Stress induces glucocorticoid-mediated apoptosis of rat Leydig cells in vivo.

Stress can disrupt endocrine signalling in the male reproductive axis through high concentrations of glucocorticoids, the hallmark of stress. Our previous work revealed that a stress level of exogenous glucocorticoids could induce apoptosis of rat Leydig cells, which are the primary source of testosterone. The aim of this study was to investigate whether stress can induce apoptosis in rat Leydig cells in vivo and, if so, whether the process is the result of a direct effect of glucocorticoids. In a chronically stressed rat model, serum corticosterone concentration was increased significantly whereas serum testosterone was decreased. The frequency of apoptotic Leydig cells in stressed rats was also increased. Adrenalectomised rats subjected to chronic stress showed an elevated serum testosterone, while the apoptotic frequency of Leydig cells was not increased. It was established that glucocorticoid-induced Leydig cell apoptosis is mediated by glucocorticoid receptors (GRs), which translocate from cytoplasm to nucleus. Adenovirus microRNA-induced downregulation of GR expression in vitro alleviated the corticosterone-induced increase in apoptosis of Leydig cells. These results indicate that the stress-induced increase in corticosterone secretion resulted in apoptosis in rat Leydig cells in vivo, and thereby decreased testosterone synthesis.

Luteinizing hormone induces expression of 11beta-hydroxysteroid dehydrogenase type 2 in rat Leydig cells.

BACKGROUND: Leydig cells are the primary source of testosterone in male vertebrates. The biosynthesis of testosterone in Leydig cells is strictly dependent on luteinizing hormone (LH). On the other hand, it can be directly inhibited by excessive glucocorticoid (Corticosterone, CORT, in rats) which is beyond the protective capability of 11beta-Hydroxysteroid dehydrogenase type 1 (11beta-HSD1) and type 2 (11beta-HSD2; encoded by gene Hsd11b2 in rats) in Leydig cells. Our previous study found that LH increases 11beta-HSD1 expression in rat Leydig cells, but the effect of LH on the expression and activity of 11beta-HSD2 is not investigated yet. METHODS: The Leydig cells were isolated from male Sprague-Dawley rats (90 days of age). After Leydig cells were incubated either for 24 h with various concentrations of LH (2.5, 5, 10 and 20 ng/mL) or for different time periods (2, 8, 12 and 24 h) with 20 ng/mL LH, the mRNA expression of 11beta-HSD2 was measured by real-time PCR. 11beta-HSD2 protein levels in Leydig cells were assayed by Western Blot and 11beta-HSD2 enzyme activity was determined by calculating the ratio of conversion of [3H]CORT to [3H]11-dehydrocorticosterone by 24 h after stimulation with 20 ng/ml LH. Four reporter gene plasmids containing various lengths of Hsd11b2 promoter region were constructed and transfected into mouse Leydig tumor cells to investigate the effect of LH on Hsd11b2 transcription. A glucocorticoid-responsive reporter gene plasmid, GRE-Luc, was constructed. To evaluate influence of LH on intracellular glucocorticoid level, rat Leydig cells were transfected with GRE-Luc, and luciferase activities were measured after incubation with CORT alone or CORT plus LH. RESULTS: We observed dose- and temporal-dependent induction of rat 11beta-HSD2 mRNA expression in Leydig cells subject to LH stimulation. The protein and enzyme activity of 11beta-HSD2 and the luciferase activity of reporter gene driven by promoter regions of Hsd11b2 were increased by LH treatment. LH decreased the glucocorticoid-induced luciferase activity of GRE-Luc reporter gene. CONCLUSION: The results of the present study suggest that LH increases the expression and enzyme activity of 11beta-HSD2, and therefore enhances capacity for oxidative inactivation of glucocorticoid in rat Leydig cells in vitro.

Mechanism of nuclear factor of activated T-cells mediated FasL expression in corticosterone -treated mouse Leydig tumor cells.

