• Development and Reproduction
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• v.26 no.3
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• pp.99-105
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• 2022

Styrene is the precursor of polystyrene. Human exposure to styrene could occur in occupational and residential settings and via food intake. Styrene is metabolized to styrene-7,8-oxide by cytochrome P450 enzyme. In the present study, we investigated the cytotoxicity mediated by styrene and styrene-7,8-oxide in TM3 testicular Leydig cells in vitro. We first monitored the nuclear fragmentation in Leydig cells after exposure to styrene or styrene-7,8-oxide. Hoechst 33258 cell staining showed that styrene exposure in TM3 Leydig cells did not exhibit nuclear fragmentation at any concentration. In contrast, nuclear fragmentation was seen in styrene-7,8-oxide-exposed cells. These results indicate that cytotoxicity-mediated cell death in Leydig cells is more susceptible to styrene-7,8-oxide than to styrene. Following styrene treatment, procaspase-3 and XIAP protein levels did not show significant changes, and cleaved (active) forms of caspase-3 were not detected. Consistent with the western blot results, the active forms of caspase-3 and XIAP proteins were not prominently altered in the cytoplasm of cells treated with styrene. In contrast to styrene, styrene-7,8-oxide induced cell death in an apoptotic fashion, as seen in caspase-3 activation and increased the expression of XIAP proteins. Taken together, the results obtained in this study demonstrate a fundamental idea that Leydig cells are capable of protecting themselves from cytotoxicity-mediated apoptosis as a result of styrene exposure in vitro. It remains unclear whether the steroid-producing function, i.e., steroidogenesis, of Leydig cells is also unaffected by exposure to styrene. Therefore, further studies are needed to elucidate the endocrine disrupting potential of styrene in Leydig cells.

• Development and Reproduction
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• v.27 no.3
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• pp.149-157
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• 2023

We investigated the involvement of autophagy with steroidogenesis in testicular Leydig cells. Human chorionic gonadotropin (hCG)-stimulated T production in Leydig cells was not remarkably altered in the presence of an autophagy inhibitor 3-methyladenine (3-MA). Although pretreatment with 3-MA demonstrated a tendency to decrease hCG-induced T production, the differences were significant only at a higher time point of 24 h following hCG. Microtubule associated protein light chain 3 (LC3)-II was detectable in the control cells in all the experiments. The hCG-induced increase in steroidogenic acute regulatory protein (StAR) and cytochrome P450 side chain cleave (P450scc) protein levels were not significantly altered by 3-MA. Leydig cells isolated from immature rat testes 12 h following hCG treatment showed relatively increased levels of LC3-II protein compared to the control group. Furthermore, LC3-II levels shown in these cells reached almost the identical to those from normal adult testes. However, LC3-II protein levels were almost comparable or even slightly lower than the controls at 48 h following hCG. Expression of StAR and P450scc was upregulated at both 12 and 48 h after hCG. We also used MA-10 cells, the mouse Leydig cell line, in this experiment. When dibutyryl cyclic-AMP was treated with MA-10 cells, P4 levels were significantly increased in the cell culture medium. However, P4 levels tended to decrease in the presence of 3-MA, but the difference was not statistically significant. This was consistent with the results of the rat Leydig cell experiments. Together, we believe that although autophagy participates in steroidogenesis and enhances steroidogenic efficacy of Leydig cells, it may not be a decisive cellular process for steroidogenesis, specifically in the mature Leydig cells.

• The Korea Journal of Herbology
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• v.26 no.1
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• pp.133-138
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• 2011

Objectives : The purpose of this study was to estimate the antioxidant activity of Sesami Semen Nigrum extract (SSN) on mouse Leydig cells, TM3. Methods : Cell viability assays were performed. The protective effects of SSN against hydrogen peroxide-induced oxidative stress in Leydig cells were examined by measuring cell viability. Lipid peroxidation levels and antioxidant enzyme concentrations such as SOD and catalase were measured. Results : Cell viability of Leydig cells increased with SSN concentration. Cell viability of Leydig cells was 136.66% when SSN concentration was $50{\mu}g/ml$. Cell viability of the hydrogen peroxide group was statistically decreased (p<0.01) compared with the control group. Antioxidant effect of SSN was measured and the protective effect of SSN concentration were 5, 10, $50{\mu}g/ml$. LPO were decreased significantly at 5, $50{\mu}g/ml$ of SSN concentrations. SOD activity was increased at 1, 10, $50{\mu}g/ml$ of SSN concentrations. Catalase activity was significantly increased at 123.7, 133.3 and 131.9 units/mg protein when SSN concentrations were 5, 10 and $50{\mu}g/ml$, respectively. Conclusions : In conclusion, Sesami Semen Nigrum extract has antioxidant activities in Leydig cells against oxidative stress.

