Role of Constitutive STAR in Leydig Cells.

Leydig cells contain significant amounts of constitutively produced steroidogenic acute regulatory protein (STAR; STARD1). Hormone-induced STAR plays an essential role in inducing the transfer of cholesterol into the mitochondria for hormone-dependent steroidogenesis. STAR acts at the outer mitochondrial membrane, where it interacts with a protein complex, which includes the translocator protein (TSPO). Mutations in STAR cause lipoid congenital adrenal hyperplasia (lipoid CAH), a disorder characterized by severe defects in adrenal and gonadal steroid production; in Leydig cells, the defects are seen mainly after the onset of hormone-dependent androgen formation. The function of constitutive STAR in Leydig cells is unknown. We generated STAR knockout (KO) MA-10 mouse tumor Leydig cells and showed that STAR KO cells failed to form progesterone in response to dibutyryl-cAMP and to TSPO drug ligands, but not to 22(R)-hydroxycholesterol, which is a membrane-permeable intermediate of the CYP11A1 reaction. Electron microscopy of STAR KO cells revealed that the number and size of lipid droplets were similar to those in wild-type (WT) MA-10 cells. However, the density of lipid droplets in STAR KO cells was drastically different than that seen in WT cells. We isolated the lipid droplets and analyzed their content by liquid chromatography-mass spectrometry. There was a significant increase in cholesteryl ester and phosphatidylcholine content in STAR KO cell lipid droplets, but the most abundant increase was in the amount of diacylglycerol (DAG); DAG 38:1 was the predominantly affected species. Lastly, we identified genes involved in DAG signaling and lipid metabolism which were differentially expressed between WT MA-10 and STAR KO cells. These results suggest that constitutive STAR in Leydig cells is involved in DAG accumulation in lipid droplets, in addition to cholesterol transport. The former event may affect cell functions mediated by DAG signaling.

Transplantation of encapsulated human Leydig-like cells: A novel option for the treatment of testosterone deficiency.

Previous studies have demonstrated that the transplantation of alginate-poly-ʟ-lysine-alginate (APA)-encapsulated rat Leydig cells (LCs) provides a promising approach for treating testosterone deficiency (TD). Nevertheless, LCs have a limited capacity to proliferate, limiting the efficacy of LC transplantation therapy. Here, we established an efficient differentiation system to obtain functional Leydig-like cells (LLCs) from human stem Leydig cells (hSLCs). Then we injected APA-encapsulated LLCs into the abdominal cavities of castrated mice without an immunosuppressor. The APA-encapsulated cells survived and partially restored testosterone production for 90 days in vivo. More importantly, the transplantation of encapsulated LLCs ameliorated the symptoms of TD, such as fat accumulation, muscle atrophy and adipocyte accumulation in bone marrow. Overall, these results suggest that the transplantation of encapsulated LLCs is a promising new method for testosterone supplementation with potential clinical applications in TD.

Interstitial Leydig Cell Tumorigenesis-Leptin and Adiponectin Signaling in Relation to Aromatase Expression in the Human Testis.

Although epidemiological studies from the last years report an increase in the incidences of Leydig cell tumors (previously thought to be a rare disease), the biochemical characteristics of that tumor important for understanding its etiology, diagnosis, and therapy still remains not completely characterized. Our prior studies reported G-protein coupled estrogen receptor signaling and estrogen level disturbances in Leydig cell tumors. In addition, we found that expressions of multi-level-acting lipid balance- and steroidogenesis-controlling proteins including peroxisome proliferator-activated receptor are altered in this tumor. In order to get deeper into the other molecular mechanisms that regulate lipid homeostasis in the Leydig cell tumor, here we investigate the presence and expression of newly-described hormones responsible for lipid homeostasis balancing (leptin and adiponectin), together with expression of estrogen synthase (aromatase). Samples of Leydig cell tumors (n = 20) were obtained from patients (31-45 years old) and used for light and transmission electron microscopic, western blotting, and immunohistochemical analyses. In addition, body mass index (BMI) was calculated. In tumor mass, abundant lipid accumulation in Leydig cells and various alterations of Leydig cell shape, as well as the presence of adipocyte-like cells, were observed. Marked lipid content and various lipid droplet size, especially in obese patients, may indicate alterations in lipid homeostasis, lipid processing, and steroidogenic organelle function in response to interstitial tissue pathological changes. We revealed significantly increased expression of leptin, adiponectin and their receptors, as well as aromatase in Leydig cell tumors in comparison to control. The majority of patients (n = 13) were overweight as indicated by their BMI. Moreover, a significant increase in expression of phospholipase C (PLC), and kinases Raf, ERK which are part of adipokine transductional pathways, was demonstrated. These data expand our previous findings suggesting that in human Leydig cell tumors, estrogen level and signaling, together with lipid status, are related to each other. Increased BMI may contribute to certain biochemical characteristics and function of the Leydig cell in infertile patients with a tumor. In addition, altered adipokine-estrogen microenvironment can have an effect on proliferation, growth, and metastasis of tumor cells. We report here various targets (receptors, enzymes, hormones) controlling lipid balance and estrogen action in Leydig cell tumors indicating their possible usefulness for diagnostics and therapy.