BACKGROUND: Fas and FasL is important mediators of apoptosis. We have previously reported that the stress levels of corticosterone (CORT, glucocorticoid in rat) increase expression of Fas/FasL and activate Fas/FasL signal pathway in rat Leydig cells, which consequently leads to apoptosis. Moreover, our another study showed that nuclear factor of activated T-cells (NFAT) may play a potential role in up-regulation of FasL during CORT-treated rat Leydig cell. It is not clear yet how NFAT is involved in CORT-induced up-regulation of FasL. The aim of the present study is to investigate the molecular mechanisms of NFAT-mediated FasL expression in CORT-treated Leydig cells. RESULTS: Western blot analysis showed that NFAT2 expression is present in mouse Leydig tumor cell (mLTC-1). CORT-induced increase in FasL expression in mLTC-1 was ascertained by Western Blot analysis and CORT-induced increase in apoptotic frequency of mLTC-1 cells was detected by FACS with annexin-V labeling. Confocal imaging of NFAT2-GFP in mLTC-1 showed that high level of CORT stimulated NFAT translocation from the cytoplasm to the nucleus. RNA interference-mediated knockdown of NFAT2 significantly attenuated CORT-induced up-regulation of FasL expression in mLTC. These results corroborated our previous finding that NFAT2 is involved in CORT-induced FasL expression in rat Leydig cells and showed that mLTC-1 is a suitable model for investigating the mechanism of CORT-induced FasL expression. The analysis of reporter constructs revealed that the sequence between -201 and +71 of mouse FasL gene is essential for CORT-induced FasL expression. The mutation analysis demonstrated that CORT-induced FasL expression is mediated via an NFAT binding element located in the -201 to +71 region. Co-transfection studies with an NFAT2 expression vector and reporter construct containing -201 to +71 region of FasL gene showed that NFAT2 confer a strong inducible activity to the FasL promoter at its regulatory region. In addition, chromatin immunoprecipitation assay further confirmed the results of reporter gene studies by showing the specific binding of NFAT2 to the -201 to +71 region. CONCLUSION: In the present study, we demonstrated that NFAT2 directly stimulates transcription of FasL in high level CORT-treated mLTC-1. In conclusion, the present study provides further evidence for our finding that CORT-induced FasL expression in Leydig cells is mediated by NFAT.

NFAT2 is implicated in corticosterone-induced rat Leydig cell apoptosis.

AIM: To investigate the activation of the nuclear factor of activated T cells (NFAT) and its function in the corticosterone (CORT)-induced apoptosis of rat Leydig cells. METHODS: NFAT in rat Leydig cells was detected by Western blotting and immunohistochemical staining. Cyclosporin A (CsA) was used to evaluate potential involvement of NFAT in the CORT-induced apoptosis of Leydig cells. Intracellular Ca(2+) was monitored in CORT-treated Leydig cells using Fluo-3/AM. After the Leydig cells were incubated with either CORT or CORT plus CsA for 12 h, the levels of NFAT2 in the nuclei and in the cytoplasm were measured by semi-quantitative Western blotting. The role of NFAT2 in CORT-induced Leydig cell apoptosis was further evaluated by observing the effects of NFAT2 overexpression and the inhibition of NFAT2 activation by CsA on FasL expression and apoptosis. RESULTS: We found that NFAT2 was the predominant isoform in Leydig cells. CsA blocked the CORT-induced apoptosis of the Leydig cells. The intracellular Ca(2+) level in the Leydig cells was significantly increased after the CORT treatment. The CORT increased the level of NFAT2 in the nuclei and decreased its level in the cytoplasm. CsA blocked the CORT-induced nuclear translocation of NFAT2 in the Leydig cells. Both CORT-induced apoptosis and FasL expression in the rat Leydig cells were enhanced by the overexpression of NFAT2 and antagonized by CsA. CONCLUSION: NFAT2 was activated in CORT-induced Leydig cell apoptosis. The effects of NFAT2 overexpression and the inhibition of NFAT2 activation suggest that NFAT2 may potentially play a pro-apoptotic role in CORT-induced Leydig cell apoptosis through the up-regulation of FasL.

Involvement of nuclear factor-kappa B on corticosterone- induced rat Leydig cell apoptosis.

