Oxidative Stress and Endoplasmic Reticulum Stress Are Involved in the Protective Effect of Alpha Lipoic Acid Against Heat Damage in Chicken Testes.
ABSTRACT: Heat stress (HS) causes testicular injury, resulting in decreased fertility. Alpha-lipoic acid (ALA) is a well-known antioxidant. The aim of this study was to investigate the protective effects of ALA on HS-induced testicular damage in chickens. Histological changes; biomarkers of oxidative stress, including glutathione peroxidase (GPx), superoxide dismutase (SOD), catalase (CAT), and malondialdehyde (MDA); markers of endoplasmic reticulum (ER) stress, including glucose-regulated protein 78 (GRP78) and CCAAT/enhancer binding protein homologous protein (CHOP); apoptosis-related modulators, including Bax, Bcl-2, and caspase 3, in testicular tissue and serum testosterone levels were evaluated in chickens under heat stress. Heat stress induces spermatogenic cell abnormalities in chicken testes. Compared to the HS group, the histomorphological abnormalities in testicular tissue were visibly ameliorated, with significant increases in the enzyme activities of GPx, SOD, and CAT, increased serum testosterone concentration, and decreased MDA levels in the ALA + HS group. Consistent with these results, compared with the HS group, the protein levels of GRP78, CHOP, caspase 3, and Bax were significantly decreased, whereas Bcl-2, StAR, and 3?-HSD protein levels were increased in the ALA + HS group. Collectively, these findings suggest that ALA significantly ameliorates the heat-induced histomorphological abnormalities in the testes and decreased testosterone production by potentiating the activities of anti-oxidative enzymes (GPx, SOD, and CAT), inhibiting ER stress-related apoptotic pathways (Bax, Bcl-2, and caspase 3), and increasing steroidogenic gene (StAR and 3?-HSD) expression in chickens.
Project description:Galactooligosaccharides (GOS) that are delivered in ovo improve intestinal microbiota composition and mitigate the negative effects of heat stress in broiler chickens. Hubbard hybrids are slow-growing chickens with a high resistance to heat. In this paper, we determined the impact of GOS delivered in ovo on slow-growing chickens that are challenged with heat. The experiment was a 2 × 2 × 2 factorial design. On day 12 of incubation, GOS (3.5 mg/egg) was delivered into the egg (n = 300). Controls (C) were mock-injected with physiological saline (n = 300). After hatching, the GOS and C groups were split into thermal groups: thermoneutral (TN) and heat stress (HS). HS (30 °C) lasted for 14 days (days 36-50 post-hatching). The spleen (n = 8) was sampled after acute (8.5 h) and chronic (14 days) HS. The gene expression of immune-related (IL-2, IL-4, IL-6, IL-10, IL-12p40, and IL-17) and stress-related genes (HSP25, HSP90AA1, BAG3, CAT, and SOD) was detected with RT-qPCR. Chronic HS up-regulated the expression of the genes: IL-10, IL-12p40, SOD (p < 0.05), and CAT (p < 0.01). GOS delivered in ovo down-regulated IL-4 (acute p < 0.001; chronic p < 0.01), IL-12p40, CAT and SOD (chronic p < 0.05). The obtained results suggest that slow-growing hybrids are resistant to acute heat and tolerant to chronic heat, which can be supported with in ovo GOS administration.
