Sirt3-dependent deacetylation of SOD2 plays a protective role against oxidative stress in oocytes from diabetic mice

Maternal diabetes has been demonstrated to adversely affect oocyte quality in mouse oocytes. However, the potential molecular mechanisms are poorly understood. Here, we established a type I diabetic mouse model and detected the increased reactive oxygen species (ROS) levels and decreased Sirt3 expression in oocytes from diabetic mice. Furthermore, we found that forced expression of Sirt3 in diabetic oocytes significantly attenuates such an excessive production of ROS. The acetylation status of lysine 68 of superoxide dismutase (SOD2K68) is dependent on Sirt3 in oocytes. In line with this, SOD2K68 acetylation levels were markedly increased in diabetic oocytes, and Sirt3 overexpression could effectively suppress this tendency. Importantly, the deacetylation-mimetic mutant SOD2K68R is capable of partly preventing the oxidative stress in oocytes from diabetic mice. In conclusion, our findings support a model where Sirt3 plays a protective role against oxidative stress in oocytes exposed to maternal diabetes through deacetylating SOD2K68.     

Loss of HDAC3 contributes to meiotic defects in aged oocytes

Maternal age‐related decline in oocyte quality is associated with meiotic defects, but the underlying mechanisms remain to be explored. Histone deacetylase 3 (HDAC3) has been shown to govern multiple cellular events via deacetylating diverse substrates. We previously found that HDAC3 could promote meiotic apparatus assembly in mouse oocytes. In the present study, we identified a substantial reduction in HDAC3 protein in oocytes from old mice. Importantly, overexpression of HDAC3 in old oocytes not only partially prevents spindle/chromosome disorganization, but also significantly lowers the incidence of aneuploidy. Meanwhile, we noticed the elevated acetylation level of α‐tubulin in oocytes derived from old mice. By employing site‐directed mutagenesis, we showed that acetylation‐mimetic mutant tubulin‐K40Q disrupts the kinetochore–microtubule attachments and results in the assembly failure of meiotic apparatus in mouse oocytes. Importantly, forced expression of tubulin‐K40R (nonacetylatable‐mimetic mutant) was capable of alleviating the defective phenotypes of oocytes from aged mice. To sum up, this study uncovers that loss of HDAC3 represents one potential mechanism mediating the effects of advanced maternal age on oocyte quality.     

Oxidative stress induces meiotic defects of oocytes in a mouse psoriasis model

Psoriasis, an immune-mediated inflammatory disease, is associated with poor pregnancy outcomes. Emerging evidence indicates that these defects are likely attributed to compromised oocyte competence. Nevertheless, little is known about the underlying associated mechanisms between psoriasis and poor oocyte quality. In this study, we construct an imiquimod-induced chronic psoriasis-like mouse model to review the effects of psoriasis on oocyte quality. We discover that oocytes from psoriasis-like mice display spindle/chromosome disorganization, kinetochore-microtubule mis-attachment, and aneuploidy. Importantly, our results show that melatonin supplement in vitro and in vivo not only increases the rate of matured oocytes but also significantly attenuates oxidative stress and meiotic defects by restoring mitochondrial function in oocytes from psoriasis-like mice. Altogether, our data uncover the adverse effects of psoriasis symptoms on oocytes, and melatonin supplement ameliorates oxidative stress and meiotic defects of oocytes from psoriatic mice.Subject terms: Meiosis, Immunology     

Induction of meiotic maturation in mouse oocytes by adenosine analogs

In this study we have examined the meiosis-inducing influence of adenosine analogs in mouse oocytes. When a varied group of nucleosides and nucleotides were tested on overnight cultures of hypoxanthine-arrested, cumulus cell-enclosed oocytes (CEO), halogenated adenosine nucleosides, but not native adenosine, exhibited a significant meiosis-inducing capability. When tested under a variety of conditions, meiotic induction by 8-bromo-adenosine (8-Br-Ado) and a second adenosine analog, methylmercaptopurine riboside (MMPR), was especially potent in denuded oocytes (DO) compared to CEO and was not dependent on the type of inhibitor chosen to maintain meiotic arrest. Germinal vesicle breakdown (GVB) was stimulated with rapid kinetics and was preceded by an increase in AMP-activated protein kinase (AMPK) activity. Moreover, compound C, an inhibitor of AMPK, blocked the meiosis-inducing activities of both adenosine analogs. When tested for an effect on meiotic progression to metaphase II (MII) in spontaneously maturing CEO, 8-Br-Ado and the AMPK activator, 5-aminoimidazole-4-carboxamide 1-beta-D-ribofuranoside (AICAR), increased the percentage of MII-stage oocytes, but MMPR decreased this number. Adenosine and inhibitors of de novo purine synthesis had no effect on the completion of maturation, while compound C suppressed this process. These results support the proposition that oocyte AMPK mediates the positive influence of AICAR and 8-Br-Ado on both the initiation and completion of meiotic maturation. The role of AMPK in MMPR action is less clear.     