The meaning of non-classical estrogen receptors and peroxisome proliferator-activated receptor for boar Leydig cell of immature testis.

Communication in biological systems involves diverse-types of cell-cell interaction including cross-talk between receptors expressed by the target cells. Recently, novel sort of estrogen receptors (G protein - coupled estrogen receptor; GPER and estrogen-related receptor; ERR) that signal directly via estrogen binding and/or via mutual interaction-regulated estrogen signaling were reported in various organs including testis. Peroxisome proliferator - activated receptor (PPAR) is responsible for maintaining of lipid homeostasis that is critical for sex steroid production in the testis. Here, we investigated the role of interaction between GPER, ERRß and PPARγ in steroidogenic Leydig cells of immature boar testis. Testicular fragments cultured ex vivo were treated with GPER or PPARγ antagonists. Then, cell ultrastructure, expression and localization of GPER, ERRß, PPARγ together with the molecular receptor mechanism, through cyclic AMP and Raf/Ras/extracellular signal activated kinases (ERK), in the control of cholesterol concentration and estrogen production by Leydig cells were studied. In the ultrastructure of antagonist-treated Leydig cells, mitochondria were not branched and not bifurcated as they were found in control. Additionally, in PPARγ-blocked Leydig cells changes in the number of lipid droplets were revealed. Independent of used antagonist, western blot revealed decreased co-expression of GPER, ERRß, PPARγ with exception of increased expression of ERRß after PPARγ blockage. Immunohistochemistry confirmed presence of all receptors partially located in the nucleus or cytoplasm of Leydig cells of both control and treated testes. Changes in receptor expression, decreased cholesterol and increased estradiol tissue concentrations occurred through decreased cAMP level (with exception after GPER blockage) as well as Raf/Ras/ERK pathway expression. These all findings indicate that GPER-ERRß-PPARγ interaction exists in immature boar testis and regulates Leydig cell function. Further detailed studies and considerations on GPER-ERRß-PPARγ as possible diagnosis/therapy target in disturbances of testis steroidogenic function are needed.

Directing differentiation of human induced pluripotent stem cells toward androgen-producing Leydig cells rather than adrenal cells.

Reduced serum testosterone (T), or hypogonadism, affects millions of men and is associated with many pathologies, including infertility, cardiovascular diseases, metabolic syndrome, and decreased libido and sexual function. Administering T-replacement therapy (TRT) reverses many of the symptoms associated with low T levels. However, TRT is linked to side effects such as infertility and increased risk of prostate cancer and cardiovascular diseases. Thus, there is a need to obtain T-producing cells that could be used to treat hypogonadism via transplantation and reestablishment of T-producing cell lineages in the body. T is synthesized by Leydig cells (LCs), proposed to derive from mesenchymal cells of mesonephric origin. Although mesenchymal cells have been successfully induced into LCs, the limited source and possible trauma to donors hinders their application to clinical therapies. Alternatively, human induced pluripotent stem cells (hiPSCs), which are expandable in culture and have the potential to differentiate into all somatic cell types, have become the emerging source of autologous cell therapies. We have successfully induced the differentiation of hiPSCs into either human Leydig-like (hLLCs) or adrenal-like cells (hALCs) using chemically defined culture conditions. Factors critical for the development of LCs were added to both culture systems. hLLCs expressed all steroidogenic genes and proteins important for T biosynthesis, synthesized T rather than cortisol, secreted steroid hormones in response to dibutyryl-cAMP and 22(R)-hydroxycholesterol, and displayed ultrastructural features resembling LCs. By contrast, hALCs synthesized cortisol rather than T. The success in generating hiPSC-derived hLLCs with broad human LC (hLC) features supports the potential for hiPSC-based hLC regeneration.