Project description:It is well established that physiological stress has an adverse effect on the male reproductive system. Experimental studies have demonstrated the promising effects of MOTILIPERM in male infertility. MOTILIPERM extract is composed of three crude medicinal herbs: Morinda officinalis How (Rubiaceae) roots, Allium cepa L. (Liliaceae) outer scales, and Cuscuta chinensis Lamark (convolvulaceae) seeds. The present study aimed to investigate the possible mechanisms responsible for the effects of MOTILIPERM on testicular dysfunction induced by immobilization stress. Fifty male Sprague Dawley rats were divided into five groups (10 rats each): a normal control group (CTR), a control group administered MOTILIPERM 200 mg/kg (M 200), an immobilization-induced stress control group (S), an immobilization-induced stress group administered MOTILIPERM 100 mg/kg (S + M 100), and MOTILIPERM 200 mg/kg (S + M 200). Stressed rats (n = 30) were subjected to stress by immobilization for 6 h by placing them in a Perspex restraint cage, while controls (n = 20) were maintained without disturbance. Rats were administrated 100 or 200 mg/kg MOTILIPERM once daily for 30 days 1 h prior to immobilization. At the end of the treatment period, we measured body and reproductive organ weight; sperm parameters; histopathological damage; reproductive hormone levels; steroidogenic acute regulatory protein (StAR); biomarkers of oxidative stress; and apoptosis markers. MOTILIPERM treatment improved testicular dysfunction by up-regulating (p < 0.05) sperm count, sperm motility, serum testosterone level, StAR protein level, Johnsen score, and spermatogenic cell density in stressed rats. MOTILIPERM decreased oxidative stress by increasing (p < 0.05) testicular superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione peroxidase-4 (GPx 4), catalase, nuclear factor erythroid 2-related factor 2 (Nrf2), and heme oxygenase 1 (HO-1) levels and decreasing (p < 0.05) malondialdehyde (MDA) and reactive oxygen species/reactive nitrogen species (ROS/RNS) levels. Furthermore, MOTILIPERM down-regulated (p < 0.05) cleaved caspase 3 and BCL2 associated X protein (Bax) levels; increased pro caspase-3 and B-cell lymphoma 2 (Bcl-2) levels; and upregulated testicular germ cell proliferation in stressed rats. The number of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive cells and serum luteinizing hormone (LH) and follicle stimulating hormone (FSH) levels also significantly (p < 0.05) decreased after pretreatment with MOTILIPERM in stressed rats. Collectively, our results suggest that, in immobilization-mediated stress-induced testicular dysfunction, MOTILIPERM sustains normal spermatogenesis via antioxidant and anti-apoptotic activities by activating the NRF/HO-1 signaling pathway.
Project description:Heat stress (HS) is a critical concern to the poultry industry as it affects both productivity and well-being. Various managerial and nutritional strategies have been proposed to mitigate the negative effects of HS in chickens, with plant-based additives showing promise. Recently, we reported the positive effect of a phytogenic feed additive (PFA) on growth performance in HS birds. Owing to the antioxidant nature of these compounds, we sought to further explore the effect of PFA on whole blood circulating chemokines, cytokines, and inflammasomes in HS broilers. Broilers (600 males, 1 d) were randomly assigned to 12 environmental chambers, subjected to 2 environmental conditions (12 h cyclic heat stress, HS, 35°C vs. thermoneutral condition [TN], 24°C) and fed 3 diets (control, PFA-C 250 ppm, PFA-C 400 ppm) in a 2 × 3 factorial design. After 21 d of cyclic HS, blood samples were collected for target gene expression analysis. HS upregulated the expression of superoxide dismutase 1 (SOD1) and downregulated glutathione peroxidase-3 (GPX-3), and there was diet × temperature interaction for SOD2, GPX-1, and GPX-3, where gene expression was increased by PFA-C250 during HS but was unchanged for PFA-C400. Plasma total antioxidant capacity (TAC) and malondialdehyde (MDA) content were increased by HS. Gene expression of interleukin-18 (IL-18) was decreased by HS, without further effect of PFA. HS increased tumor necrosis factor ? (TNF?), but this effect was mitigated by PFA-C400. C-C motif chemokine ligands 4 and 20 (CCL4 and CCL20) showed a similar pattern to TNF?, with PFA-C400 ameliorating the negative effect of HS. The nucleotide-binding, leucine-rich repeat and pyrin domain containing 3 (NLRP3) inflammasome was decreased by HS and further lowered by PFA-C400, but the nucleotide-binding oligomerization domain, leucine-rich repeat, and CARD domain containing 3 (NLRC3) and nucleotide-binding, leucine-rich repeat containing X1 (NLRX1) inflammasomes were increased by PFA under TN conditions, with no effects of HS. Heat shock proteins (HSP) and heat shock factors (HSF) were unaffected by PFA or HS. Together these data indicate that gene expression of circulating inflammatory factors are dysregulated during HS, and supplemental dietary PFA may be protective.