Regulation of histone acetylation during meiotic maturation in mouse oocytes

Histone acetylation is an important epigenetic modification implicated in the regulation of chromatin structure and, subsequently, gene expression. Global histone deacetylation was reported in mouse oocytes during meiosis but not mitosis. The regulation of this meiosis-specific deacetylation has not been elucidated. Here, we demonstrate that p34(cdc2) kinase activity and protein synthesis are responsible for the activation of histone deacetylases and the inhibition of histone acetyltransferases (HATs), respectively, resulting in deacetylation of histone H4 at lysine-12 (H4K12) during mouse oocyte meiosis. Temporal changes in the acetylation state of H4K12 were examined immunocytochemically during meiotic maturation using an antibody specific for acetylated H4K12. H4K12 was deacetylated during the first meiosis, temporarily acetylated around the time of the first polar body (PB1) extrusion, and then deacetylated again during the second meiosis. Because these changes coincided with the known oscillation pattern of p34(cdc2) kinase activity, we investigated the involvement of the kinase in H4K12 deacetylation. Roscovitine, an inhibitor of cyclin-dependent kinase activity, prevented H4K12 deacetylation during both the first and second meiosis, suggesting that p34(cdc2) kinase activity is required for deacetylation during meiosis. In addition, cycloheximide, a protein synthesis inhibitor, also prevented deacetylation. After PB1 extrusion, at which time H4K12 had been deacetylated, H4K12 was re-acetylated in the condensed chromosomes by treatment with cycloheximide but not with roscovitine. These results demonstrate that HATs are present but inactivated by newly synthesized protein(s) that is (are) not involved in p34(cdc2) kinase activity. Our results suggest that p34(cdc2) kinase activity induces the deacetylation of H4K12 and that the deacetylated state is maintained by newly synthesized protein(s) that inhibits HAT activity during meiosis.     

Selection of antioxidants against ovarian oxidative stress in mouse model

Oxidative stress (OS) plays an important role in the process of ovarian granulosa cell apoptosis and follicular atresia. The aim of this study was to select antioxidant against OS in ovary tissue. Firstly, we chose the six antioxidants and analyzed the reactive oxygen species (ROS) level in the ovary tissue. The results showed that proanthocyanidins, gallic acid, curcumin, and carotene decrease the ROS level compared with control group. We further demonstrated that both proanthocyanidins and gallic acid increase the antioxidant enzymes activity. Moreover, change in the ROS level was not observed in proanthocyanidins and gallic acid group of brain, liver, spleen, and kidney tissues. Finally, we found that proanthocyanidins and gallic acid inhibit pro‐apoptotic genes expression in granulosa cells. Taken together, proanthocyanidins and gallic acid may be the most acceptable and optimal antioxidants specifically against ovarian OS and also may be involved in the inhibition of granulosa cells apoptosis in mouse ovary.     

Downregulation of sirtuin 3 by palmitic acid increases the oxidative stress,impairment of mitochondrial function,and apoptosis in liver cells

Elevated levels of saturated fatty acids show a strong cytotoxic effect in liver cells. Sirtuin 3 (SIRT3), a mitochondrially localized member of NAD⁺‐dependent deacetylase has been shown to protect hepatocytes against the oxidative stress. The role of SIRT3 on the cytotoxicity caused by fatty acids in liver cells is not fully understood. The aim of this study was to evaluate the expression level of SIRT3, oxidative stress, and mitochondrial impairments in human hepatoma HepG2 cells exposed to palmitic acid (PA). Our results showed that PA treatment caused the deposition of lipid droplets and resulted in an increased expression of tumor necrosis factor‐α in a dose‐dependent manner. Excessive accumulation of PA induces the reactive oxygen species formation and apoptosis while dissipating the mitochondrial transmembrane potential. The level of SIRT3 expression in both nuclear and mitochondrial fractions in HepG2 cells was decreased with the increase in PA concentrations. However, in the cytosolic fraction, the SIRT3 was undetectable. In conclusion, our results showed that PA caused an increase in inflammation and oxidative stress in HepG2 cells. The exposure of PA also resulted in the decline in transmembrane potential and an increase in apoptosis. The underexpression of nuclear and mitochondrial SIRT3 by PA suggests that the PA target the process that regulates the stress‐related gene expression and mitochondrial functions.     