Do estrogens regulate lipid status in testicular steroidogenic Leydig cell?

In this study mouse Leydig cell (MA-10) were treated with G-protein coupled membrane estrogen receptor antagonist (G-15; 10 nM). Cells were analyzed by Western blotting for expression of estrogen-related receptors (ERRα, ß and γ), steroidogenic markers (lutropin receptor; LHR and 3ß-hydroxysteroid dehydrogenase; 3ß-HSD) and lipid droplet markers (perilipin; PLIN and microtubule-associated protein 1 A/1B-light chain 3; LC3). Concomitantly, microscopic analyses by light microscope (immunofluorescent staining for lipid droplets, PLIN and LC3) as well as by electron microscope (for lipid droplet ultrastructure) were utilized. For analysis of cholesterol content, cAMP level and progesterone secretion, G-15, estrogen receptor (ER) antagonist (ICI 182,780; 10 µM), 17ß-estradiol (10 mM) and, bisphenol A (BPA; 10 nM) were used alone or in combinations. We revealed no changes in ERRs expression but alterations in ERRß and γ localization in G-15-treated cells when compared to control. Partial translocation of ERRß and γ from the cell nucleus to cytoplasm was observed. Decreased expression of LHR, 3ß-HSD, PLIN and LC3 was detected. Moreover, in treated cells large lipid droplets and differences in their distribution were found. Very strong signal of co-localization for PLIN and LC3 was found in treated cells when compared to control. In ultrastructure of treated cells, degenerating lipid droplets and double membrane indicating on presence of lipophagosome were observed. We found, that only (i) BPA and G-15 did not effect on cholesterol content, (ii) BPA, G-15 and ICI did not effect on cAMP level and (iii) BPA, ICI alone and in combination, and BPA with G-15 did not modulate progesterone secretion. These findings showed complex and diverse estrogen effects on mouse Leydig cells at various steps of steroid hormone production (cholesterol storage, release and processing). Lipid homeostasis and metabolism in these cells were affected by endogenous and exogenous estrogen, interactions of receptors (GPER, ER and ERR) and GPER and ER antagonists.

Suppression of testosterone production by nanoparticulate TiO2 is associated with ERK1/2-PKA-PKC signaling pathways in rat primary cultured Leydig cells.

BACKGROUND: Nanoparticulate titanium dioxide (nano-TiO2) enters the body through various routes and causes organ damage. Exposure to nano-TiO2 is reported to cause testicular injury in mice or rats and decrease testosterone synthesis, sperm number, and motility. Importantly, nano-TiO2 suppresses testosterone production by Leydig cells (LCs) and impairs the reproductive capacity of animals. METHODS: In an attempt to establish the molecular mechanisms underlying the inhibitory effect of nano-TiO2 on testosterone synthesis, primary cultured rat LCs were exposed to varying concentrations of nano-TiO2 (0, 10, 20, and 40 µg/mL) for 24 hours, and alterations in cell viability, cell injury, testosterone production, testosterone-related factors (StAR, 3ßHSD, P450scc, SR-BI, and DAX1), and signaling molecules (ERK1/2, PKA, and PKC) were investigated. RESULTS: The data show that nano-TiO2 crosses the membrane into the cytoplasm or nucleus, triggering cellular vacuolization and nuclear condensation. LC viability decreased in a time-dependent manner at the same nano-TiO2 concentration, nano-TiO2 treatment (10, 20, and 40 µg/mL) decreased MMP (36.13%, 45.26%, and 79.63%), testosterone levels (11.40% and 44.93%), StAR (14.7%, 44.11%, and 72.05%), 3ßHSD (26.56%, 50%, and 79.69%), pERK1/2 (27.83%, 63.61%, and 78.89%), PKA (47.26%, 70.54%, and 85.61%), PKC (30%, 50%, and 71%), SR-BI (16.41%, 41.79%, and 67.16%), and P450scc (39.41%, 55.26%, and 86.84%), and upregulated DAX1 (1.31-, 1.63-, and 3.18-fold) in primary cultured rat LCs. CONCLUSION: Our collective findings indicated that nano-TiO2-mediated suppression of testosterone in LCs was associated with regulation of ERK1/2-PKA-PKC signaling pathways.

