P-025 Mitochondrial activity in sperm is associated to blastocyst development: a double-donation study

Molecularly Classified High Risk Myeloma Cells with Low ROS Levels and High Nestin Expression Have High Proliferation Index and GEP80 Score,

It is now widely accepted that the higher levels of reactive oxygen species (ROS) produced by damaged or deficient spermatozoa are associated with a loss of motility and a decreased capacity for sperm-oocyte fusion. Furthermore, earlier studies show, under physiological conditions, that some ROS may be involved in capacitation and hyperactivation of human spermatozoa. We measured ROS levels, acrosome reaction (AR) and acrosin activity (AA) in semen samples from suspected subfertile men to reveal the influence of ROS on AR and AA of human spermatozoa. Semen samples were obtained from 60 patients. Samples with > or = 1 x 10(6) leukocytes/mL were excluded from the study. ROS production was determined using a chemiluminescence technique. AR was determined using a triple stain technique. The percentage of acrosome-reacted spermatozoa after low temperature induction of the AR (test value), and the inducibility of AR (= the difference between the test value and the control), were calculated. The AA was analysed by determining the proteolytic potential of spermatozoa on gelatin plates. The mean halo diameter and percentage of halo formation in each sample were measured as AA parameters. Scatter plots of ROS levels and AR parameters showed that the percentage of acrosome reacted spermatozoa and AR inducibility were better in samples with low rather than high ROS levels. On the other hand, there were no apparent similarities between ROS and the AA parameters. Therefore, the percentage of acrosome-reacted spermatozoa and AR inducibility were significantly higher in the low than in the high ROS group (p = 0.028, p = 0.0001, respectively). In addition, there was no significant difference in AA parameters between groups. These findings suggest that lower ROS in semen may have a role in AR but excessive ROS may exert a negative influence on AR, while ROS in semen has no relationship to AA.

In donkeys, the use of frozen-thawed sperm for artificial insemination (AI) leads to low fertility rates. Furthermore, donkey sperm produce a large amount of reactive oxygen species (ROS), and post-AI inflammation induces the formation of neutrophil extracellular traps (NETosis), which further generates many more ROS. These high ROS levels may induce lipid peroxidation in the sperm plasma membrane, thus affecting its integrity. Enzymatic and non-enzymatic antioxidants, mainly found in the seminal plasma (SP), are responsible for maintaining the redox balance. However, this fluid is removed prior to cryopreservation, thereby exposing sperm cells to further oxidative stress. The exogenous addition of antioxidants to the freezing medium can reduce the detrimental effects caused by ROS generation. Therefore, the aim of this study was to evaluate how the addition of different reduced glutathione (GSH) concentrations (control, 2 mM, 4 mM, 6 mM, 8 mM, and 10 mM) to fresh sperm affect their cryotolerance. Total and progressive motility, kinematic parameters and motile sperm subpopulations were significantly (p < 0.05) different from the control in treatments containing 8 mM and 10 mM GSH, but not at lower concentrations. Plasma and acrosome membrane integrity, mitochondrial membrane potential (MMP) and intracellular superoxide levels (O2−) were not affected (p > 0.05) by any GSH concentration. Interestingly, however, the addition of 8 mM or 10 mM GSH reduced (p < 0.05) the percentages of viable sperm with high overall ROS levels compared to the control. In conclusion, frozen-thawed donkey sperm are able to tolerate high GSH concentrations, which differs from what has been observed in other species. This antioxidant capacity suggests that ROS could be important during post-AI and that the impact of using exogenous antioxidants like GSH to improve the sperm resilience to freeze-thawing is limited in this species.