Project description:BACKGROUND:Monotropein, astragalin, and spiraeoside (MAS) are active compounds extracted from medicinal herbs; monotropein from Morinda officinalis How (Rubiaceae), astragalin (kaempferol 3-O-glucoside) from Cuscuta chinensis Lamark (Convolvulaceae) and spiraeoside from the outer scales of Allium cepa L. (Liliceae) in a ratio of 6.69:0.41:3.61. Monotropein, astragalin, and spiraeoside are well-known antioxidants, anti-inflammatory, and antinociceptive agents. The current investigation aims to study the molecular mechanism of varicocele-induced male infertility and the underlying pharmacological mechanisms of MAS. METHODS:Four groups were included: control (CTR), MAS 200 group (MAS 200?mg/kg), varicocele group (VC), and VC?+?MAS 200 group (MAS 200?mg/kg). Sprague-Dawley (SD) rats were treated with 200?mg/kg MAS or vehicle once daily for 28?days. The possible signaling mechanism and effects of MAS were measured via histological staining, immunohistochemistry, western blot, and biochemical assays. RESULTS:Parameters such as sperm motility and count, Johnsen's scores, spermatogenic cell density, serum testosterone, testicular superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx) and expression of the steroidogenic acute regulatory protein (StAR) improved significantly in the VC?+?MAS 200 group compared with the VC group. MAS treatment of varicocele-induced group significantly decreased the levels of serum luteinizing hormone (LH) and follicle-stimulating hormone (FSH), as well as testicular interleukin-6 (IL6), tumor necrosis factor-? (TNF-?), ROS/RNS, and malondialdehyde (MDA). It also decreased the apoptotic index and reduced the expression of endoplasmic reticulum (ER) protein levels (Grp78, p-IRE1?, and p-JNK) and apoptotic markers such as cleaved caspase-3 and Bax/Bcl2 ratio. CONCLUSION:This study suggests that the crosstalk between oxidative stress, ER stress, and mitochondrial pathway mediates varicocele-induced testicular germ cell apoptosis. MAS promotes spermatogenesis in varicocele-induced SD rat, probably by decreasing cytokines (IL-6, TNF-?) levels, regulating abnormal sex hormones, and decreasing oxidative stress, ER stress, and apoptosis.
Project description:Scrotal hyperthermia leads to oxidative stress and apoptosis in spermatogenic cells, which subsequently causes male infertility. In this study, we examined the effects of ?-carotene and/or curcumin on heat-stress- (HS-) induced testicular injuries in mice. ICR male mice (8 weeks old) were consecutively treated with ?-carotene (10?mg/kg) and/or curcumin (20?mg/kg) orally once a day for 14 days and then subjected to single exposure with scrotal HS at 43°C for 15?min on day 7. HS induced a significant reduction in testicular weight, appearance of multinucleated giant cells, and desquamation of germ cells in destructive seminiferous tubules, as well as degenerative Leydig cells. Moreover, HS reduced the superoxide dismutase (SOD) activity and mRNA levels of mitochondrial SOD, phospholipid hydroperoxide glutathione peroxidase, B-cell lymphoma-extra-large, and 3?-hydroxysteroid dehydrogenase, with increases in lipid peroxidation levels and mRNA levels of BCL2-associated X protein and caspase-3 relative to those of the control group. However, these changes were significantly recovered by combined treatment with ?-carotene and curcumin after HS. These findings indicate that the combined treatment with ?-carotene and curcumin might be a valuable protective agent to ameliorate hyperthermic spermatogenic disorders via its potent antioxidative, antiapoptotic, and androgen synthetic effects.
Project description:Schisandra chinensis Baillon (SC) has been utilized for its antioxidants and anti-inflammatory activities in a broad variety of medical applications. However; SC uses for improving fertility in males and related disorders with proper scientific validation remain obscure. The present study aimed to investigate the effects of SC on varicocele (VC)-induced testicular dysfunction and the potential molecular mechanism associated with VC-induced germ cell apoptosis. The male Sprague-Dawley rats were equally divided into four groups consisting of 10 rats in a normal control group (CTR), a control group administered SC 200 mg/kg (SC 200), a varicocele-induced control group (VC), and a varicocele-induced group administered SC 200 mg/kg (VC + SC 200). Rats were administrated 200 mg/kg SC once daily for 28 days after induction of varicocele rats and sham controls. At the end of the treatment period, body and reproductive organ weight, sperm parameters, histopathological damages, proinflammatory cytokines, apoptosis markers, biomarkers of oxidative stress, endoplasmic reticulum (ER) stress, and steroidogenic acute regulatory protein (StAR) were evaluated. The effects of SC extract on human sperm motility were also analyzed. SC treatment reduces VC-induced testicular dysfunction by significantly increasing testicular weight, sperm count and sperm motility, serum testosterone level, Johnsen score, spermatogenic cell density, testicular superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase level, and steroidogenic acute regulatory protein (StAR) level. Furthermore, the effects of SC on malondialdehyde (MDA) level, reactive oxygen species (ROS)/reactive nitrogen species (RNS) level, apoptotic index, serum luteinizing hormone (LH) and follicle stimulating hormone (FSH) levels, Glucose-regulated protein-78 (Grp 78), phosphorylated c-Jun-N-terminal kinase (p-JNK), phosphorylated inositol-requiring transmembrane kinase/endoribonuclease 1? (p-IRE1?), cleaved caspase 3, and Bax:Bcl2 in VC-induced rats were significantly decreased. Treatment with SC extracts also increased sperm motility in human sperm. Our findings suggest that the SC ameliorate testicular dysfunction in VC-induced rats via crosstalk between oxidative stress, ER stress, and mitochondrial-mediated testicular germ cell apoptosis signaling pathways. SC promotes spermatogenesis by upregulating abnormal sex hormones and decreasing proinflammatory cytokines (interleukin-6; TNF-?).