The role of G-protein-coupled membrane estrogen receptor in mouse Leydig cell function-in vivo and in vitro evaluation.

In this study, G-coupled estrogen receptor (GPER) was inactivated, by treatment with antagonist (G-15), in testes of C57BL/6 mice: immature (3 weeks old), mature (3 months old) and aged (1.5 years old) (50 µg/kg bw), as well as MA-10 mouse Leydig cells (10 nM/24 h) alone or in combination with 17ß-estradiol or antiestrogen (ICI 182,780). In G-15-treated mice, overgrowth of interstitial tissue was found in both mature and aged testes. Depending on age, differences in structure and distribution of various Leydig cell organelles were observed. Concomitantly, modulation of activity of the mitochondria and tubulin microfibers was revealed. Diverse and complex GPER regulation at the mRNA level and protein of estrogen signaling molecules (estrogen receptor α and ß; ERα, ERß and cytochrome P450 aromatase; P450arom) in G-15 Leydig cells was found in relation to age and the experimental system utilized (in vivo and in vitro). Changes in expression patterns of ERs and P450arom, as well as steroid secretion, reflected Leydig cell heterogeneity to estrogen regulation throughout male life including cell physiological status.We show, for the first time, GPER with ERs and P450arom work in tandem to maintain Leydig cell architecture and supervise its steroidogenic function by estrogen during male life. Full set of estrogen signaling molecules, with involvement of GPER, is crucial for proper Leydig cell function where each molecule acts in a specific and/or complementary manner. Further understanding of the mechanisms by which GPER controls Leydig cells with special regard to male age, cell of origin and experimental system used is critical for predicting and preventing testis steroidogenic disorders based on perturbations in estrogen signaling.

Adjuvant potential of virgin coconut oil extract on antiretroviral therapy-induced testicular toxicity: An ultrastructural study.

The effects of Virgin coconut oil as an adjuvant to highly active antiretroviral therapy (HAART) were investigated on the testicular ultrastructure and biochemical markers in rats. Twenty male Sprague-Dawley rats, weighing 153-169 g were divided into four groups and treated as follows: control A (distilled water), B (HAART), C (HAART+Virgin coconut oil 10 ml/kg) and D (Virgin coconut oil [VCO] 10 ml/kg). Testicular segments were evaluated using transmission electron microscopy. Serum was assayed for testosterone, luteinising hormone, follicle stimulating hormone and testicular tissue for malondialdehyde and glutathione. Ultrastructure of basement membrane (Bm), mitochondria and spermatocytes was normal in the control group. HAART-treated group showed significant increase (p < .01) in Bm thickness with significant decrease in Leydig cell nuclear diameter (p < .05) and volume (p < .01) when compared with control group. Mitochondrial cristae appear collapsed, and Sertoli cells showed cytoplasmic vacuolations. HAART+VCO group showed improved ultrastructural details in Bm, and Sertoli cell and Leydig cells show abundant lipid droplets. Virgin coconut oil-treated group showed thinning of Bm with otherwise normal ultrastructural features of organelles. HAART-treated group showed significant increase (p < .01) in testosterone levels. There was no significant effect on malondialdehyde and glutathione levels. Virgin coconut oil improved testicular morphology and reversed HAART-induced ultrastructural alterations. Further studies on putative mechanism are required.

Does signaling of estrogen-related receptors affect structure and function of bank vole Leydig cells?