Confocal laser scanning microscopy analysis of bioenergetic potential and oxidative stress in fresh and frozen-thawed human ovarian tissue from oncologic patients

Poisoning caused by the mushroom Amanita phalloides, due to the toxin α-amanitin, accounts for approximately 90% of food poisoning deaths in China with no specific antidotes. To investigate the role of salidroside (Sal) in α-amanitin (α-AMA)-induced mitophagy, mouse liver cells AML-12 were exposed to α-AMA in the presence of Sal or not. Intracellular reactive oxygen species (ROS) levels were measured using a ROS detection kit, mitochondrial activity was evaluated using a mitochondrial red fluorescent probe kit or JC-1 dye, and protein expression levels of PINK1, Parkin, LC3 II, P62, Bax, Bcl-2, Caspase 3, Cleaved-Caspase 3, PARP I, and Cleaved-PARP I were detected through Western blot. Results demonstrated that α-AMA led to increased intracellular ROS levels, cell apoptosis, and decreased mitochondrial membrane potential. Notably, expression levels of mitophagy-related proteins PINK1, Parkin, and LC3 increased significantly while the P62 protein expression decreased remarkably. Furthermore, Sal reversed the α-AMA-induced decrease in cell viability and mitochondrial membrane potential and increase in intracellular ROS level. In addition, Sal promoted expression levels of PINK1, Parkin, and LC3 II while suppressing the Bax/Bcl-2 ratio, Cleaved-Caspase 3, and Cleaved-PARP I as well as P62. The results above proved that salidroside alleviates α-AMA-induced mouse liver cells damage via promoting PINK1/Parkin-mediated mitophagy and reducing cell apoptosis.

Analysis of the Metabolic State of Residual Multiple Myeloma Plasma Cells Reveals a Proliferative Subset with Glycolytic Gene Expression

Many studies have suggested that there is a close correlation among declines in internal ascorbic acid (AsA) levels, various disorders, and senescence. To clarify the relationships between age-associated changes in intracellular AsA levels and the effects of AsA administration on intracellular reactive oxygen species (ROS) levels, we investigated aging-related changes in AsA uptake, ROS levels, and the effects of AsA administration on intracellular ROS levels in young and old (senescent) human fibroblasts. Our results demonstrated that AsA uptake was increased in old cells compared with young cells, although mRNA and protein expression of sodium-dependent vitamin C transporter 2 was barely altered between the young and old cells. We also demonstrated that the intracellular superoxide anion level was higher in young cells, whereas the level of intracellular peroxides was significantly increased in old cells under both normal and oxidative stress conditions. Moreover, AsA administration markedly decreased the augmentation of intracellular peroxides in old cells, whereas there was no effect of AsA treatment in young cells under both normal and oxidative stress conditions. Therefore, our results also indicate that AsA could play an important role in regulating the intracellular ROS levels in senescent cells and that the need for AsA is enhanced by cellular senescence.