Project description:Background:DA-9401 was prepared as a mixture of Chinese medicinal herb extracts from roots of Morinda officinalis How (Rubiaceae), outer scales of Allium cepa L. (Liliceae) and seeds of Cuscuta chinensis Lamark (Convolvulaceae). The present study was designed to investigate the possible protective role of DA-9401 in adriamycin (ADR)-induced testicular toxicity associated with oxidative stress, endoplasmic reticulum (ER) stress, and apoptosis. Methods:Fifty healthy 8-week-old male Sprague-Dawley rats were equally divided into five groups. The first CTR group was treated with normal saline 2 ml/day by gavage. The second was treated with DA-100 (DA-9401 100 mg/kg/day). The third (ADR) group received ADR (2 mg/kg/once a week) intraperitoneally, while the combination of ADR and DA-9401 was given to the fourth ADR + DA-100 (100 mg/kg/day p.o) group and fifth ADR + DA-200 (200 mg/kg/day p.o) group. At the end of the 8-week treatment period, body weight, reproductive organ weights, fertility rate, pups per female were recorded, and serum were assayed for hormone concentrations. Tissues were subjected to semen analysis, histopathological changes, interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), oxidative stress markers and expression levels of endoplasmic reticulum (ER) stress markers, apoptosis markers, tight junction protein markers, steroidogenic acute regulatory protein (StAR), cation channel of sperm (CatSper) and glycogen synthase kinase-3 (GSK-3) by western blot. Results:DA-9401 administration to ADR-treated rats significantly decreased serum luteinizing hormone (LH) and follicle-stimulating hormone (FSH) levels, interleukin-6, TNF-α, MDA level, ROS/RNS level, ER stress response protein levels, tunnel positive cells, cleaved caspase-3, and Bax/Bcl2 ratio. Moreover, pretreatment with DA-9401 significantly increased body weight, reproductive organ weights, fertility rate, pups per female, Johnsen's score, spermatogenic cell density, sperm count and sperm motility, serum testosterone concentration, testicular superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx), tight junction protein markers, star protein level, CatSper, and GSK-3 level. Conclusions:ADR treatment can markedly impair testicular function and induce testicular cell death presumably by causing significant changes in oxidative stress, ER stress, and mitochondrial pathway. DA-9401 exerts beneficial effects against oxidative stress, ER stress, and mitochondria-mediated cell death pathway in testis tissue by up-regulating expression levels of tight junction protein markers, steroidogenic acute regulatory protein, GSK-3 alpha, and cation channels of sperm.
Project description:Vitrification is an economically effective method for embryo cryopreservation in human and livestock animals; however, it carries the risk of damage by the exposure to severe oxidative stress. The present study was conducted to evaluate the effect of leptin at different levels on the in vitro development of fresh and vitrified preimplantation embryos in a rabbit model. Normal embryos at morulae stage were randomly cultured for 2 h with 0, 10, 20 or 100 ng/mL of leptin, then were cultured for further 48 h as freshly or after vitrification. Thereafter, developed blastocysts form the best leptin level in fresh and vitrified embryos along with their controls were allocated to analyze the pro-oxidant (malondialdehyde, MDA; nitric oxide, NO), antioxidant (total antioxidant capacity, TAC; superoxide dismutase, SOD; glutathione peroxidase, GPx), apoptotic (Bcl-2 associated X protein, BAX; heat shock 60kD protein member 1, HSP60; tumor necrosis factor alpha, TNF?) and developmental (sex determining region Y box protein 2, SOX2; Nanog homeobox protein, NANOG; Octamer-binding protein 4, OCT4) biomarkers. Results indicate that expanding and hatching rates of embryos were significantly higher at 20 ng/mL leptin than the other levels, while vitrification had an independent suppression effect on the in vitro development rates. The MDA and NO were significantly higher, while TAC, SOD and GPx were significantly lower in the vitrified than fresh embryos. In contrast, leptin treatment significantly decreased the pro-oxidant biomarkers and increased the antioxidant biomarkers in both fresh and vitrified embryos. Vitrification significantly increased the antiapoptotic biomarkers, and decreased the developmental biomarkers in embryos. In contrast, leptin decreased the BAX and TNF?, increased the HSP60, and moreover, ameliorated the reduction of developmental biomarkers in the vitrified embryos. These results conclude that leptin could be used as antiapoptotic and antioxidant promotor to support the in vitro embryonic development, particularly under oxidative stress emerged from cryopreservation programs.