To get a deeper insight into the function of estrogen-related receptors (ERRs) and dissect underlying mechanism in Leydig cells, ERRs (type α, ß and γ) were blocked or activated in testes of adult bank voles (Myodes glareolus) which show seasonal changes in the intratesticular sex hormones level. Both actively reproducing animals (long day conditions; LD) and those with regression of the reproductive system (short day conditions; SD) received intraperitoneal injections of selective ERRα antagonist 3-[4-(2,4-Bis-trifluoromethylbenzyloxy)-3-methoxyphenyl]-2-cyano-N-(5-trifluoromethyl-1,3,4-thiadiazol-2-yl)acrylamide (XCT 790) or selective ERRß/ERRγ agonist N-(4-(Diethylaminobenzylidenyl)-N'-(4-hydroxybenzoyl)-hydrazine (DY131) (50 µ/kg bw; six doses every other day). Markedly more, XCT 790 (P < 0.05) but also DY131 affected interstitial tissue histology whose volume increased in both LD and SD males while seminiferous epithelium structure was untouched. Ultrastructure analysis revealed alterations in mitochondria number as well as endoplasmic reticulum and Golgi complexes volume and structure especially after ERRα blockage. Diverse and complex ERRs regulation at mRNA level and protein expression (P < 0.05; P < 0.01 and P < 0.001) of steroidogenic (lutropin receptor (LHR), translocator protein (TSPO), steroidogenic acute regulatory protein (StAR)) and secretory (insulin-like protein 3 (INSL3) and relaxin (RLN)) molecules were revealed in relations to endogenous estrogen level in treated males. Notably, immunolocalization of ERRs and above proteins, exclusively in Leydig cells, indicated their involvement in Leydig cell function control based on interactions with endogenous estrogen level and/or estrogen signaling via ERRs. Treatment with XCT 790 or DY131 significantly decreased (P < 0.05; P < 0.01 and P < 0.001) intratesticular estrogens concentration, with exception in SD DY131 males. In addition, androgens level was decreased, but not in LD DY131 voles. Similarly, ERRßγ activation significantly reduced (P < 0.05; P < 0.01 and P < 0.001) cAMP and calcium ions (Ca2+) concentrations particularly in DY131 voles. Overall, for the first time, we have shown that ERRs are involved in maintenance of Leydig cell architecture and supervision of its steroidogenic and secretory activity that is closely related to endogenous estrogen status in the testis. Further understanding of mechanism(s) by which individual types of ERRs can control Leydig cell function is relevant for predicting and preventing steroidogenic and spermatogenic disorders.

Probing GATA factor function in mouse Leydig cells via testicular injection of adenoviral vectors.

Testicular Leydig cells produce androgens essential for proper male reproductive development and fertility. Here, we describe a new Leydig cell ablation model based on Cre/Lox recombination of mouse Gata4 and Gata6, two genes implicated in the transcriptional regulation of steroidogenesis. The testicular interstitium of adult Gata4flox/flox ; Gata6flox/flox mice was injected with adenoviral vectors encoding Cre + GFP (Ad-Cre-IRES-GFP) or GFP alone (Ad-GFP). The vectors efficiently and selectively transduced Leydig cells, as evidenced by GFP reporter expression. Three days after Ad-Cre-IRES-GFP injection, expression of androgen biosynthetic genes (Hsd3b1, Cyp17a1 and Hsd17b3) was reduced, whereas expression of another Leydig cell marker, Insl3, was unchanged. Six days after Ad-Cre-IRES-GFP treatment, the testicular interstitium was devoid of Leydig cells, and there was a concomitant loss of all Leydig cell markers. Chromatin condensation, nuclear fragmentation, mitochondrial swelling, and other ultrastructural changes were evident in the degenerating Leydig cells. Liquid chromatography-tandem mass spectrometry demonstrated reduced levels of androstenedione and testosterone in testes from mice injected with Ad-Cre-IRES-GFP. Late effects of treatment included testicular atrophy, infertility and the accumulation of lymphoid cells in the testicular interstitium. We conclude that adenoviral-mediated gene delivery is an expeditious way to probe Leydig cell function in vivo Our findings reinforce the notion that GATA factors are key regulators of steroidogenesis and testicular somatic cell survival.Free Finnish abstract: A Finnish translation of this abstract is freely available at http://www.reproduction-online.org/content/154/4/455/suppl/DC2.

Histological alterations in Leydig cells and macrophages in azoospermic men.

The study aimed to compare the histological features of Leydig cells and macrophages in the testicular interstitium of obstructive versus nonobstructive azoospermia. Thirty-nine azoospermic men undergoing testicular sperm extraction during intracytoplasmic sperm injection were allocated into obstructive azoospermia group (GI) and nonobstructive azoospermia group (GII) which was subdivided into Sertoli cell-only syndrome (GIIA), germ cell arrest (GIIB) and hypospermatogenesis (GIIC) subgroups. Serum LH, FSH and testosterone levels were measured. Ultrastructural changes and the mean number of CD68-positive cells were estimated in the different groups. In GIIA, Leydig cells' processes came in contact with macrophages and showed smooth endoplasmic reticulum dilatation. In GIIB, Leydig cells showed apoptotic changes. Macrophages were commonly encountered in their vicinity demonstrating large number of lysosomes. In GIIC, Leydig cells showed euchromatic nuclei. Macrophages showed expulsion of their lysosomal contents in the interstitium surrounded by apoptotic bodies. The mean count of total CD68-positive macrophages was higher in cases of obstructive azoospermia with nonsignificant differences compared to nonobstructive azoospermia groups. Significant increase in FSH level was detected in GIIA compared to GI. It is concluded that structural interactions might take place between Leydig cells and macrophages in the interstitial tissue of azoospermic men.