• Clinical and Experimental Reproductive Medicine
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• v.35 no.3
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• pp.181-191
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• 2008

Objective: Environmental chemicals alter reproduction, growth, and survival by changing the normal function of the endocrine system. Bisphenol A (BPA), one of the endocrine disruptors, is known to be an estrogen receptor agonist. Therefore, we hypothesized that BPA may affect male reproduction including spermatogenesis in the mouse testis. Methods: We used 7-week-old ICR mice. The first experiment group received BPA in sesame oil (vehicle, 1 mg/kg, 10 mg/kg, and 100 mg/kg) by i.p. injection and mice were sacrificed 24 hr later. The second experiment group received BPA (vehicle, 10 ${\mu}g/kg$, 1 mg/kg, and 100 mg/kg) daily for 14 days by subcutaneous injection. Expression of cell type-specific marker genes in the testis was evaluated by RT-PCR. Histological analysis, immunofluorescence staining, and TUNEL staining were also performed. Results: RT-PCR analyses showed that expression of luteinizing hormone receptor (LHR), a marker gene for the Leydig cell, was notably decreased in the testes of high dose-exposed mice. No obvious difference in the histology of testes was noted among treatment groups. Immunostaining of LHR in the first experiment group did not show noticeable difference in LHR protein expression in Leydig cells. Immunohistochemistry also revealed heightened expression of the immunoreactive Bax in the treatment group, and this was accompanied by positive TUNEL staining in the interstitial area within testis where Leydig cells reside. Conclusions: Our result suggests that BPA affects Leydig cell functions by altering gene expression and by increasing apoptosis in the mouse testis.

• Korean Journal of Veterinary Research
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• v.42 no.3
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• pp.299-308
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• 2002

The purpose of this morphometric study was to obtain detailed quantitative information on all cell types in the testis interstitium of Korean ring-necked pheasants combined with data on changes in the steroidogenic function of the testis during the breeding and nonbreeding seasons. Animals collected during the breeding season, testis weights, sperm production, serum testosterone levels, leuteinizing hormone-stimulated testosterone secretion, and the length of the seminiferous tubules were significantly (p < 0.05) increased as compared to the nonbreeding season. Seminiferous tubules occupied 93.25% of testis volume in the breeding season. Leydig cells constituted 0.82% of the testicular volume. The mean volume of an Leydig cell was $1039{\mu}m^3$, and each testis contained about 24.53 million Leydig cells. Testes of the pheasants during the nonbreeding season displayed a 98% reduction in testis volume that was associated with a decrease in the absolute volume of seminiferous tubules (98% reduction), tubular lumen(100%), interstitium(90%), blood vessels(84%), lymphatic spaces(97%), Leydig cells(79%), mesenchymal cells(51%), and myoid cells(61%). The number of Leydig cells, mesenchymal cells, myoid cells per testis in the breeding season was higher (p < 0.05) than in the nonbreeding season. Although the average volume of a Leydig cell was 74% lower in the nonbreeding season, the average volume of a myoid and mesenchymal cell remained unchanged. These results demonstrate that there are a striking differences in the testicular structure of the Korean ring-necked pheasant in the breeding and nonbreeding seasons. Every structural parameter of the Leydig cell was pasitively correlated with both serum and LH-stimulated secretion concentrations of testosterone. Correlation of changes in hormonal status with morphometric alterations of all Leydig cell suggests that the Korean-ring necked pheasant may be used as a model to study structure-function relations in the avian testis.

• Advances in pediatric surgery
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• v.15 no.1
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• pp.73-79
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• 2009

Sertoli-Leydig cell tumor is a rare sex-cord stromal tumor of the ovary. They make up less than 0.5 % of all ovarian tumors. We experienced a case of an ovarian Sertoli-Leydig cell tumor in a 4 year-old girl who presented with nausea, vomiting, and lower abdominal pain of 2 days' duration. On physical examination, there was mild tenderness in the right lower quadrant of the abdomen. Abdominal ultrasonography and computed tomography (CT) scan revealed a pelvic mass measuring $5{\times}3cm$ that appeared to arise from the right ovary. At exploratory laparotomy, a $6{\times}5{\times}3cm$ solid right ovarian mass without torsion was found. A right salpingo-oophorectomy was performed. The postoperative course was uneventful. The child was discharged 5 days after surgery.

• Journal of Animal Reproduction and Biotechnology
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• v.36 no.3
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• pp.145-153
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• 2021

The role of transient receptor potential vanilloid receptor-1 (TRPV1) has been primarily investigated in pain sensory neurons. Relatively, little research has been performed in testicular cells. TRPV1 is abundantly expressed in Leydig cells of young adult mice. This study was conducted to determine the role of the TRPV1 channel in Leydig cells. TRPV1 modulators and testosterone were treated to the mouse Leydig cell line TM3 cells for 24 h. Capsaicin, a TRPV1 activator, dose-dependently induced cell death, whereas capsazepine, a TRPV1 inhibitor, inhibited capsaicin-induced cell death. Testosterone treatment reduced capsaicin-induced cell death. High concentrations of testosterone decreased TRPV1 mRNA and protein expression levels. However, TRPV1 modulators did not affect testosterone production. These results showed that capsaicin induced cell death of Leydig cells and that testosterone reduced capsaicin-induced cell death. Our findings suggest that testosterone may regulate the survival of Leydig cells in young adult mice by decreasing the expression level of TRPV1.