本研究旨在探讨羧甲基-β-1，3葡聚糖（CMG）对人肝癌HepG2细胞X射线或12C6+离子束辐射敏感性的影响。首先用CCK-8法检测CMG对HepG2细胞的生长抑制情况，得到半数抑制浓度（IC50）为120.6μg/mL。用浓度为0.1×IC50的CMG预处理HepG2细胞24 h，再给予2 Gy X射线或12C6+离子束辐照（CMG+辐照组）；CMG未处理组直接接受2 Gy X射线或12C6+离子束辐照（辐照组）。对比分析辐照组和CMG+辐照组细胞的克隆存活、DNA损伤、凋亡与周期分布、细胞内活性氧（ROS）水平。发现：与X射线辐照组相比，相同剂量的12C6+离子辐照组克隆存活率更小，DNA损伤和周期阻滞更加严重，细胞凋亡率和细胞内ROS水平也更高。与单独X射线或12C6+离子束辐照组相比，CMG+辐照组克隆存活率明显降低，细胞凋亡率随辐照后CMG作用时间的延长而明显增加，CMG使辐照后细胞内ROS维持在一个较高的水平，同时CMG明显加重了单独辐照诱导的DNA损伤和周期阻滞。结果表明，与X射线相比，HepG2细胞对相同剂量的12C6+离子辐射更敏感；CMG可增加HepG2细胞对X射线或12C6+离子辐射的敏感性；CMG可能通过增加受照HepG2细胞内的ROS水平，加剧辐照诱导的DNA损伤，促进辐射诱导细胞凋亡而起到辐射增敏作用。 This study aims to investigate the effect of carboxymethy-β-1, 3-glucan (CMG) on the sensitivity of human hepatoma HepG2 cells to X-rays or 12C6+ ions irradiation. First, the inhibitory effect of CMG on the growth of HepG2 cells was detected by CCK-8 assay, and the half maximal inhibitory concentration (IC50) was 120.6 μg/mL. HepG2 cells were pretreated with CMG at a concentration of 0.1×IC50 for 24 h and then irradiated with 2 Gy X-ray or 12C6+ ion beams (CMG + irradiation group). CMG untreated group was directly irradiated by 2 Gy X-rays or 12C6+ ions beam (irradiation group). The clone survival, DNA damage, cell apoptosis, cell cycle distribution, and intracellular reactive oxygen species (ROS) levels in irradiation group and CMG + irradiation group were comparatively analyzed. The results showed that the clone survival rate was lower, DNA damage and cycle arrest were more serious, and the rate of apoptosis and intracellular ROS levels were higher in 12C6+ ions irradiation group than those in the same dose of X-rays irradiation group. Compared with X-rays or 12C6+ ions irradiation group, the clone survival rate of CMG + irradiation group was significantly decreased, and the apoptosis rate significantly increased with the prolongation of CMG treatment post-irradiation; CMG maintained intracellular ROS at a higher level after irradiation, CMG also significantly aggravated radiation-induced DNA damage and cycle arrest. These results indicated that HepG2 cells were more sensitive to 12C6+ ions radiation than those at the same dose of X-rays. CMG increased the sensitivity of HepG2 cells to X-rays or 12C6+ ions irradiation by increasing intracellular ROS level, exacerbating radiation-induced DNA damage and promoting radiation-induced apoptosis in irradiated HepG2 cells.

The ubiquitous proteoglycan, biglycan (BGN) acts as an important modulator, regulating key molecular pathways of metabolism and brain function. Autophagy is documented as a defining feature of neurodegeneration in Alzheimer's disease (AD), Parkinson's disease (PD), and Huntington's disease (HD). In the present study, we found that BGN protected neuronal cells from nitric oxide (NO)-induced cell apoptosis. However, it is still unclear that whether the neuroprotective effect of BGN relates to autophagy. Here, we discovered that an NO donor, sodium nitroprusside (SNP) induced autophagy in human SH-SY5Y neuroblastoma cells, including activating LC3B and inhibiting p62. Inhibiting autophagy by 3MA aggravated NO-induced cell death, otherwise promoting autophagy by Rapamycin rescued NO-triggered cell death. Notably, BGN downregulated by NO, significantly protected SH-SY5Y cells against NO-induced neurotoxicity by inhibiting the activation of autophagy-dependent AMPK signaling pathway. Moreover, BGN overexpression also diminished NO-induced the elevation of intracellular reactive oxygen species (ROS) level, but not NO content. These findings suggest that BGN protects neuroblastoma cells from NO-induced death by suppressing autophagy-dependent AMPK-mTOR signaling and intracellular ROS level.