[The ultrastructure of Leydig cells under the influence of drinking mineral water and electromagnetic radiation under the stress conditions in the rats]. / Ul'trastruktura kletok Leidiga pri deistvii mineral'noi vody i nizkointensivnogo elektromagnitnogo izlucheniya v usloviyakh stressa u krys.

The objective of the present study was elucidate the peculiar features of low-intensity electromagnetic radiation (LI EMR) and mineral water (MW) on the ultrastructure of rat Leydig cells under conditions of immobilization stress. The experiments were carried out on outbred male rats with the use of electron microscopy. It has been demonstrated that the prophylactic consumption of drinking sulfate-containing mineral water and the application low-intensity electromagnetic radiation (with the flow power density of 1 mcW/cm2 and frequency around 1,000 Hz) or the combination of these two modalities under conditions of immobilization stress reduced the degree of ultrastructural derangement in the rat Leydig cells and stimulated the development of regenerative processes. In the cases of the single-factor impact, drinking mineral water exerted more pronounced action than low-intensity electromagnetic radiation on mitochondrial regeneration. In case of the simultaneous application of the two factors their protective action on the Leydig cells was much more conspicuous than that of either of them applied alone. It is concluded that drinking sulfate-containing mineral water in combination with the application of low-intensity electromagnetic radiation enhances resistance of the rat Leydig cells to stress.

Involvement of oxidative stress in tri-ortho-cresyl phosphate-induced autophagy of mouse Leydig TM3 cells in vitro.

BACKGROUND: As a plasticizer, plastic softener, and flame-retardant, tri-ortho-cresyl phosphate (TOCP) is and has been widely used in industry and reported to have a toxic effect on the male reproductive system in animals besides neurotoxicity and immunotoxicity. We have reported that TOCP inhibits spermatogenesis and induces autophagy of rat spermatogonial stem cells, but it is still unknown whether TOCP induces autophagy of mouse Leydig cells and its potential mechanism. METHODS: Cell viability was observed by MTT assay. Level of testosterone was measured by radioimmunoassay. Apoptosis was observed by AnnexinV-FITC/PI assay. The contents of LC3, Atg5-Atg12, and Beclin 1 were detected by Western blotting analysis. Autophagosomes were investigated by transmission electron microscopy. The contents of MDA and GSH and the activities of SOD, GSH-PX, total antioxidant status (TAS) and total oxidant status (TOS) were measured by oxidative stress kits. RESULTS: The present study shows that TOCP markedly inhibited viability and testosterone output of mouse Leydig TM3 cells but had no effect on apoptosis. However, TOCP significantly increased both LC3-II and the ratio of LC3-II to LC3-I and the contents of autophagy proteins Atg5 and Beclin 1. Transmission electron microscopy (TEM) showed that TOCP increased autophagic vacuoles of the cytoplasm, indicating that TOCP could induce autophagy of the cells. TOCP significantly induced oxidative stress of mouse Leydig TM3 cells. H2O2 also inhibited viability and induced autophagy of the cells; however, inhibition of oxidative stress by N-acetyl-L-cysteine (NAC) could rescue the inhibition of cell viability and induction of autophagy by TOCP. CONCLUSIONS: The results show oxidative stress might be involved in TOCP-induced autophagy of mouse Leydig TM3 cells.

Microscopic visualization of testosterone in mouse testis by use of imaging mass spectrometry.