A follicular fluid component prevents gonadotropin reversal of cyclic adenosine monophosphate-dependent meiotic arrest in murine oocytes

The effects of the putative maturation inhibitor in porcine follicular fluid on gonadotropinstimulated reversal of cyclic adenosine monophosphate (cAMP)-maintained meiotic arrest in mouse oocytes in vitro were assessed in this study. When cumulus cell-enclosed oocytes were cultured in a suboptimal inhibitory concentration of dibutyryl cAMP (dbcAMP), the effect of follicle-stimulating hormone (FSH) on oocyte maturation was initially inhibitory at 3 hr, but stimulatory at 6 hr. Supplementation of the medium with an ultrafiltrate of porcine follicuiar fluid (PM10-filtrate) completely suppressed FSH-promoted reversal of inhibition at 6 hr. Charcoal extraction eliminated this effect of the PM10-filtrate. FSH reversed the inhibition of maturation of cumulus cell-enclosed oocytes maintained by a high concentration of dbcAMP and suboptimal concentrations of the phosphodiesterase inhibitor, 3-isobutyl-1-methyl xanthine (IBMX), during a 21–22-hr culture period. However, the effect of a completely inhibitory concentration of IBMX was not reversed by gonadotropin. A component of serum was also found to inhibit FSH reversal of dbcAMP-maintained meiotic arrest, and this activity was removed by charcoal extraction. In addition, when oocytes were cultured in medium containing a suboptimal concentration of dbcAMP plus a low molecular weight fraction (< 1,000) of porcine follicular fluid, porcine serum, or fetal bovine serum, a synergistic inhibition of maturation was observed. Experiments with highly purified gonadotropins revealed that reversal of dbcAMP-maintained meiotic arrest occurred only in response to FSH; neither highly purified luteinizing hormone nor human chorionic gonadotropin could mimic this action of FSH. Also, this effect was mediated by the cumulus cells, since FSH could not reverse dbcAMP-maintained meiotic arrest in denuded oocytes. Furthermore, elevating cAMP levels in denuded oocytes augmented, rather than reversed, the inhibitory action of dbcAMP on oocyte maturation. These data therefore suggest that dbcAMP- or IBMX-maintained meiotic arrest in vitro is reversed by an FSH-stimulated, cAMP-dependent process mediated by the cumulus cells and demonstrate that a factor present both in follicular fluid and serum prevents this action of the gonadotropin.     

Differential response of cumulus cell-enclosed and denuded mouse oocytes in a meiotic induction model system

In this study we have examined the effects of denuded oocyte coculture with dissociated cumulus cells (CC) or intact oocyte-CC complexes on meiotic resumption. When denuded oocytes (DO) or cumulus cell-enclosed oocytes (CEO) were cultured in 40-microl drops of medium under oil, and held in meiotic arrest with 4 mM hypoxanthine plus 25 microM dbcAMP, they underwent germinal vesicle breakdown (GVB) at similar frequencies (34%-35%). Coculture of DO with complexes or dissociated CCs stimulated maturation (50% and 61% GVB, respectively), with no effect of DO on maturation of cocultured CEO (32% GVB). This coculture effect was increased with the number of CCs added to the culture drop. When either glucose or glutamine was eliminated from the medium, no meiotic induction resulted from cocultured CCs. When CEO were cultured alone in microdrops, increasing their number from 10 to 50 significantly lowered the percentage resuming maturation, an effect also reduced by removing glucose and/or glutamine from the medium. This effect was not observed with DO. When inhibitory medium was conditioned overnight with complexes, subsequent culture with DO led to higher maturation percentages than culture in unconditioned medium; however, when CEO were cultured in conditioned medium, there was either no effect or increased inhibition of maturation. Assay of glucose and pyruvate in spent medium showed that DO cultured alone consumed glucose and pyruvate, but under CC coculture conditions more glucose was consumed and significant amounts of pyruvate accumulated in the medium, changes that led to an increase in the maturation of DO. Further experiments showed that DO were more sensitive than CEO to the meiosis-inducing effect of pyruvate. These results demonstrate different responsiveness of DO and CEO to coculture conditions and question the physiological relevance of denuded oocyte/CC coculture to study meiotic induction.     