AIM: To investigate the activation of nuclear factor-kappa B (NF-kappa B) and its function in glucocorticoid-induced Leydig cell apoptosis. METHODS: The Leydig cells were isolated from male Sprague-Dawley rats (90 days of age) and were incubated with corticosterone (CORT, glucocorticoid in rat) for 6 h, 12 h and 24 h, respectively. The P65 subunit of NF-kappa B (NF-kappa B/P65) in nuclei and the inhibitor of NF-kappa B (Ikappa B) in cytoplasm were analyzed by Western-blotting. The Leydig cells were treated with anti-Fas antibody for 3 h followed by Western blotting to assay the changes of NF-kappa B/P65 in nuclei and in cytoplasm. The role of NF-kappa B in CORT-induced Leydig cell apoptosis was evaluated by observing the effects of NF-kappa B/P65 overexpression and inhibiting activation of NF-kappa B by 100 micromol/L Pyrrolidine dithiocarbamate (PDTC) on this apoptosis. RESULTS: The treatment of Leydig cells with CORT increased the levels of NF-kappa B/P65 in nuclei and decreased the levels of Ikappa B in cytoplasm. Following the Leydig cells were treated with anti-Fas antibody, the levels of NF-kappaB/P65 was increased in nuclei and decreased in cytoplasm. The CORT-induced Leydig cell apoptosis was inhibited by overexpressed NF-kappaB/P65 and was enhanced by incubation with PDTC. CONCLUSION: NF-kappa B is activated by increased FasL/Fas in CORT-induced Leydig cell apoptosis. NF-kappa B may play an anti-apoptotic role in this apoptosis.

Glucocorticoid induces apoptosis in rat leydig cells.

The aim of the present study was to investigate whether glucocorticoid induces apoptosis in rat Leydig cells. To determine whether there are developmental differences in glucocorticoid sensitivity, Leydig cells were isolated at distinct stages of their differentiation [mesenchymal-like progenitors (PLC), immature Leydig cells (ILC), and adult Leydig cells (ALC)] from 21-, 35-, and 90-d-old Sprague Dawley rats, respectively. Glucocorticoid induction of apoptosis was evaluated after both in vitro and in vivo exposures. In the first set of experiments, PLC, ILC, and ALC were treated with 100 nM corticosterone (CORT) for either 4 or 24 h in vitro and then assessed for labeling with the apoptotic marker annexin V. PLC exposed to CORT had levels of annexin V-fluorescein isothiocyanate labeling that were unchanged relative to control values at both time points (P > 0.05). In contrast, CORT-treated ILC and ALC had increased frequencies of apoptosis: in ALC, a 22.1 +/- 1.7% incidence after 4 h and 30.5 +/- 2.3% after 24 h compared with 7.4 +/- 0.8% in untreated controls (P < 0.05). Similar trends were observed for ILC. Ultrastructural analysis confirmed that the increase in annexin V labeling was associated with characteristic signs of apoptosis, including nuclear fragmentation and formation of apoptotic bodies. A second line of experiments examined whether apoptosis was evident in purified Leydig cells after administration of CORT in vivo. Male rats were subjected to bilateral adrenalectomy and were treated with CORT by ip injection twice daily at doses ranging from 2.5-7.5 mg/100 g BW starting 3 d after surgery. The frequency of Leydig cell apoptosis was measured at 12, 24, 48, and 72 h after the first injection. Administration of the 2.5-mg dose raised circulating CORT 5-10 times above normal basal concentrations, and LH levels sampled at these times were not altered in the treated animals. Increased Leydig cell apoptosis was measurable after 24 h of treatment, with an incidence of 21.1 +/- 1.8% in ALC compared with 5.7 +/- 0.8% in untreated controls (P < 0.05). Sharp reductions in immunocytochemical staining intensity were observed in the treated animals for a Leydig cell marker, 11beta-hydroxysteroid dehydrogenase, which occurred concurrently with decreased serum T levels. This was consistent with the hypothesis that CORT-mediated induction of apoptosis leads to declines in Leydig cell numbers, thereby affecting T production. These results suggest that excessive exposure to CORT initiates apoptosis in rat Leydig cells, potentially contributing to suppression of circulating T levels during stress and other conditions in which glucocorticoid concentrations are elevated.

Mechanisms of glucocorticoid-induced Leydig cell apoptosis.