Testosterone is one of the androgens synthesized from cholesterol as a precursor in the Leydig cells of testes. Since the ionization efficiency of testosterone in matrix-assisted laser desorption ionization (MALDI) is quite low, visualization of testosterone by using MALDI-imaging mass spectrometry (MALDI-IMS) has been considered difficult. To overcome this problem, we used two types of on-tissue derivatization techniques, which were achieved by pyridine sulfur trioxide and Girard's T (GT) reagent, to introduce a polar group into testosterone molecule with the aim to increase the sensitivity. Derivatization by use of GT reagent provided excellent results, superior to those obtained with pyridine sulfur trioxide, in terms of ionization efficiency, molecular specificity, and tissue damage. In GT derivatized testis tissues of mice treated with human chorionic gonadotropin (hCG), testosterone was broadly observed both inside and outside the seminiferous tubules by using an iMScope. To evaluate our imaging results, we performed quantification experiments of underivatized testosterone extracted from hCG-treated testes and control testes using LC-MS/MS. We confirmed the 256-fold concentration change between hCG-treated tissues and control tissues. We also confirmed the 228-fold change in detected peak intensities between hCG-treated tissue sections and control tissue sections in imaging results. We consider our tissue preparation methods for IMS provide high sensitivity with high precision. In addition, high-spatial definition IMS was also available, and we confirmed testosterone had mainly accumulated on the surface of the Leydig cells. Graphical abstract Girard's T-testosterone (GT-Ts) provides the fragment ion at m/z 343.24. Clear GT-Ts signal was detected in hCG treated mouse testis not only as spectra but also as a mass image.

Cellular changes in the hamster testicular interstitium with ageing and after exposure to short photoperiod.

The aim of this study was to evaluate the cellular changes that occur in the hamster testicular interstitium in two very different physiological situations involving testicular involution: ageing and exposure to a short photoperiod. The animals were divided into an 'age group' with three subgroups - young, adult and old animals - and a 'regressed group' with animals subjected to a short photoperiod. The testicular interstitium was characterised by light and electron microscopy. Interstitial cells were studied histochemically with regard to their proliferation, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP in situ nick end labelling (TUNEL+) and testosterone synthetic activity. We identified two types of Leydig cell: Type A cells showed a normal morphology, while Type B cells appeared necrotic. With ageing, pericyte proliferation decreased but there was no variation in the index of TUNEL-positive Leydig cells. In the regressed group, pericyte proliferation was greater and TUNEL-positive cells were not observed in the interstitium. The testicular interstitium suffered few ultrastructural changes during ageing and necrotic Leydig cells were observed. In contrast, an ultrastructural involution of Leydig cells with no necrosis was observed in the regressed group. In conclusion, the testicular interstitium of Mesocricetus auratus showed different cellular changes in the two groups (age and regressed), probably due to the irreversible nature of ageing and the reversible character of changes induced by short photoperiod.

Effects of Etomidate on the Steroidogenesis of Rat Immature Leydig Cells.

BACKGROUND: Etomidate is a rapid hypnotic intravenous anesthetic agent. The major side effect of etomidate is the reduced plasma concentration of corticosteroids, leading to the abnormal reaction of adrenals. Cortisol and testosterone biosynthesis has similar biosynthetic pathway, and shares several common steroidogenic enzymes, such as P450 side chain cleavage enzyme (CYP11A1) and 3ß-hydroxysteroid dehydrogenase 1 (HSD3B1). The effect of etomidate on Leydig cell steroidogenesis during the cell maturation process is not well established. METHODOLOGY: Immature Leydig cells isolated from 35 day-old rats were cultured with 30 µM etomidate for 3 hours in combination with LH, 8Br-cAMP, 25R-OH-cholesterol, pregnenolone, progesterone, androstenedione, testosterone and dihydrotestosterone, respectively. The concentrations of 5α-androstanediol and testosterone in the media were measured by radioimmunoassay. Leydig cells were cultured with various concentrations of etomidate (0.3-30 µM) for 3 hours, and total RNAs were extracted. Q-PCR was used to measure the mRNA levels of following genes: Lhcgr, Scarb1, Star, Cyp11a1, Hsd3b1, Cyp17a1, Hsd17b3, Srd5a1, and Akr1c14. The testis mitochondria and microsomes from 35-day-old rat testes were prepared and used to detect the direct action of etomidate on CYP11A1 and HSD3B1 activity. RESULTS AND CONCLUSIONS: In intact Leydig cells, 30 µM etomidate significantly inhibited androgen synthesis. Further studies showed that etomidate also inhibited the LH- stimulated androgen production. On purified testicular mitochondria and ER fractions, etomidate competitively inhibited both CYP11A1 and HSD3B1 activities, with the half maximal inhibitory concentration (IC50) values of 12.62 and 2.75 µM, respectively. In addition, etomidate inhibited steroidogenesis-related gene expression. At about 0.3 µM, etomidate significantly inhibited the expression of Akr1C14. At the higher concentration (30 µM), it also reduced the expression levels of Cyp11a1, Hsd17b3 and Srd5a1. In conclusion, etomidate directly inhibits the activities of CYP11A1 and HSD3B1, and the expression levels of Cyp11a1 and Hsd17b3, leading to the lower production of androgen by Leydig cells.