• The Korea Journal of Herbology
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• v.29 no.6
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• pp.111-116
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• 2014

Objectives : The purpose of this study was to estimate the antioxidant activity of water extract of Cnidii Fructus (CF) and Torilis Fructus (TF) in Leydig cells. Methods : Free radical scavenging activity of CF and TF against 2,2-diphenyl-1-picrylhydrazyl (DPPH) was determined spectrophotometrically. We investigated the effect of CF and TF in Leydig cells by MTT assay. The protective effects of CF and TF against hydrogen peroxide-induced oxidative stress in Leydig cells. Superoxide dismutase (SOD), and catalase activity assays were performed in Leydig cells. Results : The results showed that CF scavenged DPPH radical in a dose-dependent manner by up to 81.2%, TF scavenged DPPH radical in a dose-dependent manner by up to 63.8%. CF showed cell viability as 121.0, 132.7, 126.6% in 5, 10, $100{\mu}g/ml$ concentrations. TF showed cell viability as 127.5, 111.8% in 5, $100{\mu}g/ml$ concentraions, respectively. The hydrogen peroxide-induced cytotoxicity of Leydig cells were protected to 86.3% by CF at concentration of $10{\mu}g/ml$ and protected to 83.5% by TF at concentration of $100{\mu}g/ml$. Both CF and TF at all concentrations, SOD activity was not significantly changed. Catalase activity was significantly increased at 10, $100{\mu}g/ml$ concentrations of CF, respectively. TF's catalase activity showed no significant difference from that of the control. Conclusions : These results suggest that CF, as an antioxidant, protects Leydig cells in hydrogen peroxide-induced oxidative stress. know that "Kwangjebikeup" played a role in settlement and spreading of foreign knowledge to civilians.

• Korean Journal of Veterinary Service
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• v.39 no.3
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• pp.141-149
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• 2016

Changes in the Leydig cell from pre-puberty to adulthood were studied in Korean native cattle. Eight groups of male cattle aged 14, 17, 20, 25, 30, 35, 40 and 104 weeks (n=7 cattle per group) after birth were used. The purpose of this study was to obtain quantitative information on the Leydig cell of the Korean native cattle during postnatal development. Testes of cattle were fixed by perfusion using a fixative containing 2.5% glutaraldehyde in cacodylate buffer, processed and embedded in Epon-araldite. Using $1{\mu}m$ section stained with methylene blue-azure II, qualitative and quantitative (stereological) morphological studies were performed. The average diameter of seminiferous tubules gradually increased with age from 14 ($75.56{\mu}m$) to 104 weeks ($298.9{\mu}m$). The volume density of the seminiferous tubules increased with age from 54.2% at week 14 to 76.9% at week 104. The volume density of the interstitium represents 45.52% at week 14. This proportion progressively diminishes during development to reach a value of 23.14% at week 104. The volume density of Leydig cells decreased almost linearly from 14 (20.71%) to 104 weeks (5.28%). The absolute volume of Leydig cells per testis increased significantly from 14 to 104 weeks. The number of Leydig per testis have almost linearly increased from 14 to 104 weeks. The average volume of a Leydig cell reached maximum size by 104 weeks ($2553{\mu}m^3$). These data suggested development of Leydig cell can be classified as the fetal and immature adult Leydig cells (14~35 weeks), and the adult Leydig cells (40~104 weeks).

• Journal of Animal Reproduction and Biotechnology
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• v.38 no.4
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• pp.291-299
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• 2023

Background: Leydig cells, crucial for testosterone production, express ion channels like ANO1 that influence hormone secretion. This study investigates the expression and role of the Tandem of P domains in a weak inward rectifying K⁺ channel-related Acid-Sensitive K⁺-1 (TASK-1) channel in these cells, exploring its impact on testicular function and steroidogenesis. Methods: TASK-1 expression in Leydig cells was confirmed using immunostaining, while RT-PCR and Western Blot (WB) validated its expression in the TM3 Leydig cell line. The effect of a TASK-1 channel blocker on cell viability was assessed through live/dead staining and MTT assays. Additionally, the blocker's effect on testosterone secretion was evaluated by measuring testosterone levels. Results: Immunohistochemical analysis revealed a predominant presence of TASK-1, along with c-Kit and ANO-1, in Leydig cells adjacent to seminiferous tubules and also in Sertoli and spermatogenic cells. Expression levels of TASK-1 mRNA and protein were significantly higher in TM3 Leydig cells compared to TM4 Sertoli cells. In addition, blocking TASK-1 in TM3 cells with ML365 induced cell death but did not affect LH-induced testosterone secretion. Conclusions: These findings suggest that TASK-1 in Leydig cells is crucial for their viability and proliferation, highlighting its potential importance in testicular physiology.