Cryopreservation is known to induce oxidative stress in spermatozoa. Although melatonin has powerful antioxidant properties, little is known about its effects on human sperm quality during cryopreservation. The present study was undertaken to investigate the effects of melatonin treatment on human sperm parameters essential for fertilization. We first evaluated the effects of various concentrations of melatonin (0-15mM) on human sperm parameters such as motility, viability and levels of intracellular reactive oxygen species during cryopreservation in order to identify an optimal dose with the greatest effects for further studies. Liquefied semen samples were then divided into three aliquots: cryopreserved without melatonin (control), cryopreserved with 3mM melatonin and fresh groups. After being thawed, samples were evaluated for motility, viability, membrane integrity, intracellular reactive oxygen species levels, caspase-3 activity and AKT phosphorylation. Treatment of spermatozoa with the various concentrations of melatonin significantly increased their motility and viability and decreased their intracellular reactive oxygen species levels compared with the control group. The optimal melatonin concentration (3mM) significantly decreased the intracellular reactive oxygen species levels, caspase-3 activity and the percentage of both dead and apoptotic-like sperm cells and increased the vitality, progressive motility and total motility and AKT phosphorylation compared with the control group. Thus, melatonin exerts protective effects against cryodamage during human spermatozoa cryopreservation and may exert its effects via the PI3K/AKT signaling pathway.

The objective of this study was to investigate the effects of the combined RF radiation (837 MHz CDMA plus 1950 MHz WCDMA) signal on levels of intracellular reactive oxygen species (ROS) in neuronal cells. Exposure of the combined RF signal was conducted at specific absorption rate values of 2 W/kg of CDMA plus 2 W/kg of WCDMA for 2 h. Co-exposure to combined RF radiation with either H2O2 or menadione was also performed. The experimental exposure groups were incubator control, sham-exposed, combined RF radiation-exposed with or without either H2O2 or menadione groups. The intracellular ROS level was measured by flow cytometry using the fluorescent probe dichlorofluorescein diacetate. Intracellular ROS levels were not consistently affected by combined RF radiation exposure alone in a time-dependent manner in U87, PC12 or SH-SY5Y cells. In neuronal cells exposed to combined RF radiation with either H2O2 or menadione, intracellular ROS levels showed no statically significant alteration compared with exposure to menadione or H2O2 alone. These findings indicate that neither combined RF radiation alone nor combined RF radiation with menadione or H2O2 influences the intracellular ROS level in neuronal cells such as U87, PC12 or SH-SY5Y.

High atmospheric oxygen level during in vitro embryo culture system is reported to induce oxidative stress, leading to an increased intracellular reactive oxygen species (ROS) accumulation and lower the quality and development of the embryos. The Nrf2 is a redox sensitive transcription factor, which regulates the antioxidant machinery against oxidative stress in bovine pre-implantation embryos (Amin et al. 2014Mol. Reprod. Dev. 81, 497-513). Quercetin is a plant-derived flavonoid found in fruits and vegetables with antioxidant, anti-inflammatory, and anti-apoptotic properties. The effect of these antioxidants on the development and quality of bovine pre-implantation embryos with respect to the activation of the Nrf2 signalling pathways is not yet discovered. Here, we aimed to investigate the effect of quercetin supplementation on the activation of the Nrf2 signalling pathway and the subsequent impact on the quality of bovine embryos. For this, presumptive bovine zygotes were cultured in SOFaa embryo culture media supplemented with or without 10 µM quercetin (482 and 485 zygotes, respectively) and incubated under high oxygen level (20%) conditions. Day 7 and Day 8 blastocysts were analysed for the accumulation intracellular ROS and mitochondrial activity using H2DCFDA and MitoTracker® Red, respectively. Thereafter, blastocysts from both groups were subjected to total RNA isolation using Norgen total RNA purification plus kit (BioTek, Winooski, VT, USA). The relative abundance of Nrf2 and its downstream antioxidant genes (NQO1, PRDX1, SOD1, and CAT) was quantified using quantitative PCR. Moreover, the protein level of Nrf2 was detected using immunofluorescence staining. Data from 4 independent biological replicates were statistically analysed using a 2-tailed Student’s t-test. A significant difference in the mean value among treatments was determined at P=0.05. Results showed that quercetin supplementation in embryo culture medium significantly increased the blastocyst total cell number both at Day 7 (125±7.98 v. 100±3.74; P=0.007) and Day 8 (128±4.49 v. 95±3.15; P&amp;lt;0.0001). However, no difference was observed in the quercetin-treated group compared with control with respect to the cleavage rate (85.89±1.11v. 83.86±1.27; P=0.2) as well as the blastocyst rate at Day 7 (24.57±4.94 v. 27.23±1.68; P=0.64) and Day 8 (41.17±3.70 v. 38.87±0.85; P=0.58). Interestingly, quercetin-supplemented blastocysts showed a significant reduction in intracellular ROS level and increment in the mitochondrial activity compared with untreated counterparts. Supplementation of quercetin activated the Nrf2 transcriptional factor both at the mRNA and protein level, which subsequently activated the transcription of the aforementioned downstream antioxidant genes. In conclusion, supplementation of quercetin to embryo culture media protects embryos against oxidative stress by activating the Nrf2-mediated oxidative stress response mechanism, which leads to reduced accumulation of ROS and elevated mitochondrial activity.