Stress triggers mitochondrial biogenesis to preserve steroidogenesis in Leydig cells.

Adaptability to stress is a fundamental prerequisite for survival. Mitochondria are a key component of the stress response in all cells. For steroid-hormones-producing cells, including also Leydig cells of testes, the mitochondria are a key control point for the steroid biosynthesis and regulation. However, the mitochondrial biogenesis in steroidogenic cells has never been explored. Here we show that increased mitochondrial biogenesis is the adaptive response of testosterone-producing Leydig cells from stressed rats. All markers of mitochondrial biogenesis together with transcription factors and related kinases are up-regulated in Leydig cells from rats exposed to repeated psychophysical stress. This is followed with increased mitochondrial mass. The expression of PGC1, master regulator of mitochondrial biogenesis and integrator of environmental signals, is stimulated by cAMP-PRKA, cGMP, and ß-adrenergic receptors. Accordingly, stress-triggered mitochondrial biogenesis represents an adaptive mechanism and does not only correlate with but also is an essential for testosterone production, being both events depend on the same regulators. Here we propose that all events induced by acute stress, the most common stress in human society, provoke adaptive response of testosterone-producing Leydig cells and activate PGC1, a protein required to make new mitochondria but also protector against the oxidative damage. Given the importance of mitochondria for steroid hormones production and stress response, as well as the role of steroid hormones in stress response and metabolic syndrome, we anticipate our result to be a starting point for more investigations since stress is a constant factor in life and has become one of the most significant health problems in modern societies.

Mycotoxin zearalenone induces apoptosis in mouse Leydig cells via an endoplasmic reticulum stress-dependent signalling pathway.

Zearalenone (ZEN) is a Fusarium mycotoxin that causes several reproductive disorders and genotoxic effects. This study demonstrated the involvement of endoplasmic reticulum (ER) stress in ZEN-induced mouse Leydig cell death. Our study showed that ZEN reduced cell proliferation in a murine Leydig tumour cell line in a dose-dependent manner. The involvement of apoptosis as a major cause of ZEN-induced cell death was further confirmed by the results of a caspase-3 activity assay, which showed a ZEN dose-dependent increase in cell death. Treatment of MLTC-1 and primary mouse Leydig cells with ZEN upregulated the expression of the ER stress-typical markers GRP78, CHOP and caspase-12 protein. Further, pre-treating the cells with 4-phenylbutyrate or knocking down GRP78 using lentivirus-encoded shRNA significantly diminished ZEN-induced apoptosis and inhibited the expression of CHOP and caspase-12. In summary, these results suggest that the activation of an ER stress pathway plays a key role in ZEN-induced apoptosis in the mouse Leydig cells.

Association of cellular and molecular alterations in Leydig cells with apoptotic changes in germ cells from testes of Graomys griseoflavus×Graomys centralis male hybrids.

Spermatogenesis is disrupted in Graomys griseoflavus×Graomys centralis male hybrids. This study was aimed to determine whether morphological alterations in Leydig cells from hybrids accompany the arrest of spermatogenesis and cell death of germ cells and whether apoptotic pathways are also involved in the response of these interstitial cells. We used three groups of 1-, 2- and 3-month-old male animals: (1) G. centralis, (2) G. griseoflavus and (3) hybrids obtained by crossing G. griseoflavus females with G. centralis males. Testicular ultrastructure was analyzed by transmission electron microscopy. TUNEL was studied using an in situ cell death detection kit and the expression of apoptotic molecules by immunohistochemistry. The data confirmed arrest of spermatogenesis and intense apoptotic processes of germ cells in hybrids. These animals also showed ultrastructural alterations in the Leydig cells. Fas, FasL and calbindin D28k overexpression without an increase in DNA fragmentation was detected in the Leydig cells from hybrids. In conclusion, the sterility of Graomys hybrids occurs with ultrastructural changes in germ and Leydig cells. The enhancement of Fas and FasL is not associated with cell death in the Leydig cells. Probably the apoptosis in these interstitial cells is inhibited by the high expression of the antiapoptotic molecule calbindin D28k.