Project description:Under conditions of high ambient temperatures and/or strenuous exercise, humans and animals experience considerable heat stress (HS) leading among others to intestinal epithelial damage through induction of cellular oxidative stress. The aim of this study was to characterize the effects of ?-Lipoic Acid (ALA) on HS-induced intestinal epithelial injury using an in vitro Caco-2 cell model.A confluent monolayer of Caco-2 cells was pre-incubated with ALA (24 h) prior to control (37?°C) or HS conditions (42?°C) for 6 or 24 h and the expression of heat shock protein 70 (HSP70), heat shock factor-1 (HSF1), and the antioxidant Nrf2 were investigated. Intestinal integrity was determined by measuring transepithelial resistance, paracellular permeability, junctional complex reassembly, and E-cadherin expression and localization. Furthermore, cell proliferation was measured in an epithelial wound healing assay and the expression of the inflammatory markers cyclooxygenase-2 (COX-2) and transforming growth Factor-? (TGF-?) was evaluated.ALA pretreatment increased the HSP70 mRNA and protein expression under HS conditions, but did not significantly modulate the HS-induced activation of HSF1. The HS-induced increase in Nrf2 gene expression as well as the Nrf2 nuclear translocation was impeded by ALA. Moreover, ALA prevented the HS-induced impairment of intestinal integrity. Cell proliferation under HS conditions was improved by ALA supplementation as demonstrated in an epithelial wound healing assay and ALA was able to affect the HS-induced inflammatory response by decreasing the COX-2 and TGF-? mRNA expression.ALA supplementation could prevent the disruption of intestinal epithelial integrity by enhancing epithelial cell proliferation, and reducing the inflammatory response under HS conditions in an in vitro Caco-2 cell model.
Project description:The current study examined the efficacy of royal jelly (RJ) against cadmium chloride (CdCl?)-induced testicular dysfunction. A total of 28 Swiss male mice were allocated into four groups (n = 7), and are listed as follows: (1) the control group, who was intraperitoneally injected with physiological saline (0.9% NaCl) for 7 days; (2) the RJ group, who was orally supplemented with RJ (85 mg/kg daily equivalent to 250 mg crude RJ) for 7 days; (3) the CdCl? group, who was intraperitoneally injected with 6.5 mg/kg for 7 days; and (4) the fourth group, who was supplemented with RJ 1 h before CdCl? injection for 7 days. Cd-intoxicated mice exhibited a decrease in serum testosterone, luteinizing hormone (LH), and follicle stimulating hormone (FSH) levels. A disturbance in the redox status in the testicular tissue was recorded, as presented by the increase in lipid peroxidation and nitrate/nitrite levels and glutathione (GSH) depletion. Moreover, the activities of glutathione peroxidase (GPx), glutathione reductase (GR), superoxide dismutase (SOD), catalase (CAT), and nuclear factor (erythroid-derived 2)-like-2 factor (Nrf2) and their gene expression were inhibited. In addition, interleukin-1ß (IL-1?) and tumor necrosis factor-? (TNF-?) levels were elevated. Furthermore, Cd triggered an apoptotic cascade via upregulation of caspase-3 and Bax and downregulation of Bcl-2. Histopathological examination showed degenerative changes in spermatogenic cells, detachment of the spermatogenic epithelium from the basement membrane, and vacuolated seminiferous tubules. Decreased cell proliferation was reflected by a decrease in proliferating cell nuclear antigen (PCNA) expression. Interestingly, RJ supplementation markedly minimized the biochemical and molecular histopathological changes in testes tissue in response to Cd exposure. The beneficial effects of RJ could be attributed to its antioxidative properties.