Pyruvate utilization by mouse oocytes is influenced by meiotic status and the cumulus oophorus

In this study, the effects of meiotic status on the energy substrate dynamics of mouse oocyte-cumulus cell complexes (OCCs) and denuded oocytes (DOs) have been examined. In the first series of experiments, OCCs from PMSG-primed, immature mice were cultured in minimum essential medium in 8-microl microdrops under a variety of conditions, and the medium and oocytes were sampled for pyruvate and glucose concentration and for meiotic status. Oocytes in control medium underwent germinal vesicle breakdown within 3 hr and the OCCs displayed a time-dependent increase in pyruvate consumption, but the glucose concentration changed very little. Treatment with IBMX or dbcAMP, which maintained complete meiotic arrest, suppressed pyruvate consumption, but slightly more glucose was consumed than in controls. Hypoxanthine (HX) allowed up to 10% of the oocytes to resume maturation, and pyruvate and glucose consumption resembled that of control OCCs. FSH added to HX-containing medium stimulated significant glucose consumption and pyruvate production. In general, a reciprocal relationship was observed between glucose and pyruvate consumption. When the energy substrate dynamics were compared with meiotic status of the oocytes, pyruvate consumption was associated with the maturation process. Although HX maintained oocytes in the germinal vesicle stage, the meiotic arrest was "leaky," allowing increased pyruvate consumption. Additional experiments showed that DOs at either the prophase I or metaphase II stages consumed less pyruvate than oocytes actively engaged in meiotic maturation. DOs oxidized significantly more pyruvate than OCCs, and glycolytic metabolism of glucose lowered the oxidation rate in OCCs. Furthermore, while 5-6.2 times more pyruvate was consumed by OCCs than by DOs in the absence of glucose, oxidation did not mediate the meiosis-inducing effect of pyruvate, since less of this substrate was oxidized by OCCs than by DOs. We conclude that meiotically active oocytes have a greater requirement for pyruvate than prophase I- or metaphase II-arrested oocytes and that meiotic status can influence the metabolism not only of oocytes, but also of the OCCs.     

Autophagy of metallothioneins prevents TNF-induced oxidative stress and toxicity in hepatoma cells

Lysosomal membrane permeabilization (LMP) induced by oxidative stress has recently emerged as a prominent mechanism behind TNF cytotoxicity. This pathway relies on diffusion of hydrogen peroxide into lysosomes containing redox-active iron, accumulated by breakdown of iron-containing proteins and subcellular organelles. Upon oxidative lysosomal damage, LMP allows relocation to the cytoplasm of low mass iron and acidic hydrolases that contribute to DNA and mitochondrial damage, resulting in death by apoptosis or necrosis. Here we investigate the role of lysosomes and free iron in death of HTC cells, a rat hepatoma line, exposed to TNF following metallothionein (MT) upregulation. Iron-binding MT does not normally occur in HTC cells in significant amounts. Intracellular iron chelation attenuates TNF and cycloheximide (CHX)-induced LMP and cell death, demonstrating the critical role of this transition metal in mediating cytokine lethality. MT upregulation, combined with starvation-activated MT autophagy almost completely suppresses TNF and CHX toxicity, while impairment of both autophagy and MT upregulation by silencing of Atg7, and Mt1a and/or Mt2a, respectively, abrogates protection. Interestingly, MT upregulation by itself has little effect, while stimulated autophagy alone depresses cytokine toxicity to some degree. These results provide evidence that intralysosomal iron-catalyzed redox reactions play a key role in TNF and CHX-induced LMP and toxicity. The finding that chelation of intralysosomal iron achieved by autophagic delivery of MT, and to some degree probably of other iron-binding proteins as well, into the lysosomal compartment is highly protective provides a putative mechanism to explain autophagy-related suppression of death by TNF and CHX.     

Fructose 1,6-bisphosphate prevents oxidative stress in the isolated and perfused rat heart

Rat hearts were perfused with the Langendorff technique at constant flux in the presence of the oxidizing agents hydrogen peroxide and diamide. Fructose 1,6-bisphosphate strongly prevented the decline of heart contractility due to the infusion of these oxidizing agents. On the other hand, fructose 1,6-bisphosphate had no effect on the release of total glutathione into the perfusate but prevented the loss of lactate dehydrogenase indicating a protective effect on cell membranes. Comparing the cytosolic and mitochondrial loss of glutathione, fructose 1,6-bisphosphate exerted a beneficial action only on the mitochondrial fraction. Several mechanisms of action have been considered to explain the protective action of frutose 1,6-bisphosphate. In our experimental conditions fructose 1,6-bisphosphate might stimulate its own production giving rise to dihydroxyacetone phosphate, that, after reduction to glycerol 3-phosphate, can permeate the mitochondrial membrane with the final production of energy.     

