduced the expression of (B-cell lymphoma-2) BCL-2 mRNA and protein. BMAL1 inhibition resulted in decreased testosterone secretion and reduced expression of key genes during hormone synthesis, specifically steroidogenic acute regulatory protein (STAR), cytochrome P450 family 11 subfamily A member 1 (CYP11A1), and 3β-hydroxysteroid dehydrogenase (3β-HSD). In addition, BMAL1 knockdown reduced the expression of phosphorylated p85 and AKT as confirmed by western blot. In conclusion, BMAL1 may affect testosterone secretion and apoptosis in mouse Leydig cells through regulation of the PI3K/AKT signaling pathway.