The high levels of corticosterone (CORT) that are typically achieved during stress induce apoptotic death of Leydig cells. The intracellular mechanisms by which CORT acts on Leydig cells to induce apoptosis are unknown, and the present study tested for mediation by Fas ligand (FasL), a member of the tumor necrosis factor ligand family, in association with caspase activation. In addition, another apoptotic pathway involving in the participation of mitochondria was studied by evaluation of mitochondrial membrane potential (DeltaPsi) loss and generation of reactive oxygen species (ROS), which are early apoptotic events in many cell types. Rat Leydig cells were isolated from adrenalectomized rats on day 90 postpartum at 3, 6, 12, 24 and 48 h after the start of CORT administration (at a dose of 5 mg total/100 g body weight per day intraperitoneally in two daily injections starting 3 days after surgery). Both FasL and Fas receptor protein levels, analyzed by Western blot and fluorescent immunohistochemistry, increased at 6 h after the start of CORT administration, peaking at 24 h and declining thereafter. Leydig cell caspase-3 activity was analyzed in vitro. Low molecular weight DNA fragments that are characteristic of apoptosis were evident in Leydig cells by 12 h of exposure to 100 nM CORT in vitro, and the abundance of the fragments was more pronounced at 24 h. In the presence of a specific caspase inhibitor, Ac-DEVD-CHO, Leydig cell apoptosis was suppressed, corroborating the hypothesis that caspase-3 is involved in CORT-mediated cell death. Western blotting analysis revealed that procaspase-3 was present only at low levels in untreated control Leydig cells, and increased by 6 h of CORT administration. By 12 h, however, procaspase-3 was significantly reduced, and the cleaved, active caspase-3 forms appeared and increased through 24 h. These results indicated that FasL/Fas and caspase were implicated in CORT-mediated Leydig cell apoptosis. Decreased DeltaPsi and increased ROS generation were also measurable in Leydig cells for up to 2 days following CORT administration in vitro. These data indicate that activation of the Fas system, cleavage of procaspase-3, loss of DeltaPsi and increased ROS generation are all implicated in the process of CORT-induced Leydig cell death.

Chronic stress induces ageing-associated degeneration in rat Leydig cells.

Several studies have suggested that stress and ageing exert inhibitory effects on rat Leydig cells. In a pattern similar to the normal process of Leydig cell ageing, stress-mediated increases in glucocorticoid levels inhibit steroidogenic enzyme expression that then results in decreased testosterone secretion. We hypothesized that chronic stress accelerates the degenerative changes associated with ageing in Leydig cells. To test this hypothesis, we established a model of chronic stress to evaluate stress-induced morphological and functional alterations in Brown Norway rat Leydig cells; additionally, intracellular lipofuscin levels, reactive oxygen species (ROS) levels and DNA damage were assessed. The results showed that chronic stress accelerated ageing-related changes: ultrastructural alterations associated with ageing, cellular lipofuscin accumulation, increased ROS levels and more extensive DNA damage were observed. Additionally, testosterone levels were decreased. This study sheds new light on the idea that chronic stress contributes to the degenerative changes associated with ageing in rat Leydig cells in vivo.

Stress hormone and male reproductive function.

The Leydig cell is the primary source of testosterone in males. Levels of testosterone in circulation are determined by the steroidogenic capacities of individual Leydig cells and the total numbers of Leydig cells per testis. Stress-induced increases in serum glucocorticoid concentrations inhibit testosterone-biosynthetic enzyme activity, leading to decreased rates of testosterone secretion. It is unclear, however, whether the excessive glucocorticoid stimulation also affects total Leydig cell numbers through induction of apoptosis and thereby contributes to the stress-induced suppression of androgen levels. Exposure of Leydig cells to high concentrations of corticosterone (CORT, the endogenously secreted glucocorticoid in rodents) increases their frequency of apoptosis. Studies of immobilization stress indicate that stress-induced increases in CORT are directly responsible for Leydig cell apoptosis. Access to glucocorticoid receptors in Leydig cells is modulated by oxidative inactivation of glucocorticoid by 11 beta-hydroxysteroid dehydrogenase (11 betaHSD). Under basal levels of glucocorticoid, sufficient levels of glucocorticoid metabolism occur and there is likely to be minimal binding of the glucocorticoid receptor. We have established that Leydig cells express type 1 11 betaHSD, an oxidoreductase, and type 2, a unidirectional oxidase. Generation of redox potential through synthesis of the enzyme cofactor NADPH, a byproduct of glucocorticoid metabolism by 11 betaHSD-1, may potentiate testosterone biosynthesis, as NADPH is the cofactor used by steroidogenic enzymes such as type 3 17beta-hydroxysteroid dehydrogenase. In this scenario, inhibition of steroidogenesis will only occur under stressful conditions when high input amounts of CORT exceed the capacity of oxidative inaction by 11 betaHSD. Changes in autonomic catecholaminergic activity may contribute to suppressed Leydig cell function during stress, and may explain the rapid onset of inhibition. However, recent analysis of glucocorticoid action in Leydig cells indicates the presence of a fast, non-genomic pathway that will merit further investigation.