Hydroxytyrosol prevents dermal papilla cells inflammation under oxidative stress by inducing autophagy

Hydroxytyrosol (HT), a primary phenolic antioxidant in olive oil, can afford protection from oxidative stress (OS) in different cells, including skin cells. In particular, it regulates several inflammation‐associated processes as well as in improving the antioxidant defense system. However, there is no information about HT used in the treatment of hair loss. This work aimed at exploring the potential protective actions of HT against OS in rat dermal papilla cells. After treatment, the related expression of protein and messenger RNA were detected using morphological and molecular analyses. The results showed that HT significantly reduced intracellular reactive oxygen species level, apoptotic markers and inflammation induced by OS and enhanced cell survival by regulating autophagy. Furthermore, HT enhanced the secretion of hair growth factors in the anti‐inflammation process. These results suggest that HT has a significant protective ability against OS and encourage the use of this biological ingredient as a possible tool to prevent alopecia.     

Physical training prevents oxidative stress in L‐NAME‐induced hypertension rats

The present study investigated the effects of a 6‐week swimming training on blood pressure, nitric oxide (NO) levels and oxidative stress parameters such as protein and lipid oxidation, antioxidant enzyme activity and endogenous non‐enzymatic antioxidant content in kidney and circulating fluids, as well as on serum biochemical parameters (cholesterol, triglycerides, urea and creatinine) from Nω‐nitro‐L‐arginine methyl ester hydrochloride (L‐NAME)‐induced hypertension treated rats. Animals were divided into four groups (n = 10): Control, Exercise, L‐NAME and Exercise L‐NAME. Results showed that exercise prevented a decrease in NO levels in hypertensive rats (P < 0·05). An increase in protein and lipid oxidation observed in the L‐NAME‐treated group was reverted by physical training in serum from the Exercise L‐NAME group (P < 0·05). A decrease in the catalase (CAT) and superoxide dismutase (SOD) activities in the L‐NAME group was observed when compared with normotensive groups (P < 0·05). In kidney, exercise significantly augmented the CAT and SOD activities in the Exercise L‐NAME group when compared with the L‐NAME group (P < 0·05). There was a decrease in the non‐protein thiols (NPSH) levels in the L‐NAME‐treated group when compared with the normotensive groups (P < 0·05). In the Exercise L‐NAME group, there was an increase in NPSH levels when compared with the L‐NAME group (P < 0·05). The elevation in serum cholesterol, triglycerides, urea and creatinine levels observed in the L‐NAME group were reverted to levels close to normal by exercise in the Exercise L‐NAME group (P < 0·05). Exercise training had hypotensive effect, reducing blood pressure in the Exercise L‐NAME group (P < 0·05). These findings suggest that physical training could have a protector effect against oxidative damage and renal injury caused by hypertension. Copyright © 2012 John Wiley & Sons, Ltd.     

GTPase-activating protein Elmod2 is essential for meiotic progression in mouse oocytes

Meiotic failure in oocytes is the major determinant of human zygote-originated reproductive diseases, the successful accomplishment of meiosis largely relay on the normal functions of many female fertility factors. Elmod2 is a member of the Elmod family with the strongest GAP (GTPase-activating protein) activity; although it was identified as a possible maternal protein, its actual physiologic role in mammalian oocytes has not been elucidated. Herein we reported that among Elmod family proteins, Elmod2 is the most abundant in mouse oocytes, and that inhibition of Elmod2 by specific siRNA caused severe meiotic delay and abnormal chromosomal segregation during anaphase. Elmod2 knockdown also significantly decreased the rate of oocyte maturation (to MII, with first polar body extrusion), and significantly greater numbers of Elmod2-knockdown MII oocytes were aneuploid. Correspondingly, Elmod2 knockdown dramatically decreased fertilization rate. To investigate the mechanism(s) involved, we found that Elmod2 knockdown caused significantly more abnormal mitochondrial aggregation and diminished cellular ATP levels; and we also found that Elmod2 co-localized and interacted with Arl2, a GTPase that is known to maintain mitochondrial dynamics and ATP levels in oocytes. In summary, we found that Elmod2 is the GAP essential to meiosis progression of mouse oocytes, most likely by regulating mitochondrial dynamics.