UVB Radiation Induces Apoptosis in Keratinocytes by Activating a Pathway Linked to “BLT2-Reactive Oxygen Species”

High grade serous carcinoma (HGSC) of the ovary constitutes the majority of incidence and death of ovarian cancer, mainly because of the elusive etiology and cell of origin. Ovulation has been regarded as a strong risk factor and use of oral contraceptives (OC) as a protective factor for ovarian cancer with obvious dose (the span of ovulation life and duration of OC use) effect, but the mechanism is unknown. Meanwhile, more and more evidences have indicated the fallopian tube fimbriae to be the tissue of origin of HGSC. We hypothesize that high level of reactive oxygen species (ROS), which is indispensible for ovulation, as well as abundant mitogens in the mature follicular fluid is responsible for the initiation (DNA double strand breaks, DSB) and promotion (proliferation) of carcinogenesis to the fallopian tube secretory cells. We first tested the scrapped cells of the human fallopian tube epithelium for the extending of DSB. By flow cytometry with gating for the secretory cell marker PAX8 and DSB marker γH2AX, we found the secretory cells in the fimbriae harbored significantly more DSB than the those in the proximal tube. In an immortalized cell line of the human fimbriae secretory cell (we named FE25), we tested the mutagenic and mitogenic effects of the ROS and antioxidant in human follicular fluids at ovulation (FF). We found a balanced activity of of ROS and antioxidants in the undiluted follicular fluid with the antioxidants to be limited, such that, upon serial dilution, the intracellular ROS level raised proportionally. This dilution-raised effect of ROS was able to induce γH2Ax accumulation in the FE25 cells. Upon treating with sub-nM level of melatonin, the most potent and major antioxidant in FF, the level of intracellular ROS and γH2Ax readily decreased. Temporally, we found a two-stage effect of FF on the survival and proliferation of FE25, with an early phase of cell death, which occurred within 12 hours after FF treatment, followed by a cell expansion phase occurring at day 2. We interpret this two-stage effect to be an immediate ROS-induced DSB and cell apoptosis followed by a mitogen-induced proliferation of the survived cells. These survived cells carried the DSB and may be clonally expanded and move on to the carcinogenic road. We conclude that an imbalance favoring the ROS activity in the FF, likely from a significant dilution of FF by an unknown pathological status (such as fimbriae inflammation or ascites), may induce an oxidative stress and DSB in the fallopian tube fimbriae. Cells that survive this stress with some carrying DSB are expanded by the rich mitogens in the same FF. When enough mutations of critical genes were accumulated in a stem cell in this secretory population, cancer lesion such as STIC can thus arise. This abstract is also presented as Poster B5. Citation Format: Hsuan-Shun Huang, Che-Fang Hsu, Taolan Zhang, Tang-Yuan Chu. Oxidants and antioxidants in the follicular fluid in initiation of fimbriae carcinogenesis. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr PR03.

Inactivation of Listeria Monocytogenes at various growth temperatures by ultrasound pretreatment and cold plasma